Patent application title: Methods and Systems for Analyzing Nucleic Acid Molecules
Inventors:
IPC8 Class: AC12Q16886FI
USPC Class:
1 1
Class name:
Publication date: 2021-06-10
Patent application number: 20210172022
Abstract:
Processes and materials to detect cancer from a biopsy are described. In
some cases, cell-free nucleic acids can be sequenced, and the sequencing
result can be utilized to detect sequences derived from a neoplasm.
Detection of somatic variants occurring in phase can indicate the
presence of cancer in a diagnostic scan and a clinical intervention can
be performed.Claims:
1. A method comprising: (a) obtaining, by a computer system, sequencing
data derived from a plurality of cell-free nucleic acid molecules that is
obtained or derived from a subject; (b) processing, by the computer
system, the sequencing data to identify one or more cell-free nucleic
acid molecules of the plurality of cell-free nucleic acid molecules,
wherein each of the one or more cell-free nucleic acid molecules
comprises a plurality of phased variants relative to a reference genomic
sequence, wherein at least about 10% of the one or more cell-free nucleic
acid molecules comprises a first phased variant of the plurality of
phased variants and a second phased variant of the plurality of phased
variants that are separated by at least one nucleotide; and (c)
analyzing, by the computer system, the identified one or more cell-free
nucleic acid molecules to determine a condition of the subject.
2.-115. (canceled)
116. The method of claim 1, further comprising separating, in silico, (i) at least a portion of the identified one or more cell-free nucleic acid molecules from (ii) one or more other cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules that are not identified to comprise the plurality of phased variants.
117. The method of claim 116, further comprising analyzing, by the computer system, (i) and (ii) as different variables.
118. The method of claim 1, wherein the analyzing of the identified one or more cell-free nucleic acid molecules is not based on other cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules that are not identified to comprise the plurality of phased variants.
119. The method of claim 1, wherein the sequencing data is obtained without in silico removal or suppression of (i) background error or (ii) sequencing error.
120. The method of claim 1, wherein a number of the plurality of phased variants from the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject.
121. The method of claim 120, wherein a ratio of (i) the number of the plurality of phased variants from the identified one or more cell-free nucleic acid molecules and (ii) a number of single nucleotide variants (SNVs) from the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject.
122. The method of claim 1, wherein a frequency of the plurality of phased variants in the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject.
123. The method of claim 1, wherein the at least about 10% of the one or more cell-free nucleic acid molecules comprises at least about 50% of the one or more cell-free nucleic acid molecules.
124. The method of claim 123, wherein the at least about 10% of the one or more cell-free nucleic acid molecules comprises at least about 100% of the one or more cell-free nucleic acid molecules.
125. The method of claim 1, wherein the first and second phased variants are separated by at least 2 nucleotides.
126. The method of claim 1, wherein the first phased variant and the second phased variant are separated by at most about 160 nucleotides.
127. The method of claim 1, wherein the reference genome sequence comprises at least a portion of hg19 human genome, hg18 genome, hg17 genome, hg16 genome, or hg38 genome.
128. The method of claim 1, wherein the reference genomic sequence is derived from a sample of the subject.
129. The method of claim 1, wherein the reference genomic sequence is derived from a healthy cell of the subject.
130. The method of claim 1, wherein the plurality of cell-free nucleic acid molecules comprise a plurality of cell-free deoxyribonucleic acid (DNA) molecules.
131. The method of claim 1, wherein the condition comprises neoplasm, cancer, or tumor.
132. The method of claim 1, wherein the condition comprises a solid tumor.
133. The method of claim 1, wherein the condition comprises a lymphoma.
134. The method of claim 1, further comprising determining, by the computer, that the identified one or more cell-free nucleic acid molecules are derived from a sample associated with the condition, based on performing a statistical model analysis of the identified one or more cell-free nucleic acid molecules.
135. A method comprising: (a) providing a mixture comprising (1) a set of nucleic acid probes and (2) a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject, wherein an individual nucleic acid probe of the set of nucleic acid probes is designed to hybridize to at least a portion of a target cell-free nucleic acid molecule comprising a plurality of phased variants relative to a reference genomic sequence that are separated by at least one nucleotide, and wherein the individual nucleic acid probe comprises an activatable reporter agent, activation of the activatable reporter agent being selected from the group consisting of: (i) hybridization of the individual nucleic acid probe to the plurality of phased variants and (ii) dehybridization of at least a portion of the individual nucleic acid probe that has been hybridized to the plurality of phased variants; (b) detecting the activatable reporter agent that is activated, to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules, wherein each of the one or more cell-free nucleic acid molecules comprises the plurality of phased variants; and (c) analyzing the identified one or more cell-free nucleic acid molecules to determine a condition of the subject.
136. The method of claim 135, wherein the activatable reporter agent is activated upon hybridization of the individual nucleic acid probe to the plurality of phased variants.
137. The method of claim 135, wherein the activatable reporter agent is activated upon dehybridization of at least a portion of the individual nucleic acid probe that has been hybridized to the plurality of phased variants.
138. The method of claim 135, wherein the activatable reporter agent is a fluorophore.
139. The method of claim 135, a number of the plurality of phased variants from the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject.
140. A method comprising: (a) obtaining sequencing data derived from a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject; (b) processing the sequencing data to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules with a limit of detection of less than about 1 out of 50,000 observations from the sequencing data; and (c) analyzing the identified one or more cell-free nucleic acid molecules to determine a condition of the subject, wherein each of the one or more cell-free nucleic acid molecules comprises a plurality of phased variants relative to a reference genomic sequence.
141. The method of claim 140, wherein the limit of detection of the identification step is less than about 1 out of 300,000 observations from the sequencing data.
142. The method of claim 141, wherein the limit of detection of the identification step is less than about 1 out of 1,000,000 observations from the sequencing data.
143. The method of claim 140, wherein a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants are separated by at least one nucleotide.
144. A composition comprising a bait set comprising a set of nucleic acid probes designed to capture cell-free DNA molecules derived from at least about 5% of genomic regions set forth in (i) the genomic regions identified in Table 1, (ii) the genomic regions identified in Table 3, or (iii) the genomic regions identified to have a plurality of phased variants in Table 3.
Description:
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/931,688, filed Nov. 6, 2019, which is entirely incorporated herein by reference.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 3, 2020, is named 58626-702_601_SL.txt and is 307,199 bytes in size.
BACKGROUND
[0004] Noninvasive blood tests that can detect somatic alterations (e.g., mutated nucleic acids) based on the analysis of cell-free nucleic acids (e.g., cell-free deoxyribonucleic acid (cfDNA) and cell-free ribonucleic acid (cfRNA)) are attractive candidates for cancer screening applications due to the relative ease of obtaining biological specimens (e.g., biological fluids). Circulating tumor nucleic acids (e.g., ctDNA or ctRNA; i.e., nucleic acids derived from cancerous cells) can be sensitive and specific biomarkers in numerous cancer subtypes. However, current methods for minimal residual disease (MRD) detection from ctDNA can be limited by one or more factors, such as low input DNA amounts and high background error rates.
[0005] Recent approaches have improved ctDNA MRD performance by tracking multiple somatic mutations with error-suppressed sequencing, resulting in detection limits as low as 4 parts in 100,000 from limited cfDNA input. Detection of residual disease during or after treatment is a powerful tool, with detectable MRD representing an adverse prognostic sign even during radiographic remission. However, current limits of detection may be insufficient to universally detect residual disease in patients destined for disease relapse or progression. This `loss of detection` is exemplified in diffuse large B-cell lymphoma (DLBCL), where ctDNA detection after two cycles of curative-intent therapy is a strong prognostic marker. Despite this, almost one-third of patients experiencing disease progression do not have detectable ctDNA at this landmark, representing `false-negative` tests. Similar false-negative rates in colon cancer and breast cancer have been observed.
SUMMARY
[0006] The present disclosure provides methods and systems for analyzing cell-free nucleic acids (e.g., cfDNA, cfRNA) from a subject. Methods and systems of the present disclosure can utilize sequencing results derived from the subject to detect cancer-derived nucleic acids (e.g., ctDNA, ctRNA) for, e.g., disease diagnosis, disease monitoring, or determining treatments for the subject. Methods and systems of the present disclosure can exhibit enhanced sensitivity, specificity and/or reliability of detection of cancer-derived nucleic acids.
[0007] In one aspect, the present disclosure provides a method comprising: (a) obtaining, by a computer system, sequencing data derived from a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject; (b) processing, by the computer system, the sequencing data to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules, wherein each of the one or more cell-free nucleic acid molecules comprises a plurality of phased variants relative to a reference genomic sequence, wherein at least about 10% of the one or more cell-free nucleic acid molecules comprises a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants that are separated by at least one nucleotide; and (c) analyzing, by the computer system, the identified one or more cell-free nucleic acid molecules to determine a condition of the subject.
[0008] In some embodiments of any one of the methods disclosed herein, the at least about 10% of the cell-free nucleic acid molecules comprise at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the one or more cell-free nucleic acid molecules.
[0009] In one aspect, the present disclosure provides a method comprising: (a) obtaining, by a computer system, sequencing data derived from a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject; (b) processing, by the computer system, the sequencing data to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules, wherein each of the one or more cell-free nucleic acid molecules comprises a plurality of phased variants relative to a reference genomic sequence that are separated by at least one nucleotide; and (c) analyzing, by the computer system, the identified one or more cell-free nucleic acid molecules to determine a condition of the subject.
[0010] In one aspect, the present disclosure provides a method comprising: (a) obtaining sequencing data derived from a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject; (b) processing the sequencing data to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules with a limit of detection of less than about 1 out of 50,000 observations from the sequencing data; and (c) analyzing the identified one or more cell-free nucleic acid molecules to determine a condition of the subject.
[0011] In some embodiments of any one of the methods disclosed herein, the limit of detection of the identification step is less than about 1 out of 100,000, less than about 1 out of 500,000, less than about 1 out of 1,000,000, less than about 1 out of 1,500,000, or less than about 1 out of 2,000,000 observations from the sequencing data.
[0012] In some embodiments of any one of the methods disclosed herein, each of the one or more cell-free nucleic acid molecules comprises a plurality of phased variants relative to a reference genomic sequence. In some embodiments of any one of the methods disclosed herein, a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants are separated by at least one nucleotide.
[0013] In some embodiments of any one of the methods disclosed herein, the processes (a) to (c) are performed by a computer system.
[0014] In some embodiments of any one of the methods disclosed herein, the sequencing data is generated based on nucleic acid amplification. In some embodiments of any one of the methods disclosed herein, the sequencing data is generated based on polymerase chain reaction. In some embodiments of any one of the methods disclosed herein, the sequencing data is generated based on amplicon sequencing.
[0015] In some embodiments of any one of the methods disclosed herein, the sequencing data is generated based on next-generation sequencing (NGS). Alternatively, in some embodiments of any one of the methods disclosed herein, the sequencing data is generated based on non-hybridization-based NGS.
[0016] In some embodiments of any one of the methods disclosed herein, the sequencing data is generated without use of molecular barcoding of at least a portion of the plurality of cell-free nucleic acid molecules. In some embodiments of any one of the methods disclosed herein, the sequencing data is obtained without use of sample barcoding of at least a portion of the plurality of cell-free nucleic acid molecules.
[0017] In some embodiments of any one of the methods disclosed herein, the sequencing data is obtained without in silico removal or suppression of (i) background error or (ii) sequencing error.
[0018] In one aspect, the present disclosure provides a method of treating a condition of a subject, the method comprising: (a) identifying the subject for treatment of the condition, wherein the subject has been determined to have the condition based on identification of one or more cell-free nucleic acid molecules from a plurality of cell-free nucleic acid molecules that is obtained or derived from the subject, wherein each of the one or more cell-free nucleic acid molecules identified comprises a plurality of phased variants relative to a reference genomic sequence that are separated by at least one nucleotide, and wherein a presence of the plurality of phased variants is indicative of the condition of the subject; and (b) subjecting the subject to the treatment based on the identification in (a).
[0019] In one aspect, the present disclosure provides a method of monitoring a progress of a condition of a subject, the method comprising: (a) determining a first state of the condition of the subject based on identification of a first set of one or more cell-free nucleic acid molecules from a first plurality of cell-free nucleic acid molecules that is obtained or derived from the subject; (b) determining a second state of the condition of the subject based on identification of a second set of one or more cell-free nucleic acid molecules from a second plurality of cell-free nucleic acid molecules that is obtained or derived from the subject, wherein the second plurality of cell-free nucleic acid molecules are obtained from the subject subsequent to obtaining the first plurality of cell-free nucleic acid molecules from the subject; and (c) determining the progress of the condition based on the first state of the condition and the second state of the condition, wherein each of the one or more cell-free nucleic acid molecules comprises a plurality of phased variants relative to a reference genomic sequence that are separated by at least one nucleotide.
[0020] In some embodiments of any one of the methods disclosed herein, the progress of the condition is worsening of the condition.
[0021] In some embodiments of any one of the methods disclosed herein, the progress of the condition is at least a partial remission of the condition.
[0022] In some embodiments of any one of the methods disclosed herein, a presence of the plurality of phased variants is indicative of the first state or the second state of the condition of the subject.
[0023] In some embodiments of any one of the methods disclosed herein, the second plurality of cell-free nucleic acid molecules is obtained from the subject at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, or at least about 3 months subsequent to obtaining the first plurality of cell-free nucleic acid molecules from the subject.
[0024] In some embodiments of any one of the methods disclosed herein, the subject is subjected to a treatment for the condition (i) prior to obtaining the second plurality of cell-free nucleic acid molecules from the subject and (ii) subsequent to obtaining the first plurality of cell-free nucleic acid molecules from the subject.
[0025] In some embodiments of any one of the methods disclosed herein, the progress of the condition is indicative of minimal residual disease of the condition of the subject. In some embodiments of any one of the methods disclosed herein, the progress of the condition is indicative of tumor burden or cancer burden of the subject.
[0026] In some embodiments of any one of the methods disclosed herein, the one or more cell-free nucleic acid molecules are captured from among the plurality of cell-free nucleic acid molecules with a set of nucleic acid probes, wherein the set of nucleic acid probes is configured to hybridize to at least a portion of cell-free nucleic acid molecules comprising one or more genomic regions associated with the condition.
[0027] In one aspect, the present disclosure provides a method comprising: (a) providing a mixture comprising (1) a set of nucleic acid probes and (2) a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject, wherein an individual nucleic acid probe of the set of nucleic acid probes is designed to hybridize to at least a portion of a target cell-free nucleic acid molecule comprising a plurality of phased variants relative to a reference genomic sequence that are separated by at least one nucleotide, and wherein the individual nucleic acid probe comprises an activatable reporter agent, activation of the activatable reporter agent being selected from the group consisting of: (i) hybridization of the individual nucleic acid probe to the plurality of phased variants and (ii) dehybridization of at least a portion of the individual nucleic acid probe that has been hybridized to the plurality of phased variants; (b) detecting the activatable reporter agent that is activated, to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules, wherein each of the one or more cell-free nucleic acid molecules comprises the plurality of phased variants; and (c) analyzing the identified one or more cell-free nucleic acid molecules to determine a condition of the subject.
[0028] In one aspect, the present disclosure provides a method comprising: (a) providing a mixture comprising (1) a set of nucleic acid probes and (2) a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject, wherein an individual nucleic acid probe of the set of nucleic acid probes is designed to hybridize to at least a portion of a target cell-free nucleic acid molecule comprising a plurality of phased variants relative to a reference genomic sequence, and wherein the individual nucleic acid probe comprises an activatable reporter agent, activation of the activatable reporter agent being selected from the group consisting of: (i) hybridization of the individual nucleic acid probe to the plurality of phased variants and (ii) dehybridization of at least a portion of the individual nucleic acid probe that has been hybridized to the plurality of phased variants; (b) detecting the activatable reporter agent that is activated, to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules, wherein each of the one or more cell-free nucleic acid molecules comprises the plurality of phased variants, wherein a limit of detection of the identification step is less than about 1 out of 50,000 cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules; and (c) analyzing the identified one or more cell-free nucleic acid molecules to determine a condition of the subject.
[0029] In some embodiments of any one of the methods disclosed herein, the limit of detection of the identification step is less than about 1 out of 100,000, less than about 1 out of 500,000, less than about 1 out of 1,000,000, less than about 1 out of 1,500,000, or less than about 1 out of 2,000,000 cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules.
[0030] In some embodiments of any one of the methods disclosed herein, a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants are separated by at least one nucleotide.
[0031] In some embodiments of any one of the methods disclosed herein, the activatable reporter agent is activated upon hybridization of the individual nucleic acid probe to the plurality of phased variants.
[0032] In some embodiments of any one of the methods disclosed herein, the activatable reporter agent is activated upon dehybridization of at least a portion of the individual nucleic acid probe that has been hybridized to the plurality of phased variants.
[0033] In some embodiments of any one of the methods disclosed herein, the method further comprises mixing (1) the set of nucleic acid probes and (2) the plurality of cell-free nucleic acid molecules.
[0034] In some embodiments of any one of the methods disclosed herein, the activatable reporter agent is a fluorophore.
[0035] In some embodiments of any one of the methods disclosed herein, analyzing the identified one or more cell-free nucleic acid molecules comprises analyzing (i) the identified one or more cell-free nucleic acid molecules and (ii) other cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules that do not comprise the plurality of phased variants as different variables.
[0036] In some embodiments of any one of the methods disclosed herein, the analyzing of the identified one or more cell-free nucleic acid molecules is not based on other cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules that do not comprise the plurality of phased variants.
[0037] In some embodiments of any one of the methods disclosed herein, a number of the plurality of phased variants from the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject. In some embodiments, a ratio of (i) the number of the plurality of phased variants from the one or more cell-free nucleic acid molecules and (ii) a number of single nucleotide variants (SNVs) from the one or more cell-free nucleic acid molecules is indicative of the condition of the subject.
[0038] In some embodiments of any one of the methods disclosed herein, a frequency of the plurality of phased variants in the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject. In some embodiments, the frequency is indicative of a diseased cell associated with the condition. In some embodiments, the condition is diffuse large B-cell lymphoma, and wherein the frequency is indicative of whether the one or more cell-free nucleic acid molecules are derived from germinal center B-cell (GCB) or activated B-cell (ABC).
[0039] In some embodiments of any one of the methods disclosed herein, genomic origin of the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject.
[0040] In some embodiments of any one of the methods disclosed herein, the first and second phased variants are separated by at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 nucleotides. In some embodiments of any one of the methods disclosed herein, the first and second phased variants are separated by at most about 180, at most about 170, at most about 160, at most about 150, or at most about 140 nucleotides.
[0041] In some embodiments of any one of the methods disclosed herein, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of the one or more cell-free nucleic acid molecules comprising a plurality of phased variants comprises a single nucleotide variant (SNV) that is at least 2 nucleotides away from an adjacent SNV.
[0042] In some embodiments of any one of the methods disclosed herein, the plurality of phased variants comprises at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 phased variants within the same cell-free nucleic acid molecule.
[0043] In some embodiments of any one of the methods disclosed herein, the one or more cell-free nucleic acid molecules identified comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 50, at least 100, at least 500, or at least 1,000 cell-free nucleic acid molecules.
[0044] In some embodiments of any one of the methods disclosed herein, the reference genomic sequence is derived from a reference cohort. In some embodiments, the reference genomic sequence comprises a consensus sequence from the reference cohort. In some embodiments, the reference genomic sequence comprises at least a portion of hg19 human genome, hg18 genome, hg17 genome, hg16 genome, or hg38 genome.
[0045] In some embodiments of any one of the methods disclosed herein, the reference genomic sequence is derived from a sample of the subject.
[0046] In some embodiments of any one of the methods disclosed herein, the sample is a healthy sample. In some embodiments, the sample comprises a healthy cell. In some embodiments, the healthy cell comprises a healthy leukocyte.
[0047] In some embodiments of any one of the methods disclosed herein, the sample is a diseased sample. In some embodiments, the diseased sample comprises a diseased cell. In some embodiments, the diseased cell comprises a tumor cell. In some embodiments, the diseased sample comprises a solid tumor.
[0048] In some embodiments of any one of the methods disclosed herein, the set of nucleic acid probes is designed based on the plurality of phased variants that are identified by comparing (i) sequencing data from a solid tumor, lymphoma, or blood tumor of the subject and (ii) sequencing data from a healthy cell of the subject or a healthy cohort. In some embodiments, the healthy cell is from the subject. In some embodiments, the healthy cell is from the healthy cohort.
[0049] In some embodiments of any one of the methods disclosed herein, the set of nucleic acid probes are designed to hybridize to at least a portion of sequences of genomic loci associated with the condition. In some embodiments, the genomic loci associated with the condition are known to exhibit aberrant somatic hypermutation when the subject has the condition.
[0050] In some embodiments of any one of the methods disclosed herein, the set of nucleic acid probes are designed to hybridize to at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of (i) the genomic regions identified in Table 1, (ii) the genomic regions identified in Table 3, or (iii) the genomic regions identified to have a plurality of phased variants in Table 3.
[0051] In some embodiments of any one of the methods disclosed herein, each nucleic acid probe of the set of nucleic acid probes has at least about 70%, at least about 80%, at least about 90% sequence identity, at least about 95% sequence identity, or about 100% sequence identity to a probe sequence selected from Table 6.
[0052] In some embodiments of any one of the methods disclosed herein, the set of nucleic acid probes comprises at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of probe sequences in Table 6.
[0053] In some embodiments of any one of the methods disclosed herein, the method further comprises determining that the subject has the condition or determining a degree or status of the condition of the subject, based on the identified one or more cell-free nucleic acid molecules comprising the plurality of phased variants. In some embodiments, the method further comprises determining that the one or more cell-free nucleic acid molecules are derived from a sample associated with the condition, based on performing a statistical model analysis of the identified one or more cell-free nucleic acid molecules. In some embodiments, the statistical model analysis comprises a Monte Carlo statistical analysis.
[0054] In some embodiments of any one of the methods disclosed herein, the method further comprises monitoring a progress of the condition of the subject based on the identified one or more cell-free nucleic acid molecules.
[0055] In some embodiments of any one of the methods disclosed herein, the method further comprises performing a different procedure to confirm the condition of the subject. In some embodiments, the different procedure comprises a blood test, genetic test, medical imaging, physical exam, or tissue biopsy.
[0056] In some embodiments of any one of the methods disclosed herein, the method further comprises determining a treatment for the condition of the subject based on the identified one or more cell-free nucleic acid molecules.
[0057] In some embodiments of any one of the methods disclosed herein, the subject has been subjected to a treatment for the condition prior to (a).
[0058] In some embodiments of any one of the methods disclosed herein, the treatment comprises chemotherapy, radiotherapy, chemoradiotherapy, immunotherapy, adoptive cell therapy, hormone therapy, targeted drug therapy, surgery, transplant, transfusion, or medical surveillance.
[0059] In some embodiments of any one of the methods disclosed herein, the plurality of cell-free nucleic acid molecules comprise a plurality of cell-free deoxyribonucleic acid (DNA) molecules.
[0060] In some embodiments of any one of the methods disclosed herein, condition comprises a disease.
[0061] In some embodiments of any one of the methods disclosed herein, the plurality of cell-free nucleic acid molecules are derived from a bodily sample of the subject. In some embodiments, the bodily sample comprises plasma, serum, blood, cerebrospinal fluid, lymph fluid, saliva, urine, or stool.
[0062] In some embodiments of any one of the methods disclosed herein, the subject is a mammal. In some embodiments of any one of the methods disclosed herein, the subject is a human.
[0063] In some embodiments of any one of the methods disclosed herein, the condition comprises neoplasm, cancer, or tumor. In some embodiments, the condition comprises a solid tumor. In some embodiments, the condition comprises a lymphoma. In some embodiments, the condition comprises a B-cell lymphoma. In some embodiments, the condition comprises a sub-type of B-cell lymphoma selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma, and B-cell chronic lymphocytic leukemia.
[0064] In some embodiments of any one of the methods disclosed herein, the plurality of phased variants have been previously identified as tumor-derived from sequencing a prior tumor sample or cell-free nucleic acid sample.
[0065] In one aspect, the present disclosure provides a composition comprising a bait set comprising a set of nucleic acid probes designed to capture cell-free DNA molecules derived from at least about 5% of genomic regions set forth in (i) the genomic regions identified in Table 1, (ii) the genomic regions identified in Table 3, or (iii) the genomic regions identified to have a plurality of phased variants in Table 3.
[0066] In some embodiments of any of the compositions disclosed herein, the set of nucleic acid probes are designed to pull down cell-free DNA molecules derived from at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the genomic regions set forth in (i) the genomic regions identified in Table 1, (ii) the genomic regions identified in Table 3, or (iii) the genomic regions identified to have a plurality of phased variants in Table 3.
[0067] In some embodiments of any of the compositions disclosed herein, the set of nucleic acid probes are designed to capture the one or more cell-free DNA molecules derived from at most about 10%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, or about 100% of the genomic regions set forth in (i) the genomic regions identified in Table 1, (ii) the genomic regions identified in Table 3, or (iii) the genomic regions identified to have a plurality of phased variants in Table 3.
[0068] In some embodiments of any of the compositions disclosed herein, the bait set comprises at most 5, at most 10, at most 50, at most 100, at most 500, at most 1000, or at most 2000 nucleic acid probes.
[0069] In some embodiments of any of the compositions disclosed herein, an individual nucleic acid probe of the set of nucleic acid probes comprises a pull-down tag.
[0070] In some embodiments of any of the compositions disclosed herein, the pull-down tag comprises a nucleic acid barcode.
[0071] In some embodiments of any of the compositions disclosed herein, the pull-down tag comprises biotin.
[0072] In some embodiments of any of the compositions disclosed herein, each of the cell-free DNA molecules is between about 100 nucleotides and about 180 nucleotides in length.
[0073] In some embodiments of any of the compositions disclosed herein, the genomic regions are associated with a condition.
[0074] In some embodiments of any of the compositions disclosed herein, the genomic regions exhibit aberrant somatic hypermutation when a subject has the condition.
[0075] In some embodiments of any of the compositions disclosed herein, the condition comprises a B-cell lymphoma. In some embodiments, the condition comprises a sub-type of B-cell lymphoma selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma, and B-cell chronic lymphocytic leukemia.
[0076] In some embodiments of any of the compositions disclosed herein, the composition further comprises a plurality of cell-free DNA molecules obtained or derived from a subject.
[0077] In one aspect, the present disclosure provides a method to perform a clinical procedure on an individual, the method comprising: (a) obtaining or having obtained a targeted sequencing result of a collection of cell-free nucleic acid molecules, wherein the collection of cell-free nucleic acid molecules are sourced from a liquid or waste biopsy of an individual, and wherein the targeting sequencing is performed utilizing nucleic acid probes to pull down sequences of genomic loci known to experience aberrant somatic hypermutation in a B-cell cancer; (b) identifying or having identified a plurality of variants in phase within the cell-free nucleic acid sequencing result; (c) determining or having determined, utilizing a statistical model and the identified phased variants, that the cell-free nucleic acid sequencing result contains nucleotides derived from a neoplasm; and (d) performing a clinical procedure on the individual to confirm the presence of the B-cell cancer, based upon determining that the cell-free nucleic acid sequencing result contains nucleic acid sequences likely derived from the B-cell cancer.
[0078] In some embodiments of any of the compositions disclosed herein, the biopsy is one of blood, serum, cerebrospinal fluid, lymph fluid, urine, or stool.
[0079] In some embodiments of any of the compositions disclosed herein, the genomic loci are selected from (i) the genomic regions identified in Table 1, (ii) the genomic regions identified in Table 3, or (iii) the genomic regions identified to have a plurality of phased variants in Table 3.
[0080] In some embodiments of any of the compositions disclosed herein, the sequences of the nucleic acid probes are selected from Table 6.
[0081] In some embodiments of any of the compositions disclosed herein, the clinical is procedure is a blood test, medical imaging, or a physical exam.
[0082] In one aspect, the present disclosure provides a method to treat an individual for a B-cell cancer, the method comprising: (a) obtaining or having obtained a targeted sequencing result of a collection of cell-free nucleic acid molecules, wherein the collection of cell-free nucleic acid molecules are sourced from a liquid or waste biopsy of an individual, and wherein the targeting sequencing is performed utilizing nucleic acid probes to pull down sequences of genomic loci known to experience aberrant somatic hypermutation in a B-cell cancer; (b) identifying or having identified a plurality of variants in phase within the cell-free nucleic acid sequencing result; (c) determining or having determined, utilizing a statistical model and the identified phased variants, that the cell-free nucleic acid sequencing result contains nucleotides derived from a neoplasm; and (d) treating the individual to curtail the B-cell cancer, based upon determining that the cell-free nucleic acid sequencing result contains nucleic acid sequences derived from the B-cell cancer.
[0083] In some embodiments of any of the compositions disclosed herein, the biopsy is one of blood, serum, cerebrospinal fluid, lymph fluid, urine or stool.
[0084] In some embodiments of any of the compositions disclosed herein, the genomic loci are selected from (i) the genomic regions identified in Table 1, (ii) the genomic regions identified in Table 3, or (iii) the genomic regions identified to have a plurality of phased variants in Table 3.
[0085] In some embodiments of any of the compositions disclosed herein, the sequences of the nucleic acid probes are selected from Table 6.
[0086] In some embodiments of any of the compositions disclosed herein, the treatment is chemotherapy, radiotherapy, immunotherapy, hormone therapy, targeted drug therapy, or medical surveillance.
[0087] In one aspect, the present disclosure provides a method to detect cancerous minimal residual disease in an individual and to treat the individual for a cancer, the method comprising: (a) obtaining or having obtained a targeted sequencing result of a collection of cell-free nucleic acid molecules, wherein the collection of cell-free nucleic acid molecules are sourced from a liquid or waste biopsy of an individual, wherein the liquid or waste biopsy is sourced after a series of treatments in order to detect minimal residual disease, and wherein the targeting sequencing is performed utilizing nucleic acid probes to pull down sequences of genomic loci determined to contain a plurality of variants in phase, as determined by a prior sequencing result on a prior biopsy derived from the cancer; (b) identifying or having identified at least one set of the plurality of variants in phase within the cell-free nucleic acid sequencing result; and (c) treating the individual to curtail the cancer, based upon determining that the cell-free nucleic acid sequencing result contains nucleic acid sequences derived from the cancer.
[0088] In some embodiments of any of the compositions disclosed herein, the liquid or waste biopsy is one of blood, serum, cerebrospinal fluid, lymph fluid, urine or stool.
[0089] In some embodiments of any of the compositions disclosed herein, the treatment is chemotherapy, radiotherapy, immunotherapy, hormone therapy, targeted drug therapy, or medical surveillance.
[0090] In one aspect, the present disclosure provides a computer program product comprising a non-transitory computer-readable medium having computer-executable code encoded therein, the computer-executable code adapted to be executed to implement any one of the methods disclosed herein.
[0091] In one aspect, the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto, wherein the computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any one of the methods disclosed herein.
[0092] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
INCORPORATION BY REFERENCE
[0093] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
BRIEF DESCRIPTION OF THE DRAWINGS
[0094] Various features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also "Figure" and "FIG." herein), of which:
[0095] FIGS. 1A-1E illustrate discovery of phased variants and their mutational signatures via analysis of whole-genome sequencing data. FIG. 1A. is a cartoon depicting the difference between detection of a single nucleotide variant (SNV) (top) and multiple variants `in-phase` (phased variants, PVs; bottom) on individual cell-free DNA molecules. In theory, detection of a PV is a more specific event than detection of an isolated SNV. FIG. 1B. is a scatter plot showing the distribution of the number of PVs from WGS data for 24 different histologies of cancer, normalized by the total number of SNVs. Bars show the median value and interquartile range. (FL-NHL, follicular lymphoma; DLBCL-NHL, diffuse large B-cell lymphoma; Burkitt-NHL, Burkitt lymphoma; Lung-SCC, squamous cell lung cancer; Lung-Adeno, lung adenocarcinoma; Kidney-RCC, renal cell carcinoma; Bone-Osteosarc, osteosarcoma; Liver-HCC, hepatocellular carcinoma; Breast-Adeno, breast adenocarcinoma; Panc-Adeno, pancreatic adenocarcinoma; Head-SCC, head and neck squamous cell carcinoma; Ovary-Adeno, ovarian adenocarcinoma; Eso-Adeno, esophageal adenocarcinoma; Uterus-Adeno, uterine adenocarcinoma; Stomach-Adeno, stomach adenocarcinoma; CLL, chronic lymphocytic leukemia; ColoRect-Adeno, colorectal adenocarcinoma; Prost-Adeno, prostate adenocarcinoma; CNS-GBM, glioblastoma multiforme; Panc-Endocrine, pancreatic neuroendocrine tumor; Thy-Adeno, thyroid adenocarcinoma; CNS-PiloAstro, piloastrocytoma; CNS-Medullo, medulloblastoma.) FIG. 1C. is a heatmap demonstrating the enrichment in single base substitution (SBS) mutational signatures for PVs versus single SNVs across multiple cancer types. Blue represents signatures which are enriched in PVs in specific histologies; darker gray represents signatures where un-phased, single SNVs are enriched; and red represents SNVs occurring in isolation. Only signatures which have a significant difference between PVs and unphased SNVs after correcting for multiple hypotheses are shown; other signatures are grey. Signatures associated with smoking, AID/AICDA, and APOBEC are indicated. FIG. 1D. demonstrate bar plots showing the distribution of PVs occurring in stereotyped regions across the genome in B-lymphoid malignancies and lung adenocarcinoma. In this plot, the genome was divided into 1000 bp bins, and the fraction of samples of a given histology with a PV in each 1000 bp bin was calculated. Only bins that have at least a 2 percent recurrence frequency in any cancer subtype are shown. Key genomic loci are also labeled. FIG. 1E. is a comparison of duplex sequencing to phased variant sequencing. A schema comparing error-suppressed sequencing by duplex sequencing vs. recovery of phased variants. In duplex sequencing, recovery of a single SNV observed on both strands of an original DNA double-helix (i.e., in trans) is required. This requires independent recovery of two molecules by sequencing as the plus and minus strands of the original DNA molecule go through library preparation and PCR independently. In contrast, recovery of PVs requires multiple SNVs observed on the same single strand of DNA (i.e., in cis). Thus, recovery of only the plus or the minus strand (rather than both) is sufficient for identification of PVs.
[0096] FIGS. 2A-2F illustrate design, validation, and application of phased variant enrichment sequencing. FIG. 2A is a schematic of the design for PhasED-Seq. WGS data from DLBCL tumor samples were aggregated (left), and areas of recurrent putative PVs were identified (middle). An assay capturing the genomic regions most recurrently containing PVs was then designed (right), resulting in an .about.7500.times. enrichment in PVs compared to WGS. The top right panel shows the in silico expected number of PVs per case per kilobase of panel size (y-axis) for increasing panel sizes (x-axis). The dashed line shows the selected regions in the PhasED-Seq panel. The bottom right panel shows the total number of expected PVs per case (y-axis, assessed in silico from WGS data, for increasing panel sizes (y-axis). The dark area shows the selected regions in the PhasED-Seq panel. FIG. 2B illustrate two panels showing the yield of SNVs (left) and PVs (right) for sequencing tumor DNA and matched germline by a previously established lymphoma CAPP-Seq panel or PhasED-Seq; values are assessed in silico by limiting WGS to the targeted space of interest. PVs reported in the right panel include doublet, triplet, and quadruplet phased events. FIG. 2C shows the yield of SNVs (left) and PVs (right) from experimental sequencing of tumor and/or cell-free DNA from CAPP-Seq versus PhasED-Seq, similar to FIG. 2B. FIG. 2D is a scatterplot showing the frequency of PVs by genomic location (in 1000 bp bins) for patients with DLBCL, identified either by WGS or identified by PhasED-Seq. PVs in IGH, BCL2, MYC, and BCL6 are highlighted. FIG. 2E illustrate scatterplots comparing the frequency of PVs by genomic location (in 50 bp bins) for patients with different types of lymphomas. The colored circles show the relative frequency of PVs in 50 bp bins from a specific gene of interest; the other (gray) circles show the relative frequency of PVs in 50 bp bins from the remainder of the PhasED-Seq sequencing panel. FIG. 2F illustrate volcano plots summarizing the difference in relative frequency of PVs in specific genetic loci between types of lymphoma, including ABC-DLBCL vs. GCB-DLBCL (dark Gray, left); PMBCL vs DLBCL (dark gray, middle); and HL vs. DLBCL (dark gray, right). The x-axis demonstrates the relative enrichment in PVs in a specific locus, while the y-axis demonstrates the statistical significance of this association. (Example 10).
[0097] FIGS. 3A-3I illustrate technical performance of PhasED-Seq for disease detection. FIG. 3A illustrates bar plot showing the performance of hybrid capture sequencing for recovery of synthetic 150 bp oligonucleotides from two loci (MYC and BCL6) with increasing degree of mutation/non-reference bases. Error bars represent the 95% confidence interval (n=3 replicates of each condition in distinct samples). FIG. 3B illustrates plot demonstrating the background error-rate (Example 10) for different types of error-suppression from 12 healthy control cell-free DNA samples sequenced on the PhasED-Seq panel. PhasED-Seq 2.times.' or `doublets` represents detection of two mutations in-phase on the same DNA molecule; PhasED-Seq 3.times.' or `triplets` represents detection of three mutations in-phase on the same DNA molecule. FIG. 3C illustrates bar plot showing the depth of unique molecular recovery (e.g., depth after barcode-mediated PCR duplicate removal) from sequencing data from 12 cell-free DNA samples for different types of error-suppression, including barcode deduplication, duplex sequencing, and recovery of PVs of increasing maximal distance between SNVs in-phase. FIG. 3D illustrates bar plot showing the cumulative fraction of PVs that have a maximal distance between SNVs less than the number of base-pairs shown on the x-axis. FIG. 3E illustrates a plot demonstrating the results of a limiting dilution series simulating cell-free DNA samples containing patient-specific tumor fractions of 1.times.10.sup.-3 to 0.5.times.10.sup.-6; cfDNA from 3 independent patients samples were used in each dilution. The same sequencing data was analyzed using a variety of error-suppression methods for recovery of expected tumor fractions, including iDES, duplex sequencing, and PhasED-Seq (both for recovery of doublet and triplet molecules). Points and error-bars represent the mean, minimum, and maximum across the three patient-specific tumor mutations considered. The difference between observed and expected tumor fractions for sample <1:10,000 were compared via paired t-test. *, P<0.05, **, P<0.005, ***, P<0.0005. FIG. 3F illustrates plot demonstrating the background signal for detection of tumor-specific alleles in 12 unrelated, healthy cell-free DNA samples, and the healthy cfDNA sample used for limiting dilution series (n=13 total samples). In each sample, tumor-specific SNVs or PVs from the 3 patient samples utilized in the limiting dilution experiment shown in FIG. 3E, for a total of 39 assessments were assessed. Bars represent the arithmetic mean across all 39 assessments; statistical comparison performed by Wilcoxon rank-sum test. *, P<0.05, **, P<0.005, ***, P<0.0005. FIG. 3G illustrates plot showing the theoretical rate of detection for a sample with a given number of PV-containing regions, according to simple binomial sampling. This plot is produced by assuming a unique sequencing depth of 5000.times. (line), along with a varying number of independent 150 bp PV-containing regions, from 3 regions (blue) to 67 regions (purple). Confidence envelopes consider depth from 4000-6000.times.; a 5% false-positive rate is also assumed.
[0098] FIG. 3H illustrates plot showing the observed rate of detection (y-axis) for sample of a given true tumor fraction (x-axis), with varying numbers of PV-containing regions. For each number of tumor-reporter regions ranging from 3 to 67, this number of 150 bp windows was randomly sampled from each of 3 patient-specific PV reporter lists 25 times and used to assess tumor-detection at each dilution. Filled-in points represent `wet` dilution series experiments, while open points represent in silico dilution experiments. Points and error-bars represent the mean, minimum, and maximum across the three patient-specific PV reporter lists used in the original sampling. FIG. 3I illustrates scatter plot compares the predicted vs observed rate of detection for samples from the dilution series shown in panels FIG. 3G and FIG. 311. Additional details of this experiment are provided in Example 10.
[0099] FIGS. 4A-4G illustrate clinical application of PhasED-Seq for ultra-sensitive disease detection and response monitoring in DLBCL. FIG. 4A illustrates plot showing ctDNA levels for a patient with DLBCL responding to, and subsequently relapsing after, first-line immuno-chemotherapy. Levels measured by CAPP-Seq are shown in darker gray circles while levels measured by PhasED-Seq are shown in lighter gray circles. Open circles represent undetectable levels by CAPP-Seq. FIG. 4B illustrates a univariate scatter plot showing the mean tumor allele fraction measured by PhasED-Seq for clinical samples at time-points of minimal disease (i.e., after 1 or 2 cycles of therapy). The plot is divided by samples detected vs undetected by standard CAPP-Seq; P-value from Wilcoxon rank-sum test. FIG. 4C illustrates bar plot showing the fraction of DLBCL patients who have detectable ctDNA by CAPP-Seq after 1 or 2 cycles of treatment (dark gray bars), as well as the fraction of additional patients with detectable disease when adding PhasED-Seq to standard CAPP-Seq (medium gray bars). P-value represents a Fisher's Exact Test for detection by CAPP-Seq alone versus the combination of PhasED-Seq and CAPP-Seq in 171 samples after 1 or 2 cycles of treatment. FIG. 4D illustrates a waterfall plot showing the change in ctDNA levels measured by CAPP-Seq after 2 cycles of first-line therapy in patients with DLBCL. Patients with undetectable ctDNA by CAPP-Seq are shown as "ND" ("not detected"), in darker colors. The colors of the bars also indicate the eventual clinical outcomes for these patients. FIG. 4E illustrates a Kaplan-Meier plot showing the event-free survival for 52 DLBCL patients with undetectable ctDNA measured by CAPP-Seq after 2 cycles. FIG. 4F illustrates a Kaplan-Meier plot showing the event-free survival of 52 patients shown in FIG. 4E (undetectable ctDNA by CAPP-Seq) stratified by ctDNA detection via PhasED-Seq at this same time-point (cycle 3, day 1). FIG. 4G illustrates a Kaplan-Meier plot showing the event-free survival for 89 patients with DLBCL stratified by ctDNA at cycle 3, day 1 separated into 3 strata--patients failing to achieve a major molecular response (dark gray), patients with a major molecular response who still have detectable ctDNA by PhasED-Seq and/or CAPP-Seq (light grey), and patients who have a stringent molecular remission (undetectable ctDNA by PhasED-Seq and CAPP-Seq; medium gray).
[0100] FIGS. 5A-5C illustrate enumeration of SNVs and PVs in diverse cancers from WGS. FIG. 5A-C illustrate Univariate scatter plots showing the number of SNVs (FIG. 5A), PVs (FIG. 5B), and PVs, controlling for total number of SNVs (FIG. 5C), from WGS data for 24 different histologies of cancer. Bars show the median value and interquartile range. (FL-NHL, follicular lymphoma; DLBCL-NHL, diffuse large B cell lymphoma; Burkitt-NHL, Burkitt lymphoma; Lung-SCC, squamous cell lung cancer; Lung-Adeno, lung adenocarcinoma; Kidney-RCC, renal cell carcinoma; Bone-Osteosarc, osteosarcoma; Liver-HCC, hepatocellular carcinoma; Breast-Adeno, breast adenocarcinoma; Panc-Adeno, pancreatic adenocarcinoma; Head-SCC, head and neck squamous cell carcinoma; Ovary-Adeno, ovarian adenocarcinoma; Eso-Adeno, esophageal adenocarcinoma; Uterus-Adeno, uterine adenocarcinoma; Stomach-Adeno, stomach adenocarcinoma; CLL, chronic lymphocytic leukemia; ColoRect-Adeno, colorectal adenocarcinoma; Prost-Adeno, prostate adenocarcinoma; CNS-GBM, glioblastoma multiforme; Panc-Endocrine, pancreatic neuroendocrine tumor; Thy-Adeno, thyroid adenocarcinoma; CNS-PiloAstro, piloastrocytoma; CNS-Medullo, medulloblastoma).
[0101] FIGS. 6A-6WW illustrate contribution of mutational signatures in phased and un-phased SNVs in WGS (FIGS. 6A-6WW.) Scatterplots showing the contribution of established single base substitution (SBS) mutational signatures to SNVs seen in PVs, shown in dark colors, and SNVs seen outside of possible phased relationships, shown in light colors, from WGS. This is presented for 49 SBS mutational signatures across 24 subtypes of cancer. Mutational signatures that show a significant difference in contribution between phased and un-phased SNVs after multiple hypothesis testing correction are indicated with a *. These figures represent the raw data summarized in FIG. 1C.
[0102] FIG. 7 illustrates distribution of PVs in stereotyped regions across the genome. Bar plots show the distribution of PVs occurring in stereotyped regions across the genome of multiple cancer types. In this plot, the genome was divided into 1000 bp bins, and the fraction of samples of a given histology with a PV in each 1000 bp bin was calculated. Only bins that have at least a 2 percent recurrence frequency in any cancer subtype are shown. Histologies shown are as in FIG. 1E; activated B-cell (ABC) and germinal center B-cell (GCB) subtypes of DLBCL are also shown.
[0103] FIGS. 8A-8E illustrate quantity and genomic location of PVs from WGS in lymphoid malignancies. FIG. 8A. illustrates bar plot showing the number of independent 1000 bp regions across the genome that recurrently contain PVs for DLBCL, FL, BL, and CLL (n=68, 74, 36, and 151 respectively). FIG. 8B-D illustrate plots showing the frequency of PVs for multiple lymphoid malignancies with relationships to specific genetic loci, including FIG. 8B: BCL2, FIG. 8C: MYC, and FIG. 8D: ID3. The location of the transcript for a given gene is shown below the plot in grey; exons are shown in darker gray. * indicates a region with significantly more PVs in a given cancer histology compared to all other histologies by Fisher's Exact Test (P<0.05). FIG. 8E, similar to FIG. 8B-D, these plots show the frequency of PVs across lymphoma subtypes. Here, it is shown the IGH locus, consisting of IGHV, IGHD, and IGHJ parts, for ABC and GCB subtype DLBCLs (n=25 and 25, respectively). Coding regions for Ig parts, including Ig-constant regions and V-genes, are shown. (DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; BL, Burkitt lymphoma, CLL, chronic lymphocytic leukemia).
[0104] FIGS. 9A-9K illustrate performance of PhasED-Seq for recovery of PVs across lymphomas. FIG. 9A illustrates univariate scatter plot showing the fraction of all PVs across the genome identified by WGS (n=79) that were recovered by previously reported lymphoma CAPP-Seq panel.sup.8 (left) compared to PhasED-Seq (right). FIG. 9B illustrates the expected yield of SNVs per case identified from WGS using a previously established lymphoma CAPP-Seq panel or the PhasED-Seq panel. FIG. 9C illustrates the expected yield of PVs per case identified from WGS using a previously established lymphoma CAPP-Seq panel or the PhasED-Seq panel. Data from three independent publicly available cohorts are shown in FIGS. 9A-9C. FIGS. 9D-9F illustrate plots showing the improvement in recovery of PVs by PhasED-Seq compared to CAPP-Seq in 16 patients sequenced by both assays. This includes improvement in d) two SNVs in phase (e.g., 2.times. or `doublet PVs`), e) three SNVs in phase (3.times. or `triplet PVs`) and f) four SNVs in phase (e.g., 4.times. or `quadruplet PVs`). FIGS. 9G-9K. illustrate panels showing the number of SNVs and PVs identified for patients with different types of lymphomas. These panels show the number of g) SNVs, h) doublet PVs, i) triplet PVs, j) quadruplet PVs, and k) all PVs. *, P<0.05; **, P<0.01, ***, P<0.001. (DLBCL, diffuse large B-cell lymphoma; GCB, germinal center B-cell like DLBCL; ABC, activated B-cell like DLBCL; PMBCL, primary mediastinal B-cell lymphoma; HL, Hodgkin lymphoma).
[0105] FIGS. 10A-10Y illustrate location-specific differences in PVs between ABC-DLBCL and GCB-DLBC (FIGS. 10A-10Y.) Similar to FIG. 2D, these scatterplots compare the frequency of PVs by genomic location (in 50 bp bins) for patients with different types of lymphomas; in this figure, the difference between ABC-DLBCL and GCB-DLBCL is shown. The red circles show the relative frequency of PVs in 50 bp bins from a specific gene of interest; the other (grey) circles show the relative frequency of PVs in 50 bp bins from the remainder of the PhasED-Seq sequencing panel. Only genes with a statistically significant difference in PVs between ABC-DLBCL and GCB-DLBCL are shown. P-values represent a Wilcoxon rank-sum test of 50 bp bins from a given gene against all other 50 bp bins; see Example 10.
[0106] FIGS. 11A-11X illustrate Location-specific differences in PVs between DLBCL and PMBCL (FIGS. 11A-11X). Similar to FIG. 2D, these scatterplots compare the frequency of PVs by genomic location (in 50 bp bins) for patients with different types of lymphomas; in this figure, the difference between DLBCL and PMBCL is shown. The blue circles show the relative frequency of PVs in 50 bp bins from a specific gene of interest; the other (gray) circles show the relative frequency of PVs in 50 bp bins from the remainder of the PhasED-Seq sequencing panel. Only genes with a statistically significant difference in PVs between DLBCL and PMBCL are shown. P-values represent a Wilcoxon rank-sum test of 50 bp bins from a given gene against all other 50 bp bins; see Example 10.
[0107] FIGS. 12A-12NN illustrate Location-specific differences in PVs between DLBCL and HL. Similar to FIG. 2D, scatterplots of FIGS. 12A-12NN compare the frequency of PVs by genomic location (in 50 bp bins) for patients with different types of lymphomas; in this figure, the difference between DLBCL and HL is shown. The green circles show the relative frequency of PVs in 50 bp bins from a specific gene of interest; the other (grey) circles show the relative frequency of PVs in 50 bp bins from the remainder of the PhasED-Seq sequencing panel. Only genes with a statistically significant difference in PVs between DLBCL and HL are shown. P-values represent a Wilcoxon rank sum test of 50 bp bins from a given gene against all other 50 bp bins; see Example 10.
[0108] FIG. 13 illustrates differences in PVs between lymphoma types in mutations in the IGH locus. This figure shows the frequency of PVs from PhasED-Seq across the @IGH locus for different types of B-cell lymphomas. The bottom track shows the structure of the @IGH locus and gene-parts, including Ig-constant genes and V-genes. The next (outlined) track shows the frequency of PVs in this genomic region from WGS data (ICGC cohort). The remainder of the tracks show the frequency of PVs from PhasED-Seq targeted sequencing data, including 1) DLBCL, GCB-DLBCL, ABC-DLBCL, PMBCL, and HL. The regions targeted by the PhasED-Seq panel are shown at the top. Selected immunoglobulin parts with PVs enriched in specific histologies are labeled (i.e., IGHV4-34, S.epsilon., S.gamma.3 and S.gamma.1).
[0109] FIGS. 14A-14E illustrate Technical aspects of PhasED-Seq by hybrid-capture sequencing. FIG. 14A shows a plot of the theoretical energy of binding for typical 150-mers across the genome with increasing fraction of bases mutated from the reference genome. Mutations were spread throughout the 150-mer either clustered to one end of the sequence, clustered in the middle of the sequence, or randomly throughout the sequence. Point and error-bars represent the median and interquartile ranges from 10,000 in silico simulations. FIG. 14B illustrates a plot showing two histograms of summary metrics of the mutation rate of 151-bp windows across the PhasED-Seq panel across all patients in this study. The light gray histogram shows the maximum percent mutated in any 151-bp window for all patients in this study; the dark gray histogram shows the 95.sup.th percentile mutation rate across all mutated 151-bp windows. FIG. 14C is a plot showing the percentile of mutation rate across all mutated 151-bp windows across all patients in this study. FIG. 14D illustrates heatmaps showing the relative error rate (as log 10(error rate)) for single SNVs (left, "RED"), doublet PVs (middle, "YELLOW"), and triplet PVs (right, "BLUE"). FIG. 14D demonstrates that analysis based on the plurality of phased variants (e.g., double or triplet PVs) yields a lower error rate than analysis based on single SNVs. In addition, FIG. 14D demonstrates that analysis using a higher number of phased variant sets (e.g., triplet PVs labeled as "BLUE") yields a lower error rate than analysis based on a lower number of phased variant sets (e.g., doublet PVs labeled as "YELLOW"). The error rate of single SNVs from sequencing with multiple error suppression methods is shown, including barcode deduplication, iDES, and duplex sequencing. Error rates are summarized by the type of mutation. In the case of triplet PVs, the x and y-axis of the heatmap represent the first and second type of base alteration in the PV; the third alteration is averaged over all 12 possible base changes. FIG. 14E illustrates a plot showing the error rate for doublet/2.times. PVs as a function of the genomic distance between the component SNVs.
[0110] FIGS. 15 and 16A-16B illustrates comparison of ctDNA quantitation by PhasED-Seq to CAPP-Seq and clinical applications. FIG. 15 illustrates the detection-rate of ctDNA from pretreatment samples across 107 patients with large-B cell lymphomas by standard CAPP-Seq (green), as well as PhasED-Seq using doublets (light blue), triplets (medium blue), and quadruplets (dark blue). The specificity of ctDNA detection is also shown. In the lower two plots, the false-detection rate in 40 withheld healthy control cfDNA samples is shown. The size of each bar in these two plots shows the detection-rate for patient-specific cfDNA mutations in these 40-withheld controls, across all 107 cases. FIG. 16A illustrates table summarizing the sensitivity and specificity for ctDNA detection in pretreatment samples by CAPP-Seq and PhasED-Seq using doublets, triplets, and quadruplets, shown in panel A. Sensitivity is calculated across all 107 cases, while specificity is calculated across the 40 withheld control samples, assessing for each of the 107 independent patient-specific mutation lists, for a total of 4280 independent tests. FIG. 16B illustrates a scatterplot showing the quantity of ctDNA (measured as log 10(haploid genome equivalents/mL)) as measured by CAPP-Seq vs. PhasED-Seq in individual samples. Samples taken prior to cycle 1 of RCHOP therapy (i.e., pretreatment), prior to cycle 2, and prior to cycle 3, are shown in independent colors (blue, green, and red respectively; 278 total samples). Undetectable levels fall on the axes. Spearman correlation and P-value are shown.
[0111] FIGS. 17A-17D illustrate detection of ctDNA after two cycles of systemic therapy. FIG. 17A illustrates a scatter plot showing the log-fold change in ctDNA after 2 cycles of therapy (i.e., the Major Molecular Response or MMR) measured by CAPP-Seq or PhasED-Seq for patients receiving RCHOP therapy. Dotted lines show the previously established threshold of a 2.5-log reduction in ctDNA for MMR. Undetectable samples fall on the axes; the correlation coefficient represents a Spearman rho for the 33 samples detected by both CAPP-Seq and PhasED-Seq. FIG. 17B illustrates 2 by 2 tables summarizing the detection rate of ctDNA samples after 2 cycles of therapy by PhasED-Seq vs CAPP-Seq. Patients with eventual disease progression are shown in bottom panel, while patients without eventual disease progression are shown in upper panel. FIG. 17C illustrates bar-plots showing the area under the receiver operator curve (AUC) for classification of patients for event-free survival at 24 months based on CAPP-Seq (light colors) or PhasED-Seq (dark colors) after 2 cycles of therapy. Classification of all patient (n=89, left) and only patients achieving a MMR (n=69, right) are both shown. FIG. 17D illustrates Kaplan-Meier plots showing the event-free survival of 69 patients achieving a MMR stratified by ctDNA detection with CAPP-Seq (top) or PhasED-Seq (bottom).
[0112] FIGS. 18A-18H illustrate detection of ctDNA after one cycle of systemic therapy. FIG. 18A illustrates scatterplot showing the log-fold change in ctDNA after 1 cycle of therapy (i.e., the Early Molecular Response or EMR) measured by CAPP-Seq or PhasED-Seq for patients receiving RCHOP therapy. Dotted lines show the previously established threshold of a 2-log reduction in ctDNA for EMR. Undetectable samples fall on the axes; the correlation coefficient represents a Spearman rho for the 45 samples detected by both CAPP-Seq and PhasED-Seq. FIG. 18B illustrates 2 by 2 tables summarizing the detection rate of ctDNA samples after 1 cycle of therapy by PhasED-Seq vs CAPP-Ceq. Patients with eventual disease progression are shown in red, while patients without eventual disease progression are shown in blue. FIG. 18C illustrates bar-plots showing the area under the receiver operator curve (AUC) for classification of patients for event-free survival at 24 months based on CAPP-Seq (light colors) or PhasED-Seq (dark colors) after 1 cycle of therapy. Classification of all patient (n=82, left) and only patients achieving an EMR (n=63, right) are both shown. FIG. 18D illustrates Kaplan-Meier plots showing the event-free survival of 63 patients achieving an EMR stratified by ctDNA detection with CAPP-Seq (top) or PhasED-Seq (bottom). FIG. 18E illustrates waterfall plot showing the change in ctDNA levels measured by CAPP-Seq after 1 cycle of first-line therapy in patients with DLBCL. Patients with undetectable ctDNA by CAPP-Seq are shown as "ND" ("not detected"), in darker colors. The colors of the bars also indicate the eventual clinical outcomes for these patients. FIG. 18F illustrates a Kaplan-Meier plot showing the event-free survival for 33 DLBCL patients with undetectable ctDNA measured by CAPP-Seq after 1 cycle of therapy. FIG. 18G illustrates a Kaplan-Meier plot showing the event-free survival of 33 patients shown in FIG. 18F (undetectable ctDNA by CAPP-Seq) stratified by ctDNA detection via PhasED-Seq at this same time-point (cycle 2, day 1). FIG. 18H illustrates a Kaplan-Meier plot showing the event-free survival for 82 patients with DLBCL stratified by ctDNA at cycle 2, day 1 separated into 3 strata--patients failing to achieve an early molecular response, patients with an early molecular response who still have detectable ctDNA by PhasED-Seq and/or CAPP-Seq, and patients who have a stringent molecular remission (undetectable ctDNA by PhasED-Seq and CAPP-Seq).
[0113] FIG. 19 illustrates a fraction of patients where PhasED-Seq would achieve a lower LOD than duplex sequencing tracking SNVs based on PCAWG data (whole genome sequencing) from which the number of SNVs and phased variants (PVs) in different tumor types was quantified.
[0114] FIG. 20 illustrates improved LODs achieved in lung cancers (adenocarcinoma, abbreviated `A`, and squamous cell carcinoma, abbreviated `5`), compared to duplex sequencing of whole genome sequencing data.
[0115] FIG. 21 illustrates empiric data from an experiment where WGS was performed on tumor tissue and custom panels were designed for 5 patients with solid tumors (5 lung cancers) to examine and compare the LODs of custom CAPP-Seq vs PhasED-Seq, showing a .about.10.times. lower LOD using PhasED-Seq in 5/5 patients.
[0116] FIG. 22A illustrates proof of principle example patient vignette comparing using custom CAPP-Seq and PhasED-Seq for disease surveillance in lung cancer showing earlier detection of relapse using PhasED-Seq.
[0117] FIG. 22B illustrates proof of principle example patient vignette comparing using custom CAPP-Seq and PhasED-Seq for early detection of disease in breast cancer, showing earlier detection of disease with PhasED-Seq.
[0118] FIGS. 23A-23B illustrate that the method describe herein (e.g. method depicted yielding FIG. 3E and FIG. 3F) does not require barcode meditated error suppression.
[0119] FIG. 24 illustrates a flow diagram of a process to perform a clinical intervention and/or treatment on an individual based on detecting circulating-tumor nucleic acid sequences in a sequencing result in accordance with an embodiment.
[0120] FIGS. 25A-25C show example flowcharts of methods for determining a condition of a subject based on one or more cell-free nucleic acid molecules comprising a plurality of variants.
[0121] FIG. 25D shows an example flowchart of a method for treating a condition of a subject based on one or more cell-free nucleic acid molecules comprising a plurality of variants.
[0122] FIG. 25E shows an example flowchart of a method for determining a progress (e.g., progression or regression) of a condition of a subject based on one or more cell-free nucleic acid molecules comprising a plurality of variants.
[0123] FIGS. 25F and 25G show example flowcharts of methods for determining a condition of a subject based on one or more cell-free nucleic acid molecules comprising a plurality of variants.
[0124] FIGS. 26A and 26B schematically illustrate different fluorescent probes for identifying one or more cell-free nucleic acid molecules comprising a plurality of phased variants.
[0125] FIG. 27 shows a computer system that is programmed or otherwise configured to implement methods provided herein.
DETAILED DESCRIPTION
[0126] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
[0127] The term "about" or "approximately" generally mean within an acceptable error range for the particular value, which may depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term "about" meaning within an acceptable error range for the particular value may be assumed.
[0128] The term "phased variants," "variants in phase," "PV," or "somatic variants in phase," as used interchangeably herein, generally refers to two or more mutations (e.g., SNVs or indels) that occur in cis (i.e., on the same strand of a nucleic acid molecule) within a single cell-free nucleic acid molecule. In some cases, a cell-free nucleic acid molecule can be a cell-free deoxyribonucleic acid (cfDNA) molecule. In some cases, a cfDNA molecule can be derived from a diseased tissue, such as a tumor (e.g., a circulating tumor DNA (ctDNA) molecule).
[0129] The term "biological sample" or "bodily sample," as used interchangeably herein, generally refers to a tissue or fluid sample derived from a subject. A biological sample can be directly obtained from the subject. Alternatively, a biological sample can be derived from the subject (e.g., by processing an initial biological sample obtained from the subject). The biological sample can be or can include one or more nucleic acid molecules, such as DNA or ribonucleic acid (RNA) molecules. The biological sample can be derived from any organ, tissue or biological fluid. A biological sample can comprise, for example, a bodily fluid or a solid tissue sample. An example of a solid tissue sample is a tumor sample, e.g., from a solid tumor biopsy. Non-limiting examples of bodily fluids include blood, serum, plasma, tumor cells, saliva, urine, cerebrospinal fluid, lymphatic fluid, prostatic fluid, seminal fluid, milk, sputum, stool, tears, and derivatives of these. In some cases, one or more cell-free nucleic acid molecules as disclosed herein can be derived from a biological sample
[0130] The term "subject," as used herein, generally refers to any animal, mammal, or human. A subject can have, potentially have, or be suspected of having one or more conditions, such as a disease. In some cases, a condition of the subject can be cancer, a symptom(s) associated with cancer, or asymptomatic with respect to cancer or undiagnosed (e.g., not diagnosed for cancer). In some cases, the subject can have cancer, the subject can show a symptom(s) associated with cancer, the subject can be free from symptoms associated with cancer, or the subject may not be diagnosed with cancer. In some examples, the subject is a human.
[0131] The term "cell-free DNA" or "cfDNA," as used interchangeably herein, generally refers to DNA fragments circulating freely in a blood stream of a subject. Cell-free DNA fragments can have dinucleosomal protection (e.g., a fragment size of at least 240 base pairs ("bp")). These cfDNA fragments with dinucleosomal protection were likely not cut between the nucleosome, resulting in a longer fragment length (e.g., with a typical size distribution centered around 334 bp). Cell-free DNA fragments can have mononucleosomal protection (e.g., a fragment size of less than 240 base pairs ("bp")). These cfDNA fragments with mononucleosomal protection were likely cut between the nucleosome, resulting in a shorter fragment length (e.g., with a typical size distribution centered around 167 bp).
[0132] The term "sequencing data," as used herein, generally refers to "raw sequence reads" and/or "consensus sequences" of nucleic acids, such as cell-free nucleic acids or derivatives thereof. Raw sequence reads are the output of a DNA sequencer, and typically include redundant sequences of the same parent molecule, for example after amplification. "Consensus sequences" are sequences derived from redundant sequences of a parent molecule intended to represent the sequence of the original parent molecule. Consensus sequences can be produced by voting (wherein each majority nucleotide, e.g., the most commonly observed nucleotide at a given base position, among the sequences is the consensus nucleotide) or other approaches such as comparing to a reference genome. In some cases, consensus sequences can be produced by tagging original parent molecules with unique or non-unique molecular tags, which allow tracking of the progeny sequences (e.g., after amplification) by tracking of the tag and/or use of sequence read internal information.
[0133] The term "reference genomic sequence," as used herein, generally refers to a nucleotide sequence against which a subject's nucleotide sequences are compared.
[0134] The term "genomic region," as used herein, generally refers to any region (e.g., range of base pair locations) of a genome, e.g., an entire genome, a chromosome, a gene, or an exon. A genomic region can be a contiguous or a non-contiguous region. A "genetic locus" (or "locus") can be a portion or entirety of a genomic region (e.g., a gene, a portion of a gene, or a single nucleotide of a gene).
[0135] The term "likelihood," as used herein, generally refers to a probability, a relative probability, a presence or an absence, or a degree.
[0136] The term "liquid biopsy," as used herein, generally refers to a non-invasive or minimally invasive laboratory test or assay (e.g., of a biological sample or cell-free nucleic acids). The "liquid biopsy" assays can report detections or measurements (e.g., minor allele frequencies, gene expression, or protein expression) of one or more marker genes associated with a condition of a subject (e.g., cancer or tumor-associated marker genes).
A. Introduction
[0137] Modifications (e.g., mutations) of genomic DNA can be manifested in a formation and/or progression of one or more conditions (e.g., a disease, such as cancer or tumor) of a subject. The present disclosure provides methods and systems for analyzing cell-free nucleic acid molecules, such as cfDNA, from a subject to determine the presence or absence of a condition of the subject, prognosis of a diagnosed condition of the subject, progress of the condition of the subject over time, therapeutic treatment of a diagnosed condition of the subject, or predicted treatment outcome for a condition of the subject.
[0138] Analysis of cell-free nucleic acids, such as cfDNA, have been developed with broad applications in, e.g., prenatal testing, organ transplantation, infectious disease, and oncology. In the context of detecting or monitoring a disease of a subject, such as cancer, circulating tumor DNA (ctDNA) can be a sensitive and specific biomarker in numerous cancer types. In some cases, ctDNA can be used to detect the presence of minimal residual disease (MRD) or tumor burden after treatment, such as chemotherapies or surgical resection of solid tumors. However, the limit of detection (LOD) for ctDNA analysis can be restricted by a number of factors including (i) low input DNA amounts from a typical blood collection and (ii) background error rates from sequencing.
[0139] In some cases, ctDNA-based cancer detection can be improved by tracking multiple somatic mutations with error-suppressed sequencing, e.g., with LOD of about 2 parts in 100,000 from cfDNA input while using off-the-shelf panels or personalized assays. However, in some cases, current LOD of ctDNA of interest can be insufficient to universally detect MRD in patients destined for disease relapse or progression. For example, such `loss of detection` can be exemplified in diffuse large B-cell lymphoma (DLBCL). For DLBCL, interim ctDNA detection after only two cycles of curative-intent therapy can represent a major molecular response (MMR), and can be a strong prognostic marker for ultimate clinical outcomes. Despite this, nearly one-third of patients ultimately experiencing disease progression do not have detectable ctDNA at this interim landmark using available techniques (e.g., Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq)), thus representing `false-negative` measurements. Such high false-negative rates have also been observed in DLBCL patients by alternative methods, such as monitoring ctDNA through immunoglobulin gene rearrangements. Therefore, there exists a need for improved methods of ctDNA-based cancer detection with greater sensitivity.
[0140] Somatic variants detected on both of the complementary strands of parental DNA duplexes can be used to lower the LOD of ctDNA detection, thereby advantageously increasing the sensitivity of ctDNA detection. Such `duplex sequencing` can reduce background error profile due to the requirement of two concordant events for detection of a single nucleotide variant (SNV). However, the duplex sequencing approach alone can be limited by inefficient recovery of DNA duplexes as recovery of both original strands can occur in a minority of all recovered molecules. Thus, duplex sequencing may be suboptimal and inefficient for real-world ctDNA detection with limited amount of starting sample, where input DNA from practical blood volumes (e.g., between about 4,000 to about 8,000 genomes per standard 10 milliliter (mL) blood collection tube) is limited and maximal recovery of genomes is essential.
[0141] Thus, there remains a significant unmet need for detection and analysis of ctDNA with low LOD (e.g., thereby yielding high sensitivity) for determining, for example, presence or absence of a disease of a subject, prognosis of the disease, treatment for the disease, and/or predicted outcome of the treatment.
B. Methods and Systems for Determining or Monitoring a Condition
[0142] The present disclosure describes methods and systems for detecting and analyzing cell free nucleic acids with a plurality of phased variants as a characteristic of a condition of a subject. In some aspects, the cell-free nucleic acid molecules can comprise cfDNA molecules, such as ctDNA molecules. The methods and systems disclosed herein can utilize sequencing data derived from a plurality of cell-free nucleic acid molecules of the subject to identify a subset of the plurality of cell-free nucleic acid molecules having the plurality of phased variants, thereby to determine the condition of the subject. The methods and systems disclosed herein can directly detect and, in some cases, pull down (or capture) such subset of the plurality of cell-free nucleic acid molecules that exhibit the plurality of phased variants, thereby to determine the condition of the subject with or without sequencing. The methods and systems disclosed herein can reduce background error rate often involved during detection and analysis of cell-free nucleic acid molecules, such as cfDNA.
[0143] In some aspects, methods and systems for cell-free nucleic acid sequencing and detection of cancer are provided. In some embodiments, cell-free nucleic acids (e.g., cfDNA or cfRNA) can be extracted from a liquid biopsy of an individual and prepared for sequencing. Sequencing results of the cell-free nucleic acids can be analyzed to detect somatic variants in phase (i.e., phased variants, as disclosed herein) as an indication of circulating-tumor nucleic acid (ctDNA or ctRNA) sequences (i.e., sequences that derived or are originated from nucleic acids of a cancer cell). Accordingly, in some cases, cancer can be detected in the individual by extracting a liquid biopsy from the individual and sequencing the cell-free nucleic acids derived from that liquid biopsy to detect circulating-tumor nucleic acid sequences, and the presence of circulating-tumor nucleic acid sequences can indicate that the individual has a cancer (e.g., a specific type of cancer). In some cases, a clinical intervention and/or treatment can be determined and/or performed on the individual based on the detection of the cancer.
[0144] As disclosed herein, a presence of somatic variants in phase can be a strong indication that the nucleic acids containing such phased variants are derived from a bodily sample with a condition, such as a cancerous cell (or alternatively, that the nucleic acids are from derived from a bodily sample obtained or derived from a subject with a condition, such as cancer). Detection of phased somatic variants can enhance the signal-to-noise ratio of cell-free nucleic acid detection methods (e.g., by reducing or eliminating spurious "noise" signals) as it may be unlikely that phased mutations would occur within a small genetic window that is approximately the size of a typical cell-free nucleic acid molecule (e.g., about 170 bp or less).
[0145] In some aspects, a number of genomic regions can be used as hotspots for detection of phased variants, especially in various cancers, e.g., lymphomas. In some cases, enzymes (e.g., AID, Apobec3a) can stereotypically mutagenize DNA in specific genes and locations, leading to development of particular cancers. Accordingly, cell-free nucleic acids derived from such hotspot genomic regions can be captured or targeted (e.g., with or without deep sequencing) for cancer detection and/or monitoring. Alternatively, capture or targeted sequencing can performed on regions in which phased variants have been previously detected from a cancerous source (e.g., tumor) of a particular individual in order to detect cancer in that individual.
[0146] In some aspects, capture sequencing on cell-free nucleic acids can be performed as a screening diagnostic. In some cases, a screening diagnostic can be developed and used to detect circulating-tumor nucleic acids for cancers that have stereotypical regions of phased variants. In some cases, capture sequencing on cell-free nucleic acids is performed as a diagnostic to detect MRD or tumor burden to determine if a particular disease is present during or after treatment. In some cases, capture sequencing on cell-free nucleic acids can be performed as a diagnostic to determine progress (e.g., progression or regression) of a treatment.
[0147] In some aspects, cell-free nucleic acid sequencing results can be analyzed to detect whether phased somatic single nucleotide variants (SNVs) or other mutations or variants (e.g., indels) exist within the cell-free nucleic acid sample. In some cases, the presence of particular somatic SNVs or other variants can be indicative of circulating-tumor nucleic acid sequences, and thus indicative of a tumor present in the subject. In some cases, a minimum of two variants can be detected in phase on a cell-free nucleic acid molecule. In some cases, a minimum of three variants can be detected in phase on a cell-free nucleic acid molecule. In some cases, a minimum of four variants can be detected in phase on a cell-free nucleic acid molecule. In some cases, a minimum of five or more variants can be detected in phase on a cell-free nucleic acid molecule. In some cases, the greater number of phased variants detected on a cell-free nucleic acid molecule, the greater the likelihood that the cell-free nucleic acid molecule is derived from cancer, as opposed to detecting an innocuous sequence of somatic variants that arise from molecular preparation of the sequence library or random biological errors. Accordingly, the likelihood of false-positive detection can decrease with detection of more variants in phase within a molecule (e.g., thereby increasing specificity of detection).
[0148] In some aspects, a cell-free nucleic acid sequencing result can be analyzed to detect whether an insertion or deletion of one or more nucleobases (i.e., indel) exist within the cell-free nucleic acid sample, e.g., relative to a reference genomic sequence. Without wishing to be bound by theory, in some cases, presence of indels in a cell-free nucleic acid molecule (e.g., cfDNA) can be indicative of a condition of a subject, e.g., a disease such as cancer. In some cases, a genetic variation as a result of an indel can be treated as a variant or mutation, and thus two indels can be treated a two phased variants, as disclosed herein. In some examples, within a cell-free nucleic acid molecule, a first genetic variation from a first indel (a first phase variant) and a second genetic variation from a second indel (a second phase variant) can be separated from each other by at least 1 nucleotide.
[0149] Within a single cell-free nucleic acid molecule (e.g., a single cfDNA molecule), as disclosed herein, a first phased variant can be a SNV and a second phased variant can be a part of a different small nucleotide polymorphism, e.g., another SNV or a part of a multi-nucleotide variant (MNV). A multi-nucleotide variant can be a cluster of two or more (e.g., at least 2, 3, 4, 5, or more) adjacent variants existing within the same stand of nucleic acid molecule. In some cases, the first phased variant and the second phased variant can be parts of the same MNV within the single cell-free nucleic acid molecule. In some cases, the first phased variant and the second phased variant can be from two different MNVs within the single cell-free nucleic acid molecule.
[0150] In some aspects, a statistical method can be utilized to calculate the likelihood that detected phased variants are from a cancer and not random or artificial (e.g., from sample prep or sequencing error). In some cases, a Monte Carlo sampling method can be utilized to determine the likelihood that detected phased variants are from a cancer and not random or artificial.
[0151] Aspects of the present disclosure provide identification or detection of cell-free nucleic acids (e.g., cfDNA molecule) with a plurality of phased variants, e.g., from a liquid biopsy of a subject. In some cases, a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants can be directly adjacent to each other (e.g., neighboring SNVs). In some cases, a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants can be separated by at least one nucleotide. The spacing between the first phased variant and the second phased variant can be limited by the length of the cell-free nucleic acid molecule.
[0152] Within a single cell-free nucleic acid molecule (e.g., a single cfDNA molecule), as disclosed herein, a first phased variant and a second phased variant can be separated from each other by at least or up to about 1 nucleotide, at least or up to about 2 nucleotides, at least or up to about 3 nucleotides, at least or up to about 4 nucleotides, at least or up to about 5 nucleotides, at least or up to about 6 nucleotides, at least or up to about 7 nucleotides, at least or up to about 8 nucleotides, at least or up to about 9 nucleotides, at least or up to about 10 nucleotides, at least or up to about 11 nucleotides, at least or up to about 12 nucleotides, at least or up to about 13 nucleotides, at least or up to about 14 nucleotides, at least or up to about 15 nucleotides, at least or up to about 20 nucleotides, at least or up to about 25 nucleotides, at least or up to about 30 nucleotides, at least or up to about 35 nucleotides, at least or up to about 40 nucleotides, at least or up to about 45 nucleotides, at least or up to about 50 nucleotides, at least or up to about 60 nucleotides, at least or up to about 70 nucleotides, at least or up to about 80 nucleotides, at least or up to about 90 nucleotides, at least or up to about 100 nucleotides, at least or up to about 110 nucleotides, at least or up to about 120 nucleotides, at least or up to about 130 nucleotides, at least or up to about 140 nucleotides, at least or up to about 150 nucleotides, at least or up to about 160 nucleotides, at least or up to about 170 nucleotides, or at least or up to about 180 nucleotides. Alternatively or in addition to, within a single cell-free nucleic acid molecule, a first phased variant and a second phased variant may not or need not be separated by one or more nucleotides and thus can be directly adjacent to one another.
[0153] A single cell-free nucleic acid molecule (e.g., a single cfDNA molecule), as disclosed herein, can comprise at least or up to about 2 phased variants, at least or up to about 3 phased variants, at least or up to about 4 phased variants, at least or up to about 5 phased variants, at least or up to about 6 phased variants, at least or up to about 7 phased variants, at least or up to about 8 phased variants, at least or up to about 9 phased variants, at least or up to about 10 phased variants, at least or up to about 12 phased variants, at least or up to about 12 phased variants, at least or up to about 13 phased variants, at least or up to about 14 phased variants, at least or up to about 15 phased variants, at least or up to about 20 phased variants, or at least or up to about 25 phased variants within the same molecule.
[0154] From a plurality of cell-free nucleic acid molecules obtained (e.g., from a liquid biopsy of a subject), two or more (e.g., 10 or more, 1,000 or more, 10,000 or more) cell-free nucleic acid molecules can be identified to have an average of at least or up to about 2 phased variants, at least or up to about 3 phased variants, at least or up to about 4 phased variants, at least or up to about 5 phased variants, at least or up to about 6 phased variants, at least or up to about 7 phased variants, at least or up to about 8 phased variants, at least or up to about 9 phased variants, at least or up to about 10 phased variants, at least or up to about 12 phased variants, at least or up to about 12 phased variants, at least or up to about 13 phased variants, at least or up to about 14 phased variants, at least or up to about 15 phased variants, at least or up to about 20 phased variants, or at least or up to about 25 phased variants per each cell-free nucleic acid molecule identified to comprise a plurality of phased variants.
[0155] In some cases, a plurality of cell-free nucleic acid molecules (e.g., cfDNA molecules) can be obtained from a biological sample of a subject (e.g., solid tumor or liquid biopsy). Out of the plurality of cell-free nucleic acid molecules, at least or up to 1, at least or up to 2, at least or up to 3, at least or up to 4, at least or up to 5, at least or up to 6, at least or up to 7, at least or up to 8, at least or up to 9, at least or up to 10, at least or up to 15, at least or up to 20, at least or up to 25, at least or up to 30, at least or up to 35, at least or up to 40, at least or up to 45, at least or up to 50, at least or up to 60, at least or up to 70, at least or up to 80, at least or up to 90, at least or up to 100, at least or up to 150, at least or up to 200, at least or up to 300, at least or up to 400, at least or up to 500, at least or up to 600, at least or up to 700, at least or up to 800, at least or up to 900, at least or up to 1,000, at least or up to 5,000, at least or up to, 10,000, at least or up to 50,000, or at least or up to 100,000 cell-free nucleic acid molecules can be identified, such that each identified cell-free nucleic acid molecule comprises the plurality of phased variants, as disclosed herein.
[0156] In some cases, a plurality of cell-free nucleic acid molecules (e.g., cfDNA molecules) can be obtained from a biological sample of a subject (e.g., solid tumor or liquid biopsy). Out of the plurality of cell-free nucleic acid molecules, at least or up to 1, at least or up to 2, at least or up to 3, at least or up to 4, at least or up to 5, at least or up to 6, at least or up to 7, at least or up to 8, at least or up to 9, at least or up to 10, at least or up to 15, at least or up to 20, at least or up to 25, at least or up to 30, at least or up to 35, at least or up to 40, at least or up to 45, at least or up to 50, at least or up to 60, at least or up to 70, at least or up to 80, at least or up to 90, at least or up to 100, at least or up to 150, at least or up to 200, at least or up to 300, at least or up to 400, at least or up to 500, at least or up to 600, at least or up to 700, at least or up to 800, at least or up to 900, or at least or up to 1,000 cell-free nucleic acid molecules can be identified from a target genomic region (e.g., a target genomic locus), such that each identified cell-free nucleic acid molecule comprises the plurality of phased variants, as disclosed herein.
[0157] FIGS. 1A and 1E schematically illustrate examples of (i) a cfDNA molecule comprising a SNV and (ii) another cfDNA molecule comprising a plurality of phased variants. Each variant identified within the cfDNA can indicate a presence of one more genetic mutations in the cell that the cfNDA is originated from. In alternative embodiments, one or more of the phased variants may be an insertion or deletion (indel) instead of an SNV.
[0158] In one aspect, the present disclosure provides a method for determining a condition of a subject, as shown by flowchart 2510 in FIG. 25A. The method can comprise (a) obtaining, by a computer system, sequencing data derived from a plurality of cell-free nucleic acid molecules that is obtained or derived from the subject (process 2512). The method can further comprise (b) processing, by the computer system, the sequencing data to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules, wherein each of the one or more cell-free nucleic acid molecules identified comprises a plurality of phased variants relative to a reference genomic sequence (process 2514). In some cases, at least a portion of the one or more cell-free nucleic acid molecules can comprise a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants that are separated by at least one nucleotide, as disclosed herein. The method can optionally comprise (c) analyzing, by the computer system, at least a portion of the identified one or more cell-free nucleic acid molecules to determine the condition of the subject (process 2516).
[0159] In some cases, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 95%, at least or up to about 99%, or about 100% of the one or more cell-free nucleic acid molecules can comprise a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants that are separated by at least one nucleotide, as disclosed herein. In some examples, a plurality of phased variants within a single cfDNA molecule can comprise (i) a first plurality of phased variants that are separated by at least one nucleotide from one another and (ii) a second plurality of phased variants that are adjacent to one another (e.g., two phased variants within a MNV). In some examples, a plurality of phased variants within a single cfDNA molecule can consist of phased variants that are separate by at least one nucleotide from one another.
[0160] In one aspect, the present disclosure provides a method for determining a condition of the subject, as shown by flowchart 2520 in FIG. 25B. The method can comprise (a) obtaining, by a computer system, sequencing data derived from a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject (process 2522). The method can further comprise (b) processing, by the computer system, the sequencing data to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules, wherein each of the one or more cell-free nucleic acid molecules comprises a plurality of phased variants relative to a reference genomic sequence (process 2524). In some cases, a first phased variant of the plurality of phased variant and a second phased variant of the plurality of phased variant can be separated by at least one nucleotide, as disclosed herein. The method can optionally comprise (c) analyzing, by the computer system, at least a portion of the identified one or more cell-free nucleic acid molecules to determine the condition of the subject (process 2526).
[0161] In one aspect, the present disclosure provides a method for determining a condition of a subject, as shown by flowchart 2530 in FIG. 25C. The method can comprise (a) obtaining sequencing data derived from a plurality of cell-free nucleic acid molecules that is obtained or derived from the subject (process 2532). The method can further comprise (b) processing the sequencing data to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules with a LOD being less than about 1 out of 50,000 observations (or cell-free nucleic acid molecules) from the sequencing data (process 2534). In some cases, each of the one or more cell-free nucleic acid molecules comprises a plurality of phased variants relative to a reference genomic sequence. The method can optionally comprise (c) analyzing at least a portion of the identified one or more cell-free nucleic acid molecules to determine the condition of the subject (process 2536).
[0162] In some cases, the LOD of the operation of identifying the one or more cell-free nucleic acid molecules, as disclosed herein, can be less than about 1 out of 60,000, less than 1 out of 70,000, less than 10 out of 80,000, less than 1 out of 90,000, less than 1 out of 100,000, less than 1 out of 150,000, less than 1 out of 200,000, less than 1 out of 300,000, less than 1 out of 400,000, less than 1 out of 500,000, less than 1 out of 600,000, less than 1 out of 700,000, less than 1 out of 800,000, less than 1 out of 900,000, less than 1 out of 1,000,000, less than 1 out of 1,000,000, less than 1 out of 1,100,000, less than 1 out of 1,200,000, less than 1 out of 1,300,000, less than 1 out of 1,400,000, less than 1 out of 1,500,000, or less than 1 out of 2,000,000 observations from the sequencing data.
[0163] In some cases, at least one cell-free nucleic acid molecule of the identified one or more cell-free nucleic acid molecules can comprise a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants that are separated by at least one nucleotide, as disclosed herein.
[0164] In some cases, one or more of the operations (a) through (c) of the subject method can be performed by a computer system. In an example, all of the operations (a) through (c) of the subject method can be performed by the computer system.
[0165] The sequencing data, as disclosed herein, can be obtained from one or more sequencing methods. A sequencing method can be a first-generation sequencing method (e.g., Maxam-Gilbert sequencing, Sanger sequencing). A sequencing method can be a high-throughput sequencing method, such as next-generation sequencing (NGS) (e.g., sequencing by synthesis). A high-throughput sequencing method can sequence simultaneously (or substantially simultaneously) at least about 10,000, at least about 100,000, at least about 1 million, at least about 10 million, at least about 100 million, at least about 1 billion, or more polynucleotide molecules (e.g., cell-free nucleic acid molecules or derivatives thereof). NGS can be any generation number of sequencing technologies (e.g., second-generation sequencing technologies, third-generation sequencing technologies, fourth-generation sequencing technologies, etc.). Non-limiting examples of high-throughput sequencing methods include massively parallel signature sequencing, polony sequencing, pyrosequencing, sequencing-by-synthesis, combinatorial probe anchor synthesis (cPAS), sequencing-by-ligation (e.g., sequencing by oligonucleotide ligation and detection (SOLiD) sequencing), semiconductor sequencing (e.g., Ion Torrent semiconductor sequencing), DNA nanoball sequencing, and single-molecule sequencing, sequencing-by-hybridization.
[0166] In some embodiments of any one of the methods disclosed herein, the sequencing data can be obtained based on any of the disclosed sequencing methods that utilizes nucleic acid amplification (e.g., polymerase chain reaction (PCR)). Non-limiting examples of such sequencing methods can include 454 pyrosequencing, polony sequencing, and SoLiD sequencing. In some cases, amplicons (e.g., derivatives of the plurality of cell-free nucleic acid molecules that is obtained or derived from the subject, as disclosed herein) that correspond to a genomic region of interest (e.g., a genomic region associated with a disease) can be generated by PCR, optionally pooled, and subsequently sequenced to generating sequencing data. In some examples, because the regions of interest are amplified into amplicons by PCR before being sequenced, the nucleic acid sample is already enriched for the region of interest, and thus any additional pooling (e.g., hybridization) may not and need not be needed prior to sequencing (e.g., non-hybridization based NGS). Alternatively, pooling via hybridization can further be performed for additional enrichment prior to sequencing. Alternatively, the sequencing data can be obtained without generating PCR copies, e.g., via cPAS sequencing.
[0167] A number of embodiments utilize capture hybridization techniques to perform targeted sequencing. When performing sequencing on cell-free nucleic acids, in order to enhance resolution on particular genomic loci, library products can be captured by hybridization prior to sequencing. Capture hybridization can be particularly useful when trying to detect rare and/or somatic phased variants from a sample at particular genomic loci. In some situations, detection of rare and/or somatic phased variants is indicative of the source of nucleic acids, including nucleic acids derived from a cancer source. Accordingly, capture hybridization is a tool that can enhance detection of circulating-tumor nucleic acids within cell-free nucleic acids.
[0168] Various types of cancers repeatedly experience aberrant somatic hypermutation in particular genomic loci. For instance, the enzyme activation-induced deaminase induces aberrant somatic hypermutation in B-cells, which leads to various B-cell lymphomas, including (but not limited to) diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt lymphoma (BL), and B-cell chronic lymphocytic leukemia (CLL). Accordingly, in numerous embodiments, probes are designed to pull down (or capture) genomic loci known to experience aberrant somatic hypermutation in a lymphoma. FIG. 1D and Table 1 describe a number of regions that experience aberrant somatic hypermutation in DLBCL, FL, BL and CLL. Provided in Table 6 is list of nucleic acid probes that can be utilized to pull down (or capture) genomic loci to detect aberrant somatic hypermutation in B-cell cancers.
[0169] Capture sequencing can also be performed utilizing personalized nucleic acid probes designed to detect the existence of an individual's cancer. An individual having a cancer can have their cancer biopsied and sequenced to detect somatic phased variants that have accumulated in the cancer. Based on the sequencing result, in accordance with a number of embodiments, nucleic acid probes are designed and synthesized capable of pulling down the genomic loci inclusive of the positions of where the phased variants. These personalized designed and synthesized nucleic acid probes can be utilized to detect circulating-tumor nucleic acids from a liquid biopsy of that individual. Accordingly, the personalized nucleic acid probes can be useful for determining treatment response and/or detecting MRD after treatment.
[0170] In some embodiments of any one of the methods disclosed herein, the sequencing data can be obtained based on any sequencing method that utilizes adapters. Nucleic acid samples (e.g., the plurality of cell-free nucleic acid molecules from the subject, as disclosed herein) can be conjugated with one or more adapters (or adapter sequences) for recognizing (e.g., via hybridization) of the sample or any derivatives thereof (e.g., amplicons). In some examples, the nucleic acid samples can be tagged with a molecular barcode, e.g., such that each cell-free nucleic acid molecule of the plurality of cell-free nucleic acid molecules can have a unique barcode. Alternatively or in addition to, the nucleic acid samples can be tagged with a sample barcode, e.g., such that the plurality of cell-free nucleic acid molecules from the subject (e.g., a plurality of cell-free nucleic acid molecules obtained from a specific bodily tissue of the subject) can have the same barcode.
[0171] In alternative embodiments, the methods of identifying one or more cell-free nucleic acid molecules comprising the plurality of phased variants, as disclosed herein, can be performed without molecular barcoding, without sample barcoding, or without molecular barcoding and sample barcoding, at least in part due to high specificity and low LOD achieved by relying on identifying the phased variants as opposed to, e.g., a single SNV.
[0172] In some embodiments of any one of the methods disclosed herein, the sequencing data can be obtained and analyzed without in silico removal or suppression of (i) background error and/or (ii) sequencing error, at least in part due to high specificity and low LOD achieved by relying on identifying the phased variants as opposed to, e.g., a single SNV or indel.
[0173] In some embodiments of any one of the methods disclosed herein, using the plurality of variants as a condition to identify target cell-free nucleic acid molecules with specific mutations of interest without in silico methods of error suppression can yield a background error-rate that is lower than that of (i) barcode-deduplication, (ii) integrated digital error suppression, or (iii) duplex sequencing by at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, at least about 400-fold, at least about 600-fold, at least about 800-fold, or at least about 1,000-fold. This approach may advantageously increase signal-to-noise ratio (thereby increasing sensitivity and/or specificity) of identifying target cell-free nucleic acid molecules with specific mutations of interest.
[0174] In some embodiments of any one of the methods disclosed herein, increasing a minimum number of phased variants (e.g., increasing from at least two phased variants to at least three phased variants) per cell-free nucleic acid molecule required as a condition to identify target cell-free nucleic acid molecules with specific mutations of interest can reduce the background error-rate by at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold. This approach may advantageously increase signal-to-noise ratio (thereby increasing sensitivity and/or specificity) of identifying target cell-free nucleic acid molecules with specific mutations of interest.
[0175] In one aspect, the present disclosure provides a method of treating a condition of a subject, as shown in flowchart 2540 in FIG. 25D. The method can comprise (a) identifying the subject for treatment of the condition, wherein the subject has been determined to have the condition based on identification of one or more cell-free nucleic acid molecules from a plurality of cell-free nucleic acid molecules that is obtained or derived from the subject (Process 2542). Each of the identified one or more cell-free nucleic acid molecules can comprise a plurality of phased variants relative to a reference genomic sequence. At least a portion (e.g., partial or all) of the plurality of phased variants can be separated by at least one nucleotide, such that a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants are separated by at least one nucleotide, as disclosed herein. In some cases, a presence of the plurality of phased variants is indicative of the condition (e.g., a disease, such as cancer) of the subject. The method can further comprise (b) subjecting the subject to the treatment based on the step (a) (process 2544). Examples of such treatment of the condition of the subject are disclosed elsewhere in the present disclosure.
[0176] In one aspect, the present disclosure provides a method of monitoring a progress (e.g., progression or regression) of a condition of a subject, as shown in flowchart 2550 in FIG. 25E. The method can comprise (a) determining a first state of the condition of the subject based on identification of a first set of one or more cell-free nucleic acid molecules from a first plurality of cell-free nucleic acid molecules that is obtained or derived from the subject (process 2552). The method can further comprise (b) determining a second state of the condition of the subject based on identification of a second set of one or more cell-free nucleic acid molecules from a second plurality of cell-free nucleic acid molecules that is obtained or derived from the subject (process 2554). The second plurality of cell-free nucleic acid molecules can be obtained from the subject subsequent to obtaining the first plurality of cell-free nucleic acid molecules from the subject. The method can optionally comprise (c) determining the progress (e.g., progression or regression) of the condition based at least in part on the first state of the condition and the second state of the condition (process 2556). In some cases, each of the one or more cell-free nucleic acid molecules identified (e.g., each of the first set of one or more cell-free nucleic acid molecules identified, each of the second set of one or more cell-free nucleic acid molecules identified) can comprise a plurality of phased variants relative to a reference genomic sequence. At least a portion (e.g., partial or all) of the one or more cell-free nucleic acid molecules identified can be separated by at least one nucleotide, as disclosed herein. In some cases, presence of the plurality of phased variants can be indicative of a state of the condition of the subject
[0177] In some cases, the first plurality of cell-free nucleic acid molecules from the subject can be obtained (e.g., via blood biopsy) and analyzed to determine (e.g., diagnose) a first state of the condition (e.g., a disease, such as cancer) of the subject. The first plurality of cell-free nucleic acid molecules can be analyzed via any of the methods disclosed herein (e.g., with or without sequencing) to identify the first set of one or more cell-free nucleic acid molecules comprising the plurality of phased variants, and the presence or characteristics of the first set of one or more cell-free nucleic acid molecules can be used to determine the first state of the condition (e.g., an initial diagnosis) of the subject. Based on the determined first state of the condition, the subject can be subjected to one or more treatments (e.g., chemotherapy) as disclosed herein. Subsequent to the one or more treatments, he second plurality of cell-free nucleic acid molecules can be obtained from the subject.
[0178] In some cases, the subject can be subjected to at least or up to about 1 treatment, at least or up to about 2 treatments, at least or up to about 3 treatments, at least or up to about 4 treatments, at least or up to about 5 treatments, at least or up to about 6 treatments, at least or up to about 7 treatments, at least or up to about 8 treatments, at least or up to about 9 treatments, or at least or up to about 10 treatments based on the determined first state of the condition. In some cases, the subject can be subjected to a plurality of treatments based on the determined first state of the condition, and a first treatment of the plurality of treatments and a second treatment of the plurality of treatments can be separated by at least or up to about 1 day, at least or up to about 7 days, at least or up to about 2 weeks, at least or up to about 3 weeks, at least or up to about 4 weeks, at least or up to about 2 months, at least or up to about 3 months, at least or up to about 4 months, at least or up to about 5 months, at least or up to about 6 months, at least or up to about 12 months, at least or up to about 2 years, at least or up to about 3 years, at least or up to about 4 years, at least or up to about 5 years, or at least or up to about 10 years. The plurality of treatments for the subject can be the same. Alternatively, the plurality of treatments can be different by drug type (e.g., different chemotherapeutic drugs), drug dosage (e.g., increasing dosage, decreasing dosage), presence or absence of a co-therapeutic agent (e.g., chemotherapy and immunotherapy), modes of administration (e.g., intravenous vs oral administrations), frequency of administration (e.g., daily, weekly, monthly), etc.
[0179] In some cases, the subject may not and need not be treated for the condition between determination of the first state of the condition and determination of the second state of the condition. For example, without any intervening treatment, the second plurality of cell-free nucleic acid molecules may be contained (e.g., via liquid biopsy) from the subject to confirm whether the subject still exhibits indications of the first state of the condition.
[0180] In some cases, the second plurality of cell-free nucleic acid molecules from the subject can be obtained (e.g., via blood biopsy) at least or up to about 1 day, at least or up to about 7 days, at least or up to about 2 weeks, at least or up to about 3 weeks, at least or up to about 4 weeks, at least or up to about 2 months, at least or up to about 3 months, at least or up to about 4 months, at least or up to about 5 months, at least or up to about 6 months, at least or up to about 12 months, at least or up to about 2 years, at least or up to about 3 years, at least or up to about 4 years, at least or up to about 5 years, or at least or up to about 10 years after obtaining the first plurality of cell-free nucleic acid molecules from the subject.
[0181] In some cases, at least or up to about 2, at least or up to about 3, at least or up to about 4, at least or up to about 5, at least or up to about 6, at least or up to about 7, at least or up to about 8, at least or up to about 9, or at least or up to about 10 different samples comprising a plurality of nucleic acid molecules (e.g., at least the first plurality of cell-free nucleic acid molecules and the second plurality of cell-free nucleic acid molecules) can be obtained over time (e.g., once every month for 6 months, once every two months for a year, once every three months for a year, once every 6 months for one or more years, etc.) to monitor the progress of the condition of the subject, as disclosed herein.
[0182] In some cases, the step of determining the progress of the condition based on the first state of the condition and the second state of the condition can comprise comparing one or more characteristics of the first state and the second state of the condition, such as, for example, (i) a total number of cell-free nucleic acid molecules identified to comprise the plurality of phased variants in each state (e.g., per equal weight or volume of the biological sample of origin, per equal number of initial cell-free nucleic acid molecules analyzed, etc.), (ii) an average number of the plurality of phased variants per each cell-free nucleic acid molecule identified to comprise a plurality of phased variants (i.e., two or more phased variants), or (iii) a number of cell-free nucleic acid molecules identified to comprise the plurality of phased variants divided by a total number of cell-free nucleic acid molecules that comprise a mutation that overlaps with some of the plurality of phased variants (i.e., phased variant allele frequency). Based on such comparison, MRD of the condition (e.g., cancer or tumor) of the subject can be determined. For example, tumor burden or cancer burden of the subject can be determined based on such comparison.
[0183] In some cases, the progress of the condition can be progression or worsening of the condition. In an example, the worsening of the condition can comprise developing of a cancer from an earlier stage to a later stage, such as from stage I cancer to stage III cancer. In another example, the worsening of the condition can comprise increasing size (e.g., volume) of a solid tumor. Yet in a different example, the worsening of the condition can comprise cancer metastasis from once location to another location within the subject's body.
[0184] In some examples, (i) a total number of cell-free nucleic acid molecules identified to comprise the plurality of phased variants from the second state of the condition of the subject can be higher than (ii) a total number of cell-free nucleic acid molecules identified to comprise the plurality of phased variants from the first state of the condition of the subject by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 200-fold, at least or up to about 300-fold, at least or up to about 400-fold, or at least or up to about 500-fold.
[0185] In some examples, (i) an average number of the plurality of phased variants per each cell-free nucleic acid molecule identified to comprise a plurality of phased variants from the second state of the condition of the subject can be higher than (ii) an average number of the plurality of phased variants per each cell-free nucleic acid molecule identified to comprise a plurality of phased variants from the first state of the condition of the subject by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 200-fold, at least or up to about 300-fold, at least or up to about 400-fold, or at least or up to about 500-fold.
[0186] In some cases, the progress of the condition can be regression or at least a partial remission of the condition. In an example, the at least the partial remission of the condition can comprise downstaging of a cancer from a later stage to an earlier stage, such as from stage IV cancer to stage II cancer. Alternatively, the at least the partial remission of the condition can be full remission from cancer. In another example, the at least the partial remission of the condition can comprise decreasing size (e.g., volume) of a solid tumor.
[0187] In some examples, (i) a total number of cell-free nucleic acid molecules identified to comprise the plurality of phased variants from the second state of the condition of the subject can be lower than (ii) a total number of cell-free nucleic acid molecules identified to comprise the plurality of phased variants from the first state of the condition of the subject by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 200-fold, at least or up to about 300-fold, at least or up to about 400-fold, or at least or up to about 500-fold.
[0188] In some examples, (i) an average number of the plurality of phased variants per each cell-free nucleic acid molecule identified to comprise a plurality of phased variants from the second state of the condition of the subject can be lower than (ii) an average number of the plurality of phased variants per each cell-free nucleic acid molecule identified to comprise a plurality of phased variants from the first state of the condition of the subject by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 200-fold, at least or up to about 300-fold, at least or up to about 400-fold, or at least or up to about 500-fold.
[0189] In some cases, the progress of the condition can remain substantially the same between the two states of the condition of the subject. In some examples, (i) a total number of cell-free nucleic acid molecules identified to comprise the plurality of phased variants from the second state of the condition of the subject can be about the same as (ii) a total number of cell-free nucleic acid molecules identified to comprise the plurality of phased variants from the first state of the condition of the subject. In some examples, (i) an average number of the plurality of phased variants per each cell-free nucleic acid molecule identified to comprise a plurality of phased variants from the second state of the condition of the subject can about the same as (ii) an average number of the plurality of phased variants per each cell-free nucleic acid molecule identified to comprise a plurality of phased variants from the first state of the condition of the subject.
[0190] In some embodiments of any one of the methods disclosed herein, the one or more cell-free nucleic acid molecules comprising the plurality of phased variants can be identified from the plurality of cell-free nucleic acid molecules by one or more sequencing methods. Alternatively or in addition to, the one or more cell-free nucleic acid molecules comprising the plurality of phased variants can be identified by being pulled down from (or captured from among) the plurality of cell-free nucleic acid molecules with a set of nucleic acid probes. The pull down (or capture) method via the set of nucleic acid probes can be sufficient to identify the one or more cell-free nucleic acid molecules of interest without sequencing. In some cases, the set of nucleic acid probes can be configured to hybridize to at least a portion of cell-free nucleic acid (e.g., cfDNA) molecules from one or more genomic regions associated with the condition of the subject. As such, a presence of one or more cell-free nucleic acid molecules that have been pulled down by the set of nucleic acid probes can be an indication that the one or more cell-free nucleic acid molecules are derived from the condition (e.g., ctDNA or ctRNA). Additional details of the set of nucleic probes are disclosed elsewhere the present disclosure.
[0191] In some embodiments of any one of the methods disclosed herein, based the sequencing data derived from the plurality of cell-free nucleic acid molecules (e.g., cfDNA) that is obtained or derived from the subject, (i) the one or more cell-free nucleic acid molecules identified to comprise the plurality of phased variants can be separated, in silico, from (ii) one or more other cell-free nucleic acid molecules that are not identified to comprise the plurality of phased variants (or one or more other cell-free nucleic acid molecules that do not comprise the plurality of phased variants). In some cases, the method can further comprise generating an additional data comprising sequencing information of only (i) the one or more cell-free nucleic acid molecules identified to comprise the plurality of phased variants. In some cases, the method can further comprise generating a different data comprising sequencing information of only (ii) the one or more other cell-free nucleic acid molecules that are not identified to comprise the plurality of phased variants (or the one or more other cell-free nucleic acid molecules that do not comprise the plurality of phased variants).
[0192] In one aspect, the present disclosure provides a method for determining a condition of the subject, as shown by flowchart 2560 in FIG. 25F. The method can comprise (a) providing a mixture comprising (1) a set of nucleic acid probes and (2) a plurality of cell-free nucleic acid molecules obtained or derived from the subject (process 2562). In some cases, an individual nucleic acid probe of the set of nucleic acid probes can be designed to hybridize to a target cell-free nucleic acid molecule comprising a plurality of phased variants relative to a reference genomic sequence that are separated by at least one nucleotide. As such, a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants can be separated by at least one nucleotide, as disclosed herein. In some cases, the individual nucleic acid probe can comprise an activatable reporter agent. The activatable reporter agent can be activated by either one of (i) hybridization of the individual nucleic acid probe to the plurality of phased variants and (ii) dehybridization of at least a portion of the individual nucleic acid probe that has been hybridized to the plurality of phased variants. The method can further comprise (b) detecting the reporter agent that is activated, to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules (process 2564). Each of the one or more cell-free nucleic acid molecules can comprise the plurality of phased variants. The method can optionally comprise (c) analyzing at least a portion of the identified one or more cell-free nucleic acid molecules to determine the condition of the subject (process 2566).
[0193] In one aspect, the present disclosure provides a method for determining a condition of the subject, as shown by flowchart 2570 in FIG. 25G. The method can comprise (a) providing a mixture comprising (1) a set of nucleic acid probes and (2) a plurality of cell-free nucleic acid molecules obtained or derived from the subject (process 2572). In some cases, an individual nucleic acid probe of the set of nucleic acid probes can be designed to hybridize to a target cell-free nucleic acid molecule comprising a plurality of phased variants relative to a reference genomic sequence. In some cases, the individual nucleic acid probe can comprise an activatable reporter agent. The activatable reporter agent can be activated by either one of (i) hybridization of the individual nucleic acid probe to the plurality of phased variants and (ii) dehybridization of at least a portion of the individual nucleic acid probe that has been hybridized to the plurality of phased variants. The method can further comprise (b) detecting the reporter agent that is activated, to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules (process 2574). Each of the one or more cell-free nucleic acid molecules can comprise the plurality of phased variants, and a LOD of the identification step can be less than about 1 out of 50,000 cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules, as disclosed herein. The method can optionally comprise (c) analyzing at least a portion of the identified one or more cell-free nucleic acid molecules to determine the condition of the subject (process 2576).
[0194] In some cases, a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants are separated by at least one nucleotide, as disclosed herein.
[0195] In some cases, the LOD of the step of identifying the one or more cell-free nucleic acid molecules, as disclosed herein, can be less than about 1 out of 60,000, less than 1 out of 70,000, less than 10 out of 80,000, less than 1 out of 90,000, less than 1 out of 100,000, less than 1 out of 150,000, less than 1 out of 200,000, less than 1 out of 300,000, less than 1 out of 400,000, less than 1 out of 500,000, less than 1 out of 600,000, less than 1 out of 700,000, less than 1 out of 800,000, less than 1 out of 900,000, less than 1 out of 1,000,000, less than 1 out of 1,000,000, less than 1 out of 1,100,000, less than 1 out of 1,200,000, less than 1 out of 1,300,000, less than 1 out of 1,400,000, less than 1 out of 1,500,000, less than 1 out of 2,000,000, less than 1 out of 2,500,000, less than 1 out of 3,000,000, less than 1 out of 4,000,000, or less than 1 out of 5,000,000 cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules. Generally, a detection method with a lower LOD has a greater sensitivity of such detection.
[0196] In some embodiments of any one of the methods disclosed herein, the method can further comprise mixing (1) the set of nucleic acid probes and (2) the plurality of cell-free nucleic acid molecules.
[0197] In some embodiments of any one of the methods disclosed herein, the activatable reporter agent of a nucleic acid probe can be activated upon hybridization of the individual nucleic acid probe to the plurality of phased variants. Non-limiting examples of such nucleic acid probe can include a molecular beacon, eclipse probe, amplifluor probe, scorpions PCR primer, and light upon extension fluorogenic PCR primer (LUX primer).
[0198] For example, the nucleic acid probe can be a molecular beacon, as shown in FIG. 26A. The molecular beacon can be fluorescently labeled (e.g., dye-labeled) oligonucleotide probe that comprises complementarity to a target cell-free nucleic acid molecule 2603 in a region that comprises the plurality of phased variants. The molecular beacon can have a length between about 25 nucleotides to about 50 nucleotides. The molecular beacon can also be designed to be partially self-complimentary, such that it form a hairpin structure with a stem 2601a and a loop 2601b. The 5' and 3' ends of the molecular beacon probe can have complementary sequences (e.g., about 5-6 nucleotides) that form the stem structure 2601a. The loop portion 2601b of the hairpin can be designed to specifically hybridize to a portion (e.g., about 15-30 nucleotides) of the target sequence comprising two or more phased variants. The hairpin can be designed to hybridize to a portion that comprises at least 2, 3, 4, 5, or more phased variants. A fluorescent reporter molecule can be attached to the 5' end of the molecular beacon probe, and a quencher that quenches fluorescence of the fluorescent reporter can be attached to the 3' end of the molecular beacon probe. Formation of the hairpin therefore can bring the fluorescent reporter and quencher together, such that no fluorescence is emitted. However, during annealing operation of amplification reaction of the plurality of cell-free nucleic acid molecules that is obtained or derived from the subject, the loop portion of the molecular beacon can bind to its target sequence, causing the stem to denature. Thus, the reporter and quencher can be separated, abolishing quenching, and the fluorescent reporter is activated and detectable. Because fluorescence of the fluorescent reporter is emitted from the molecular beacon probe only when the probe is bound to the target sequence, the amount or level of fluorescence detected can be proportional to the amount of target in the reaction (e.g., (i) a total number of cell-free nucleic acid molecules identified to comprise the plurality of phased variants in each state or (ii) an average number of the plurality of phased variants per each cell-free nucleic acid molecule identified to comprise a plurality of phased variants, as disclosed herein).
[0199] In some embodiments of any one of the methods disclosed herein, the activatable reporter agent can be activated upon dehybridization of at least a portion of the individual nucleic acid probe that has been hybridized to the plurality of phased variants. In other words, once the individual nucleic acid probe is hybridized to target cell-free nucleic acid molecule's portion that comprises the plurality of phased variants, dehybridization of at least a portion of the individual nucleic acid prob and the target cell-free nucleic acid can activate the activatable reporter agent. Non-limiting examples of such nucleic acid probe can include a hydrolysis probe (e.g., TaqMan prob), dual hybridization probes, and QZyme PCR primer.
[0200] For example, the nucleic acid probe can be a hydrolysis probe, as shown in FIG. 26B. The hydrolysis probe 2611 can be a fluorescently labeled oligonucleotide probe that can specifically hybridize to a portion (e.g., between about 10 and about 25 nucleotides) of the target cell-free nucleic acid molecule 2613, wherein the hybridized portion comprises two or more phased variants. The hydrolysis probe 2611 can be labeled with a fluorescent reporter at the 5' end and a quencher at the 3' end. When the hydrolysis probe is intact (e.g., not cleaved), the fluorescence of the reporter is quenched due to its proximity to the quencher (FIG. 26B). During annealing operation of amplification reaction of the plurality of cell-free nucleic acid molecules obtained or derived from the subject, 5'.fwdarw.3' exonuclease activity of certain thermostable polymerases (e.g., Taq or Tth) The amplification reaction of the plurality of cell-free nucleic acid molecules obtained or derived from the subject can include a combined annealing/extension operation during which the hydrolysis probe hybridizes to the target cell-free nucleic acid molecule, and the dsDNA-specific 5'.fwdarw.3' exonuclease activity of a thermostable polymerase (e.g., Taq or Tth) cleaves off the fluorescent reporter from the hydrolysis probe. As a result, the fluorescent reporter is separated from the quencher, resulting in a fluorescence signal that is proportional to the amount of target in the sample (e.g., (i) a total number of cell-free nucleic acid molecules identified to comprise the plurality of phased variants in each state or (ii) an average number of the plurality of phased variants per each cell-free nucleic acid molecule identified to comprise a plurality of phased variants, as disclosed herein).
[0201] In some embodiments of any one of the methods disclosed herein, the reporter agent can comprise a fluorescent reporter. Non-limiting examples of a fluorescent reporter include fluorescein amidite (FAM, 2-[3-(dimethylamino)-6-dimethyliminio-xanthen-9-yl]benzoate TAN/IRA, (2E)-2-[(2E,4E)-5-(2-tert-butyl-9-ethyl-6,8,8-trimethyl-pyrano[3,2-g]quin- olin-1-ium-4-yl)penta-2,4-dienylidene]-1-(6-hydroxy-6-oxo-hexyl)-3,3-dimet- hyl-indoline-5-sulfonate Dy 750, 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein, 4,5,6,7-Tetrachlorofluorescein TET.TM., sulforhodamine 101 acid chloride succinimidyl ester Texas Red-X, ALEXA Dyes, Bodipy Dyes, cyanine Dyes, Rhodamine 123 (hydrochloride), Well RED Dyes, MAX, and TEX 613. In some cases, the reporter agent further comprises a quencher, as disclosed herein. Non-limiting examples of a quencher can include Black Hole Quencher, Iowa Black Quencher, and 4-dimethylaminoazobenzene-4'-sulfonyl chloride (DABCYL).
[0202] In some embodiments of any one of the methods disclosed herein, any PCR reaction utilizing the set of nucleic acid probes can be performed using real-time PCR (qPCR). Alternatively, the PCR reaction utilizing the set of nucleic acid probes can be performed using digital PCR (dPCR).
[0203] Provided in FIG. 24 is an example flowchart of a process to perform a clinical intervention and/or treatment based on detecting circulating-tumor nucleic acids in an individual's biological sample. In several embodiments, detection of circulating-tumor nucleic acids is determined by the detection of somatic variants in phase in a cell-free nucleic acid sample. In many embodiments, detection of circulating-tumor nucleic acids indicates cancer is present, and thus appropriate clinical intervention and/or treatment can be performed.
[0204] Referring to FIG. 24, process 2400 can begin with obtaining, preparing, and sequencing (2401) cell-free nucleic acids obtained from a non-invasive biopsy (e.g., liquid or waste biopsy), utilizing a capture sequencing approach across regions shown to harbor a plurality of genetic mutations or variants occurring in phase. In several embodiments, cfDNA and/or cfRNA is extracted from plasma, blood, lymph, saliva, urine, stool, and/or other appropriate bodily fluid. Cell-free nucleic acids can be isolated and purified by any appropriate means. In some embodiments, column purification is utilized (e.g., QIAamp Circulating Nucleic Acid Kit from Qiagen, Hilden, Germany). In some embodiments, isolated RNA fragments can be converted into complementary DNA for further downstream analysis.
[0205] In some embodiments, a biopsy is extracted prior to any indication of cancer. In some embodiments, a biopsy is extracted to provide an early screen in order to detect a cancer. In some embodiments, a biopsy is extracted to detect if residual cancer exists after a treatment. In some embodiments, a biopsy is extracted during treatment to determine whether the treatment is providing the desired response. Screening of any particular cancer can be performed. In some embodiments, screening is performed to detect a cancer that develops somatic phased variants in stereotypical regions in the genome, such as (for example) lymphoma. In some embodiments, screening is performed to detect a cancer in which somatic phased variants were discovered utilizing a prior extracted cancer biopsy.
[0206] In some embodiments, a biopsy is extracted from an individual with a determined risk of developing cancer, such as those with a familial history of the disorder or have determined risk factors (e.g., exposure to carcinogens). In many embodiments, a biopsy is extracted from any individual within the general population. In some embodiments, a biopsy is extracted from individuals within a particular age group with higher risk of cancer, such as, for example, aging individuals above the age of 50. In some embodiments, a biopsy is extracted from an individual diagnosed with and treated for a cancer.
[0207] In some embodiments, extracted cell-free nucleic acids are prepared for sequencing. Accordingly, cell-free nucleic acids are converted into a molecular library for sequencing. In some embodiments, adapters and/or primers are attached onto cell-free nucleic acids to facilitate sequencing. In some embodiments, targeted sequencing of particular genomic loci is to be performed, and thus particular sequences corresponding to the particular loci are captured via hybridization prior to sequencing (e.g., capture sequencing). In some embodiments, capture sequencing is performed utilizing a set of probes that pull down (or capture) regions that have been discovered to commonly harbor phased variants for a particular cancer (e.g., lymphoma). In some embodiments, capture sequencing is performed utilizing a set of probes that pull down (or capture) regions that have been discovered to harbor phased variants as determined prior by sequencing a biopsy of the cancer. More detailed discussion of capture sequencing and probes is provided in the section entitled "Capture Sequencing."
[0208] In some embodiments, any appropriate sequencing technique can be utilized that can detect phased variants indicative of circulating-tumor nucleic acids. Sequencing techniques include (but are not limited to) 454 sequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent sequencing, single-read sequencing, paired-end sequencing, etc.
[0209] Process 2400 analyzes (2403) the cell-free nucleic acid sequencing result to detect circulating-tumor nucleic acid sequences, as determined by detection of somatic variants occurring in phase. Because cancers are actively growing and expanding, neoplastic cells are often releasing biomolecules (especially nucleic acids) into the vasculature, lymph, and/or waste systems. In addition, due to biophysical constraints in their local environment, neoplastic cells are often rupturing, releasing their inner cell contents into the vasculature, lymph, and/or waste systems. Accordingly, it is possible to detect distal primary tumors and/or metastases from a liquid or waste biopsy.
[0210] Detection of circulating-tumor nucleic acid sequences indicates that a cancer is present in the individual being examined. Accordingly, based on detection of circulating-tumor nucleic acids, a clinical intervention and/or treatment may be performed (2405). In a number of embodiments, a clinical procedure is performed, such as (for example) a blood test, genetic test, medical imaging, physical exam, a tumor biopsy, or any combination thereof. In several embodiments, diagnostics are preformed to determine the particular stage of cancer. In a number of embodiments, a treatment is performed, such as (for example) chemotherapy, radiotherapy, chemoradiotherapy, immunotherapy, hormone therapy, targeted drug therapy, surgery, transplant, transfusion, medical surveillance, or any combination thereof. In some embodiments, an individual is assessed and/or treated by medical professional, such as a doctor, physician, physician's assistant, nurse practitioner, nurse, caretaker, dietician, or similar.
[0211] Various embodiments of the present disclosure are directed towards utilizing detection of cancer to perform clinical interventions. In a number of embodiments, an individual has a liquid or waste biopsy screened and processed by methods described herein to indicate that the individual has cancer and thus an intervention is to be performed. Clinical interventions include clinical procedures and treatments. Clinical procedures include (but are not limited to) blood tests, genetic test, medical imaging, physical exams, and tumor biopsies. Treatments include (but are not limited to) chemotherapy, radiotherapy, chemoradiotherapy, immunotherapy, hormone therapy, targeted drug therapy, surgery, transplant, transfusion, and medical surveillance. In several embodiments, diagnostics are performed to determine the particular stage of cancer. In some embodiments, an individual is assessed and/or treated by medical professional, such as a doctor, physician, physician's assistant, nurse practitioner, nurse, caretaker, dietician, or similar.
[0212] In several embodiments as described herein a cancer can be detected utilizing a sequencing result of cell-free nucleic acids derived from blood, serum, cerebrospinal fluid, lymph fluid, urine or stool. In many embodiments, cancer is detected when a sequencing result has one or more somatic variants present in phase within a short genetic window, such as the length of a cell-free molecule (e.g., about 170 bp). In numerous embodiments, a statistical method is utilized to determine whether the presence of phased variants is derived from a cancerous source (as opposed to molecular artifact or other biological source). Various embodiments utilize a Monte Carlo sampling method as the statistical method to determine whether a sequencing result of cell-free nucleic acids includes sequences of circulating-tumor nucleic acids based on a score as determined by the presence of phased variants. Accordingly, in a number of embodiments, cell-free nucleic acids are extracted, processed, and sequenced, and the sequencing result is analyzed to detect cancer. This process is especially useful in a clinical setting to provide a diagnostic scan.
[0213] An exemplary procedure for a diagnostic scan of an individual for a B-cell cancer is as follows:
[0214] (a) extract liquid or waste biopsy from individual,
[0215] (b) prepare and perform targeted sequencing of cell-free nucleic acids from biopsy utilizing nucleic acid probes specific for the B-cell cancer,
[0216] (c) detect phased variants in a sequencing results that are indicative of circulating-tumor nucleic acid sequences, and
[0217] (d) perform clinical intervention based on detection of circulating-tumor nucleic acid sequences.
[0218] An exemplary procedure for a personalized diagnostic scan of an individual for a cancer that has been previously sequenced to detect phased variants in particular genomic loci is as follows:
extract cancer biopsy from individual sequence cancer biopsy to detect phased variants that have accumulated in the cancer
[0219] (a) design and synthesize nucleic acid probes for genomic loci that include the positions of the detected phased variants,
[0220] (b) extract liquid or waste biopsy from individual,
[0221] (c) prepare and perform targeted sequencing of cell-free nucleic acids from biopsy utilizing the designed and synthesized nucleic acid probes,
[0222] (d) detect phased variants in a sequencing results that are indicative of circulating-tumor nucleic acid sequences, and
[0223] (e) perform clinical intervention based on detection of circulating-tumor nucleic acid sequences.
[0224] In some embodiments of any one of the methods disclosed herein, at least a portion of the identified one or more cell-free nucleic acid molecules comprising the plurality of phased variants can be further analyzed for determining the condition of the subject. In such analysis, (i) the identified one or more cell-free nucleic acid molecules and (ii) other cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules that do not comprise the plurality of phased variants can be analyzed as different variables. In some cases, a ratio of (i) a number the identified one or more cell-free nucleic acid molecules and (ii) a number of the other cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules that do not comprise the plurality of phased variants can be used a factor to determine the condition of the subject. In some cases, comparison of (i) a position(s) of the identified one or more cell-free nucleic acid molecules relative to the reference genomic sequence and (ii) a position(s) of the other cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules that do not comprise the plurality of phased variants relative to the reference genomic sequence can be used a factor to determine the condition of the subject.
[0225] Alternatively, in some cases, the analysis of the identified one or more cell-free nucleic acid molecules comprising the plurality of phased variants for determining the condition of the subject may not and need not be based on the other cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules that do not comprise the plurality of phased variants. As disclosed herein, non-limiting examples of information or characteristics of the one or more cell-free nucleic acid molecules comprising the plurality of phased variants can include (i) a total number of such cell-free nucleic acid molecules and (ii) an average number of the plurality of phased variations per each nucleic acid molecule in the population of identified cell-free nucleic acid molecules.
[0226] Thus, in some embodiments of any one of the methods disclosed herein, a number of the plurality of phased variants from the one or more cell-free nucleic acid molecules that have been identified to have the plurality of phased variants can be indicative of the condition of the subject. In some cases, a ratio of (i) the number of the plurality of phased variants from the one or more cell-free nucleic acid molecules and (ii) a number of single nucleotide variants from the one or more cell-free nucleic acid molecules can be indicative of the condition of the subject. For instance, a particular condition (e.g., follicular lymphoma) can exhibit a signature ratio that is different than that of another condition (e.g., breast cancer). In some examples, for cancer or solid tumor, the ratio as disclosed herein can be between about 0.01 and about 0.20. In some examples, for cancer or solid tumor, the ratio as disclosed herein can be about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, or about 0.20. In some examples, for cancer or solid tumor, the ratio as disclosed herein can be at least or up to about 0.01, at least or up to about 0.02, at least or up to about 0.03, at least or up to about 0.04, at least or up to about 0.05, at least or up to about 0.06, at least or up to about 0.07, at least or up to about 0.08, at least or up to about 0.09, at least or up to about 0.10, at least or up to about 0.11, at least or up to about 0.12, at least or up to about 0.13, at least or up to about 0.14, at least or up to about 0.15, at least or up to about 0.16, at least or up to about 0.17, at least or up to about 0.18, at least or up to about 0.19, or at least or up to about 0.20.
[0227] In some embodiments of any one of the methods disclosed herein, a frequency of the plurality of phased variants in the one or more cell-free nucleic acid molecules that have been identified can be indicative of the condition of the subject. In some cases, based on the sequencing data disclosed herein, an average frequency of the plurality of phased variant per a predetermined bin length (e.g., a bin of about 50 base pairs) within each of the identified cell-free nucleic acid molecule can be indicative of the condition of the subject. In some cases, based on the sequencing data disclosed herein, an average frequency of the plurality of phased variant per a predetermined bin length (e.g., a bin of about 50 base pairs) within each of the identified cell-free nucleic acid molecule that is associated with a particular gene (e.g., BCL2, PIM1) can be indicative of the condition of the subject. The size of the bin can be about 30, about 40, about 50, about 60, about 70, or about 80.
[0228] In some examples, a first condition (e.g., Hodgkin lymphoma or HL) can exhibit a first average frequency and a second condition (e.g., DLBCL) can exhibit a different average frequency, thereby allowing identification and/or determination of whether the subject has or is suspected of having a particular condition. In some examples, a first sub-type of a disease can exhibit a first average frequency and a second sub-type of the same disease can exhibit a different average frequency, thereby allowing identification and/or determination of whether the subject has or is suspected of having a particular sub-type of the disease. For example, the subject can have DLBCL, and one or more cell-free nucleic acid molecules derived from germinal center B-cell (GCB) DLBCL or activated B-cell (ABC) DLBCL can have different average frequency of the plurality of phased variant per a predetermined bin length, as disclosed herein.
[0229] In some example, a condition of the subject may have a predetermined number of phased variants spanning predetermined genomic loci (i.e., a predetermined frequency of phased variants). When the predetermined frequency of phased variants match a frequency of the plurality of phased variants in the one or more cell-free nucleic acid molecules that have been identified from a plurality of cell-free nucleic acid molecules from the subject, it may indicate that the subject has such condition.
[0230] In some embodiments of any one of the methods disclosed herein, the one or more cell-free nucleic acid molecules identified to comprise the plurality of phased variants can be analyzed to determine their genomic origin (e.g., which gene locus they are from). The genomic origin of the one or more cell-free nucleic acid molecules that have been identified can be indicative of the condition of the subject, as different disease can have the plurality of phased variants in different signature genes. For example, a subject can have GCB DLBCL, and one or more cell-free nucleic acid molecules originated from GCBs of the subject can have the phased variants prevalent in BCL2 gene, while one or more cell-free nucleic acid molecules originated from ABCs of the same subject may not comprise as many phased variants in the BCL2 gene as those from GCBs. On the other hand, a subject can have ABC DLBCL, and one or more cell-free nucleic acid molecules originated from ABCs of the subject can have the phased variants prevalent in PIM1 gene, while one or more cell-free nucleic acid molecules originated from GCBs of the same subject may not comprise as many phased variants in the PIM1 gene as those from ABCs.
[0231] In some embodiments of any one of the methods disclosed herein, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 95%, at least or up to about 99%, or about 100% of the one or more cell-free nucleic acid molecules comprising the plurality of phased variants can comprise a single nucleotide variant (SNV) that is at least 2 nucleotides away from an adjacent SNV.
[0232] In some embodiments of any one of the methods disclosed herein, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, or at least or up to about 50% of the one or more cell-free nucleic acid molecules comprising the plurality of phased variants can comprise a single nucleotide variant (SNV) that is at least 3 nucleotides away from an adjacent SNV.
[0233] In some embodiments of any one of the methods disclosed herein, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, or at least or up to about 50% of the one or more cell-free nucleic acid molecules comprising the plurality of phased variants can comprise a single nucleotide variant (SNV) that is at least 4 nucleotides away from an adjacent SNV.
[0234] In some embodiments of any one of the methods disclosed herein, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, or at least or up to about 50% of the one or more cell-free nucleic acid molecules comprising the plurality of phased variants can comprise a single nucleotide variant (SNV) that is at least 5 nucleotides away from an adjacent SNV.
[0235] In some embodiments of any one of the methods disclosed herein, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, or at least or up to about 50% of the one or more cell-free nucleic acid molecules comprising the plurality of phased variants can comprise a single nucleotide variant (SNV) that is at least 6 nucleotides away from an adjacent SNV.
[0236] In some embodiments of any one of the methods disclosed herein, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, or at least or up to about 50% of the one or more cell-free nucleic acid molecules comprising the plurality of phased variants can comprise a single nucleotide variant (SNV) that is at least 7 nucleotides away from an adjacent SNV.
[0237] In some embodiments of any one of the methods disclosed herein, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, or at least or up to about 50% of the one or more cell-free nucleic acid molecules comprising the plurality of phased variants can comprise a single nucleotide variant (SNV) that is at least 8 nucleotides away from an adjacent SNV.
[0238] In some embodiments of any one of the methods disclosed herein, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, or at least or up to about 50% of the one or more cell-free nucleic acid molecules comprising the plurality of phased variants can comprise a single nucleotide variant (SNV) that is at least 9 nucleotides away from an adjacent SNV.
[0239] In some embodiments of any one of the methods disclosed herein, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, or at least or up to about 50% of the one or more cell-free nucleic acid molecules comprising the plurality of phased variants can comprise a single nucleotide variant (SNV) that is at least 10 nucleotides away from an adjacent SNV.
C. Reference Genomic Sequence
[0240] In some embodiments of any one of the methods disclosed herein, the reference genomic sequence can be at least a portion of a nucleic acid sequence database (i.e., a reference genome), which database is assembled from genetic data and intended to represent the genome of a reference cohort. In some cases, a reference cohort can be a collection of individuals from a specific or varying genotype, haplotype, demographics, sex, nationality, age, ethnicity, relatives, physical condition (e.g., healthy or having been diagnosed to have the same or different condition, such as a specific type of cancer), or other groupings. A reference genomic sequence as disclosed herein can be a mosaic (or a consensus sequence) of the genomes of two or more individuals. The reference genomic sequence can comprise at least a portion of a publicly available reference genome or a private reference genome. Non-limiting examples of a human reference genome include hg19, hg18, hg17, hg16, and hg38.
[0241] In some examples, the reference genomic sequence can comprise at least or up to about 500 nucleobases, at least or up to about 1 kilobase (kb), at least or up to about 2 kb, at least or up to about 3 kb, at least or up to about 4 kb, at least or up to about 5 kb, at least or up to about 6 kb, at least or up to about 7 kb, at least or up to about 8 kb, at least or up to about 9 kb, at least or up to about 10 kb, at least or up to about 20 kb, at least or up to about 30 kb, at least or up to about 40 kb, at least or up to about 50 kb, at least or up to about 60 kb, at least or up to about 70 kb, at least or up to about 80 kb, at least or up to about 90 kb, at least or up to about 100 kb, at least or up to about 200 kb, at least or up to about 300 kb, at least or up to about 400 kb, at least or up to about 500 kb, at least or up to about 600 kb, at least or up to about 700 kb, at least or up to about 800 kb, at least or up to about 900 kb, at least or up to about 1,000 kb, at least or up to about 2,000 kb, at least or up to about 3,000 kb, at least or up to about 4,000 kb, at least or up to about 5,000 kb, at least or up to about 6,000 kb, at least or up to about 7,000 kb, at least or up to about 8,000 kb, at least or up to about 9,000 kb, at least or up to about 10,000 kb, at least or up to about 20,000 kb, at least or up to about 30,000 kb, at least or up to about 40,000 kb, at least or up to about 50,000 kb, at least or up to about 60,000 kb, at least or up to about 70,000 kb, at least or up to about 80,000 kb, at least or up to about 90,000 kb, or at least or up to about 100,000 kb.
[0242] In some cases, the reference genomic sequence can be whole reference genome or a portion (e.g., a portion relevant to the condition of interest) of the genome. For example, the reference genomic sequence can consist of at least 1, 2, 3, 4, 5, or more genes that experience aberrant somatic hypermutation under certain types of cancer. In some cases, the reference genomic sequence can be a whole chromosomal sequence, or a fragment thereof. In some cases, the reference genomic sequence can comprise two or more (e.g., at least 2, 3, 4, 5, or more) different portions of the reference genome that are not adjacent to one another (e.g., within the same chromosome or from different chromosomes).
[0243] In some embodiments of any one of the methods disclosed herein, the reference genomic sequence can be at least a portion of a reference genome of a selected individual, such as a healthy individual or the subject of any of the methods as disclosed herein.
[0244] In some cases, the reference genomic sequence can be derived from an individual who is not the subject (e.g., a healthy control individual). Alternatively, in some cases, the reference genomic sequence can be derived from a sample of the subject. In some examples, the sample can be a healthy sample of the subject. The healthy sample of the subject can be any subject cell that is healthy, e.g., a healthy leukocyte. By comparing sequencing data of the plurality of cell-free nucleic acid molecules (e.g., cfDNA molecules) of the subject against at least a portion of the genomic sequence of a healthy cell of the same subject, one or more cell-free nucleic acid molecules that comprise the plurality of phased variants can be identified and analyzed, as disclosed herein. In some examples, the sample can be a diseased sample of the subject, such as a diseased cell (e.g., a tumor cell) or a solid tumor. The reference genomic sequence can be obtained from sequencing at least a portion of a diseased cell of the subject or from sequencing a plurality of cell-free nucleic acid molecules obtained from the solid tumor of the subject. Once the subject is diagnosed to have a particular condition (e.g., a disease), the reference genomic sequence of the subject that comprises the plurality of phased variants can be used to determine whether the subject still exhibits the same phased variants at future time points. In this context, any new phased variants identified between the "diseased" reference genomic sequence of the subject and new cell-free nucleic acid molecules obtained or derived from the subject can indicate a reduced degree of aberrant somatic hypermutation in particular genomic loci (e.g., at least a partial remission).
[0245] In various embodiments, diagnostic scans can be performed for any neoplasm type, including (but not limited to) acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), anal cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, breast cancer, Burkitt's lymphoma, cervical cancer, chronic lymphocytic leukemia (CLL) chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, diffuse large B-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, fallopian tube cancer, follicular lymphoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, hairy cell leukemia, hepatocellular cancer, Hodgkin lymphoma, hypopharyngeal cancer, Kaposi sarcoma, Kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell cancer, mesothelioma, mouth cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors, pharyngeal cancer, pituitary tumor, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, skin cancer, small cell lung cancer, small intestine cancer, squamous neck cancer, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, uterine cancer, vaginal cancer, and vascular tumors.
[0246] In a number of embodiments, a diagnostic scan is utilized to provide an early detection of cancer. In some embodiments, a diagnostic scan detects cancer in individuals having stage I, II, or III cancer. In some embodiments, a diagnostic scan is utilized to detect MRD or tumor burden. In some embodiments, a diagnostic scan is utilized to determine progress (e.g., progression or regression) of treatment. Based on the diagnostic scan, a clinical procedure and/or treatment may be performed.
D. Nucleic Acid Probes
[0247] In some embodiments of any one of the methods disclosed herein, the set of nucleic acid probes can be designed based on the any of the subject reference genomic sequences of the present disclosure. In some cases, the set of nucleic acid probes can be designed based on the plurality of phased variants that have been identified by comparing (i) sequencing data from a solid tumor of the subject and (ii) sequencing data from a healthy cell of the subject or a healthy cohort, as disclosed herein. The set of nucleic acid probes can be designed based on the plurality of phased variants that have been identified by comparing (i) sequencing data from a solid tumor of the subject and (ii) sequencing data from a healthy cell of the subject. The set of nucleic acid probes can be designed based on the plurality of phased variants that have been identified by comparing (i) sequencing data from a solid tumor of the subject and (ii) sequencing data from a healthy cell of a healthy cohort.
[0248] In some embodiments of any one of the methods disclosed herein, the set of nucleic acid probes are designed to hybridize to sequences of genomic loci associated with the condition. As disclosed herein, the genomic loci associated with the condition can be determined to experience or exhibit aberrant somatic hypermutation when the subject has the condition. Alternatively, the set of nucleic acid probes are designed to hybridize to sequences of stereotyped regions.
[0249] In some embodiments of any one of the methods disclosed herein, the set of nucleic acid probes can be designed to hybridize to at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% of the genomic regions identified in Table 1.
[0250] In some embodiments of any one of the methods disclosed herein, the set of nucleic acid probes can be designed to hybridize to at least a portion of cell-free nucleic acid (e.g., cfDNA) molecules derived from at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% of the genomic regions identified in Table 1.
[0251] In some embodiments of any one of the methods disclosed herein, each nucleic acid probe of the set of nucleic acid probes can have at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% sequence identity, at least about 95% sequence identity, at least about 99%, or about 100% sequence identity to a probe sequence selected from Table 6.
[0252] In some embodiments of any one of the methods disclosed herein, the set of nucleic acid probes can comprise at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% of probe sequences in Table 6.
[0253] In some embodiments of any one of the methods disclosed herein, the set of nucleic acid probes can be designed to cover one or more target genomic regions comprising at least or up to about 500 nucleobases, at least or up to about 1 kilobase (kb), at least or up to about 2 kb, at least or up to about 3 kb, at least or up to about 4 kb, at least or up to about 5 kb, at least or up to about 6 kb, at least or up to about 7 kb, at least or up to about 8 kb, at least or up to about 9 kb, at least or up to about 10 kb, at least or up to about 20 kb, at least or up to about 30 kb, at least or up to about 40 kb, at least or up to about 50 kb, at least or up to about 60 kb, at least or up to about 70 kb, at least or up to about 80 kb, at least or up to about 90 kb, at least or up to about 100 kb, at least or up to about 200 kb, at least or up to about 300 kb, at least or up to about 400 kb, or at least or up to about 500 kb.
[0254] In some embodiments of any one of the methods disclosed herein, a target genomic region (e.g., a target genomic locus) of the one or more target genomic regions can comprise at most about 200 nucleobases, at most about 300 nucleobases, 400 nucleobases, at most about 500 nucleobases, at most about 600 nucleobases, at most about 700 nucleobases, at most about 800 nucleobases, at most about 900 nucleobases, at most about 1 kb, at most about 2 kb, at most about 3 kb, at most about 4 kb, at most about 5 kb, at most about 6 kb, at most about 7 kb, at most about 8 kb, at most about 9 kb, at most about 10 kb, at most about 11 kb, at most about 12 kb, at most about 13 kb, at most about 14 kb, at most about 15 kb, at most about 16 kb, at most about 17 kb, at most about 18 kb, at most about 19 kb, at most about 20 kb, at most about 25 kb, at most about 30 kb, at most about 35 kb, at most about 40 kb, at most about 45 kb, at most about 50 kb, or at most about 100 kb.
[0255] In some embodiments of any one of the methods disclosed herein, the set of nucleic acid probes can comprise at least or up to about 10, at least or up to about 20, at least or up to about 30, at least or up to about 40, at least or up to about 50, at least or up to about 60, at least or up to about 70, at least or up to about 80, at least or up to about 90, at least or up to about 100, at least or up to about 200, at least or up to about 300, at least or up to about 400, at least or up to about 500, at least or up to about 600, at least or up to about 700, at least or up to about 800, at least or up to about 900, at least or up to about 1,000, at least or up to about 2,000, at least or up to about 3,000, at least or up to about 4,000, or at least or up to about 5,000 different nucleic acid probes designed to hybridize to different target nucleic acid sequences.
[0256] In some embodiments of any one of the methods disclosed herein, the set of nucleic acid probes can have a length of at least or up to about 50, at least or up to about 55, at least or up to about 60, at least or up to about 65, at least or up to about 70, at least or up to about 75, at least or up to about 80, at least or up to about 85, at least or up to about 90, at least or up to about 95, or at least or up to about 100 nucleotides.
[0257] In one aspect, the present disclosure provides a composition comprising a bait set comprising any one of the set of nucleic acid probes disclosed herein. The composition comprising such bait set can be used for any of the methods disclosed herein. In some cases, the set of nucleic acid probes can be designed to pull down (or capture) cfDNA molecules. In some cases, the set of nucleic acid probes can be designed to pull down (or capture) cfRNA molecules.
[0258] In some embodiments, the bait set can comprise a set of nucleic acid probes designed to pull down cell-free nucleic acid (e.g., cfDNA) molecules derived from genomic regions set forth in Table 1. The set of nucleic acid probes can be designed to pull down cell-free nucleic acid molecules derived from at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 6%, at least or up to about 7%, at least or up to about 8%, at least or up to about 9%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 95%, at least or up to about 99%, or about 100% of the genomic regions set forth in Table 1. In some cases, the set of nucleic acid probes can be designed to pull down cfDNA molecules. In some cases, the set of nucleic acid probes can be designed to pull down cfRNA molecules.
[0259] In some embodiments of any one of the compositions disclosed herein, an individual nucleic acid probe (or each nucleic acid probe) of the set of nucleic acid probes can comprise a pull-down tag. The pull-down tag can be used to enrich a sample (e.g., a sample comprising the plurality of nucleic acid molecules obtained or derived from the subject) for a specific subset (e.g., for cell-free nucleic acid molecules comprising the plurality of phased variants as disclosed herein).
[0260] In some cases, pull-down tag can comprise a nucleic acid barcode (e.g., on either or both sides of the nucleic acid probe). By utilizing beads or substrates comprising nucleic acid sequences having complementarity to the nucleic acid barcode, the nucleic acid barcode can be used to pull-down and enrich for any nucleic acid probe that is hybridized to a target cell-free nucleic acid molecule. Alternatively or in addition to, the nucleic acid barcode can be used to identify the target cell-free nucleic acid molecule from any sequencing data (e.g., sequencing by amplification) obtained by using any of the set of nucleic acid probes disclosed herein.
[0261] In some cases, the pull-down tag can comprise an affinity target moiety that can be specifically recognized and bound by an affinity binding moiety. The affinity binding moiety specifically can bind the affinity target moiety to form an affinity pair. In some cases, by utilizing beads or substrates comprising the affinity binding moiety, the affinity target moiety can be used to pull-down and enrich for any nucleic acid probe that is hybridized to a target cell-free nucleic acid molecule. Alternatively, the pull-down tag can comprise the affinity binding moiety, while the beads/substrates can comprise the affinity target moiety. Non-limiting examples of the affinity pair can include biotin/avidin, antibody/antigen, biotin/streptavidin, metal/chelator, ligand/receptor, nucleic acid and binding protein, and complementary nucleic acids. In an example, the pull-down tag can comprise biotin.
[0262] In some embodiments of any one of the compositions disclosed herein, a length of a target cell-free nucleic acid (e.g., cfDNA) molecule that is to be pulled down by any subject nucleic acid probe can be about 100 nucleotides to about 200 nucleotides. The length of the target cell-free nucleic acid molecule can be at least about 100 nucleotides. The length of the target cell-free nucleic acid molecule can be at most about 200 nucleotides. The length of the target cell-free nucleic acid molecule can be about 100 nucleotides to about 110 nucleotides, about 100 nucleotides to about 120 nucleotides, about 100 nucleotides to about 130 nucleotides, about 100 nucleotides to about 140 nucleotides, about 100 nucleotides to about 150 nucleotides, about 100 nucleotides to about 160 nucleotides, about 100 nucleotides to about 170 nucleotides, about 100 nucleotides to about 180 nucleotides, about 100 nucleotides to about 190 nucleotides, about 100 nucleotides to about 200 nucleotides, about 110 nucleotides to about 120 nucleotides, about 110 nucleotides to about 130 nucleotides, about 110 nucleotides to about 140 nucleotides, about 110 nucleotides to about 150 nucleotides, about 110 nucleotides to about 160 nucleotides, about 110 nucleotides to about 170 nucleotides, about 110 nucleotides to about 180 nucleotides, about 110 nucleotides to about 190 nucleotides, about 110 nucleotides to about 200 nucleotides, about 120 nucleotides to about 130 nucleotides, about 120 nucleotides to about 140 nucleotides, about 120 nucleotides to about 150 nucleotides, about 120 nucleotides to about 160 nucleotides, about 120 nucleotides to about 170 nucleotides, about 120 nucleotides to about 180 nucleotides, about 120 nucleotides to about 190 nucleotides, about 120 nucleotides to about 200 nucleotides, about 130 nucleotides to about 140 nucleotides, about 130 nucleotides to about 150 nucleotides, about 130 nucleotides to about 160 nucleotides, about 130 nucleotides to about 170 nucleotides, about 130 nucleotides to about 180 nucleotides, about 130 nucleotides to about 190 nucleotides, about 130 nucleotides to about 200 nucleotides, about 140 nucleotides to about 150 nucleotides, about 140 nucleotides to about 160 nucleotides, about 140 nucleotides to about 170 nucleotides, about 140 nucleotides to about 180 nucleotides, about 140 nucleotides to about 190 nucleotides, about 140 nucleotides to about 200 nucleotides, about 150 nucleotides to about 160 nucleotides, about 150 nucleotides to about 170 nucleotides, about 150 nucleotides to about 180 nucleotides, about 150 nucleotides to about 190 nucleotides, about 150 nucleotides to about 200 nucleotides, about 160 nucleotides to about 170 nucleotides, about 160 nucleotides to about 180 nucleotides, about 160 nucleotides to about 190 nucleotides, about 160 nucleotides to about 200 nucleotides, about 170 nucleotides to about 180 nucleotides, about 170 nucleotides to about 190 nucleotides, about 170 nucleotides to about 200 nucleotides, about 180 nucleotides to about 190 nucleotides, about 180 nucleotides to about 200 nucleotides, or about 190 nucleotides to about 200 nucleotides. The length of the target cell-free nucleic acid molecule can be about 100 nucleotides, about 110 nucleotides, about 120 nucleotides, about 130 nucleotides, about 140 nucleotides, about 150 nucleotides, about 160 nucleotides, about 170 nucleotides, about 180 nucleotides, about 190 nucleotides, or about 200 nucleotides. In some examples, the length of the target cell-free nucleic acid molecule can range between about 100 nucleotides and about 180 nucleotides.
[0263] In some embodiments of any one of the compositions disclosed herein, the genomic regions can be associated with a condition. The genomic regions can be determined to exhibit aberrant somatic hypermutation when a subject has the condition. For example, the condition can comprise B-cell lymphoma or a sub-type thereof, such as diffuse large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma, and B-cell chronic lymphocytic leukemia. Additional details of the condition are provided below.
[0264] In some embodiments of any one of the compositions disclosed herein, the composition further comprises the plurality of cell-free nucleic acid (e.g., cfDNA) molecules obtained or derived from the subject.
E. Diagnostic or Therapeutic Applications
[0265] A number of embodiments are directed towards performing a diagnostic scan on cell-free nucleic acids of an individual and then based on results of the scan indicating cancer, performing further clinical procedures and/or treating the individual. In accordance with various embodiments, numerous types of neoplasms can be detected.
[0266] In some embodiments of any one of the methods disclosed herein, the method can comprise determining that the subject has the condition or determining a degree or status of the condition of the subject, based on the one or more cell-free nucleic acid molecules comprising the plurality of phased variants. In some cases, the method can further comprise determining that the one or more cell-free nucleic acid molecules (each identified to comprise a plurality of phased variants) are derived from a sample associated with the condition (e.g., cancer), based on a statistical model analysis (i.e., molecular analysis). For example, the method can comprise using one or more algorithms (e.g., Monte Carlos simulation) to determine a first probability of a cell-free nucleic acid identified to have a plurality of phased variants being associated with or originated from a first condition (e.g., 80%) and a second probability of the same cell-free nucleic acid being associated with or originated from a second condition (or from a healthy cell) (e.g., 20%). In some cases, the method can comprise determining a likelihood or probability that the subject has one or more conditions based on analysis of the one or more cell-free nucleic acid molecules each identified to comprise a plurality of phased variants (i.e., macro- or global analysis). For example, the method can comprise using one or more algorithms (e.g., comprising one or more mathematical models as disclosed herein, such as binomial sampling) to analyze a plurality of cell-free nucleic acid molecules each identified to comprise a plurality of phased variants, thereby to determine a first probability of the subject having a first condition (e.g., 80%) and a second probability of the subject having a second condition (or being healthy) (e.g., 20%).
[0267] The statistical model analysis as disclosed herein can be an approximate solution by a numerical approximation such as a binomial model, a ternary model, a Monte Carlo simulation, or a finite difference method. In an example, the statistical model analysis as used herein can be a Monte Carlo statistical analysis. In another example, the statistical model analysis as used herein can be a binomial or ternary model analysis.
[0268] In some embodiments of any one of the methods disclosed herein, the method can comprise monitoring a progress of the condition of the subject based on the one or more cell-free nucleic acid molecules identified, such that each of the identified cell-free nucleic acid molecule comprises a plurality of phased variants. In some cases, the progress of the condition can be worsening of the condition, as described in the present disclosure (e.g., developing from stage I cancer to stage III cancer). In some cases, the progress of the condition can be at least a partial remission of the condition, as described in the present disclosure (e.g., downstaging from stage IV cancer to stage II cancer). Alternatively, in some cases, the progress of the condition can remain substantially the same between two different time points, as described in the present disclosure. In an example, the method can comprise determining likelihoods or probabilities of different progresses of the condition of the subject. For example, the method can comprise using one or more algorithms (e.g., comprising one or more mathematical models as disclosed herein, such as binomial sampling) to determine a first probability of the subject's condition being worse than before (e.g., 20%), a second probability of at least partial remission of the condition (e.g., 70%), and a third probability that the subject's condition is the same as before (e.g., 10%).
[0269] In some embodiments of any one of the methods disclosed herein, the method can comprise comprising performing a different procedure (e.g., follow-up diagnostic procedures) to confirm the condition of the subject, which condition has been determined and/or monitored progress thereof, as provided in the present disclosure. Non-limiting examples of a different procedure can include physical exam, medical imaging, genetic test, mammography, endoscopy, stool sampling, pap test, alpha-fetoprotein blood test, CA-125 test, prostate-specific antigen (PSA) test, biopsy extraction, bone marrow aspiration, and tumor marker detection tests. Medical imaging includes (but is not limited to) X-ray, magnetic resonance imaging (MM), computed tomography (CT), ultrasound, and positron emission tomography (PET). Endoscopy includes (but is not limited to) bronchoscopy, colonoscopy, colposcopy, cystoscopy, esophagoscopy, gastroscopy, laparoscopy, neuroendoscopy, proctoscopy, and sigmoidoscopy.
[0270] In some embodiments of any one of the methods disclosed herein, the method can comprise determining a treatment for the condition of the subject based on the one or more cell-free nucleic acid molecules identified, each identified cell-free nucleic acid molecule comprising a plurality of phased variants. In some cases, the treatment can be determined based on (i) the determined condition of the subject and/or (ii) the determined progress of the condition of the subject. In addition, the treatment can be determined based on one or more additional factors of the following: sex, nationality, age, ethnicity, and other physical conditions of the subject. In some examples, the treatment can be determined based on one or more features of the plurality of phased variants of the identified cell-free nucleic acid molecules, as disclosed herein.
[0271] In some embodiments of any one of the methods disclosed herein, the subject may not have been subjected to any treatment for the condition, e.g., the subject may not have been diagnosed with the condition (e.g., a lymphoma). In some embodiments of any one of the methods disclosed herein, the subject may been subjected to a treatment for the condition prior to any subject method of the present disclosure. In some cases, the methods disclosed herein can be performed to monitor progress of the condition that the subject has been diagnosed with, thereby to (i) determine efficacy of the previous treatment and (ii) assess whether to keep the treatment, modify the treatment, or cancel the treatment in favor of a new treatment.
[0272] In some embodiments of any one of the methods disclosed herein, non-limiting examples of a treatment (e.g., prior treatment, new treatment to be determined based on the methods of the present disclosure, etc.) can include chemotherapy, radiotherapy, chemoradiotherapy, immunotherapy, adoptive cell therapy (e.g., chimeric antigen receptor (CAR) T cell therapy, CAR NK cell therapy, modified T cell receptor (TCR) T cell therapy, etc.) hormone therapy, targeted drug therapy, surgery, transplant, transfusion, or medical surveillance.
[0273] In some embodiments of any one of the methods disclosed herein, the condition can comprise a disease. In some embodiments of any one of the methods disclosed herein, the condition can comprise neoplasm, cancer, or tumor. In an example, the condition can comprise a solid tumor. In another example, the condition can comprise a lymphoma, such as B-cell lymphoma (BCL). Non-limiting examples of BCL can include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt lymphoma (BL), B-cell chronic lymphocytic leukemia (CLL), Marginal zone B-cell lymphoma (MZL), and Mantle cell lymphoma (MCL).
[0274] As disclosed herein, a treatment for a condition of subject can comprise administering the subject with one or more therapeutic agents. The one or more therapeutic drugs can be administered to the subject by one or more of the following: orally, intraperitoneally, intravenously, intraarterially, transdermally, intramuscularly, liposomally, via local delivery by catheter or stent, subcutaneously, intraadiposally, and intrathecally.
[0275] Non-limiting examples of the therapeutic drugs can include cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, for example, anti-CD20 antibodies, anti-PD1 antibodies (e.g., Pembrolizumab) platelet derived growth factor inhibitors (e.g., GLEEVEC.TM. (imatinib mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets PDGFR-.beta., BlyS, APRIL, BCMA receptor(s), TRAIL/Apo2, other bioactive and organic chemical agents, and the like.
[0276] Non-limiting examples of a cytotoxic agent can include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu), chemotherapeutic agents, e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin.
[0277] Non-limiting examples of a chemotherapeutic agent can include alkylating agents such as thiotepa and CYTOXAN.RTM. cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics; dynemicin, including dynemicin A; an espiramicina; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; eflornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, roridin A and anguidine); urethan; vindesine (ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, for example taxanes including TAXOL.RTM. paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.TM. Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM. docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.); oxaliplatin; leucovovin; vinorelbine (NAVELBINE.RTM.); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA.RTM.); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN.TM.) combined with 5-FU and leucovorin.
[0278] Examples of a chemotherapeutic agent can also include "anti-hormonal agents" or "endocrine therapeutics" that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX.RTM. tamoxifen), EVISTA.RTM. raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON.RTM. toremifene; anti-progesterones; estrogen receptor down-regulators (ERDs); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON.RTM. and ELIGARD) leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE.RTM. megestrol acetate, AROMASIN.RTM. exemestane, formestanie, fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole. In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.), DIDROCAL.RTM. etidronate, NE-58095, ZOMETA.RTM. zoledronic acid/zoledronate, FOSAMAX.RTM. alendronate, AREDIA.RTM. pamidronate, SKELID.RTM. tiludronate, or ACTONEL.RTM. risedronate; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGFR); vaccines such as THERATOPE.RTM. vaccine and gene therapy vaccines, for example, ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM. rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[0279] Examples of a chemotherapeutic agent can also include antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN.RTM., Genentech); cetuximab (ERBITUX.RTM., Imclone); panitumumab (VECTIBIX.RTM., Amgen), rituximab (RITUXAN.RTM., Genentech/Biogen Idec), pertuzumab (OMNITARG.RTM., 2C4, Genentech), trastuzumab (HERCEPTIN.RTM., Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG.RTM., Wyeth). Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, feMzumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and the anti-interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories) which is a recombinant exclusively human-sequence, full-length IgG1.lamda.. antibody genetically modified to recognize interleukin-12 p40 protein.
[0280] Examples of a chemotherapeutic agent can also include "tyrosine kinase inhibitors" such as an EGFR-targeting agent (e.g., small molecule, antibody, etc.); small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib mesylate (GLEEVEC.RTM., available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT.RTM., available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035,4-(3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to HER-encoding nucleic acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinib mesylate (GLEEVEC.RTM.); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone); and rapamycin (sirolimus, RAPAMUNE.RTM.).
[0281] Examples of a chemotherapeutic agent can also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.
[0282] Examples of a chemotherapeutic agent can also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene acetate: immune selective anti-inflammatory peptides (ImSAIDs) such as phenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG) (IMULAN BioTherapeutics, LLC); anti-rheumatic drugs such as azathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumor necrosis factor alpha (TNF.alpha.) blockers such as etanercept (ENBREL.RTM.), infliximab (REMICADE.RTM.), adalimumab (HUMIRA.RTM.), certolizumab pegol (CIMZIA.RTM.), golimumab (SIMPONI.RTM.), Interleukin 1 (IL-1) blockers such as anakinra (KINERET.RTM.), T-cell costimulation blockers such as abatacept (ORENCIA.RTM.), Interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA.RTM.); Interleukin 13 (IL-13) blockers such as lebrikizumab; Interferon alpha (IFN) blockers such as rontalizumab; beta 7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as Anti-M1 prime; Secreted homotrimeric LTa3 and membrane bound heterotrimer LTa/.beta.2 blockers such as Anti-lymphotoxin alpha (LTa); miscellaneous investigational agents such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or famesyl transferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin); podophyllotoxin; tegafur (UFTORAL.RTM.); bexarotene (TARGRETIN.RTM.); bisphosphonates such as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.), etidronate (DIDROCAL.RTM.), NE-58095, zoledronic acid/zoledronate (ZOMETA.RTM.), alendronate (FOSAMAX.RTM.), pamidronate (AREDIA.RTM.), tiludronate (SKELID.RTM.), or risedronate (ACTONEL.RTM.); and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE.RTM. vaccine; perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib), proteosome inhibitor (e.g., PS341); CCI-779; tipifamib (R11577); orafenib, ABT510; Bc1-2 inhibitor such as oblimersen sodium (GENASENSE.RTM.); pixantrone; famesyltransferase inhibitors such as lonafamib (SCH 6636, SARASAR.TM.); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.
[0283] In accordance with many embodiments, once a diagnosis of cancer is indicated, a number of treatments can be performed, including (but not limited to) surgery, resection, chemotherapy, radiation therapy, immunotherapy, targeted therapy, hormone therapy, stem cell transplant, and blood transfusion. In some embodiments, an anti-cancer and/or chemotherapeutic agent is administered, including (but not limited to) alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, endocrine/hormonal agents, bisphophonate therapy agents and targeted biological therapy agents. Medications include (but are not limited to) cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolomide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserelin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, zoledronate, tykerb, daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin mitoxantrone, bevacizumab, cetuximab, ipilimumab, ado-trastuzumab emtansine, afatinib, aldesleukin, alectinib, alemtuzumab, atezolizumab, avelumab, axtinib, belimumab, belinostat, bevacizumab, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib, cabozantinib, canakinumab, carfilzomib, certinib, cetuximab, cobimetinib, crizotinib, dabrafenib, daratumumab, dasatinib, denosumab, dinutuximab, durvalumab, elotuzumab, enasidenib, erlotinib, everolimus, gefitinib, ibritumomab tiuxetan, ibrutinib, idelalisib, imatinib, ipilimumab, ixazomib, lapatinib, lenvatinib, midostaurin, necitumumab, neratinib, nilotinib, niraparib, nivolumab, obinutuzumab, ofatumumab, olaparib, olaratumab, osimertinib, palbociclib, panitumumab, panobinostat, pembrolizumab, pertuzumab, ponatinib, ramucirumab, regorafenib, ribociclib, rituximab, romidepsin, rucaparib, ruxolitinib, siltuximab, sipuleucel-T, sonidegib, sorafenib, temsi rolimus, tocilizumab, tofacitinib, tositumomab, trametinib, trastuzumab, vandetanib, vemurafenib, venetoclax, vismodegib, vorinostat, and ziv-aflibercept. In accordance with various embodiments, an individual may be treated, by a single medication or a combination of medications described herein. A common treatment combination is cyclophosphamide, methotrexate, and 5-fluorouracil (CMF).
[0284] In some embodiments of any one of the methods disclosed herein, any of the cell-free nucleic acid molecules (e.g., cfDNA, cfRNA) can be derived from a cell. For example, a cell sample or tissue sample may be obtained from a subject and processed to remove all cells from the sample, thereby producing cell-free nucleic acid molecules derived from the sample.
[0285] In some embodiments of any one of the methods disclosed herein, a reference genomic sequence can be derived from a cell of an individual. The individual can be a healthy control or the subject who is being subjected to the methods disclosed herein for determining or monitoring progress of a condition.
[0286] A cell can be a healthy cell. Alternatively, a cell can be a diseased cell. A diseased cell can have altered metabolic, gene expression, and/or morphologic features. A diseased cell can be a cancer cell, a diabetic cell, and an apoptotic cell. A diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood disorders, cancers, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, cardiac disease, and the like.
[0287] A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. A cell can be a prokaryotic cell or derived from a prokaryotic cell. A cell can be a bacterial cell or can be derived from a bacterial cell. A cell can be an archaeal cell or derived from an archaeal cell. A cell can be a eukaryotic cell or derived from a eukaryotic cell. A cell can be a pluripotent stem cell. A cell can be a plant cell or derived from a plant cell. A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a microbe cell or derived from a microbe cell. A cell can be a fungi cell or derived from a fungi cell. A cell can be from a specific organ or tissue.
[0288] Non-limiting examples of a cell(s) can include lymphoid cells, such as B cell, T cell (Cytotoxic T cell, Natural Killer T cell, Regulatory T cell, T helper cell), Natural killer cell, cytokine induced killer (CIK) cells; myeloid cells, such as granulocytes (Basophil granulocyte, Eosinophil granulocyte, Neutrophil granulocyte/Hypersegmented neutrophil), Monocyte/Macrophage, Red blood cell (Reticulocyte), Mast cell, Thrombocyte/Megakaryocyte, Dendritic cell; cells from the endocrine system, including thyroid (Thyroid epithelial cell, Parafollicular cell), parathyroid (Parathyroid chief cell, Oxyphil cell), adrenal (Chromaffin cell), pineal (Pinealocyte) cells; cells of the nervous system, including glial cells (Astrocyte, Microglia), Magnocellular neurosecretory cell, Stellate cell, Boettcher cell, and pituitary (Gonadotrope, Corticotrope, Thyrotrope, Somatotrope, Lactotroph); cells of the Respiratory system, including Pneumocyte (Type I pneumocyte, Type II pneumocyte), Clara cell, Goblet cell, Dust cell; cells of the circulatory system, including Myocardiocyte, Pericyte; cells of the digestive system, including stomach (Gastric chief cell, Parietal cell), Goblet cell, Paneth cell, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromaffm cell, APUD cell, liver (Hepatocyte, Kupffer cell), Cartilage/bone/muscle; bone cells, including Osteoblast, Osteocyte, Osteoclast, teeth (Cementoblast, Ameloblast); cartilage cells, including Chondroblast, Chondrocyte; skin cells, including Trichocyte, Keratinocyte, Melanocyte (Nevus cell); muscle cells, including Myocyte; urinary system cells, including Podocyte, Juxtaglomerular cell, Intraglomerular mesangial cell/Extraglomerular mesangial cell, Kidney proximal tubule brush border cell, Macula densa cell; reproductive system cells, including Spermatozoon, Sertoli cell, Leydig cell, Ovum; and other cells, including Adipocyte, Fibroblast, Tendon cell, Epidermal keratinocyte (differentiating epidermal cell), Epidermal basal cell (stem cell), Keratinocyte of fingernails and toenails, Nail bed basal cell (stem cell), Medullary hair shaft cell, Cortical hair shaft cell, Cuticular hair shaft cell, Cuticular hair root sheath cell, Hair root sheath cell of Huxley's layer, Hair root sheath cell of Henle's layer, External hair root sheath cell, Hair matrix cell (stem cell), Wet stratified barrier epithelial cells, Surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, Urinary epithelium cell (lining urinary bladder and urinary ducts), Exocrine secretory epithelial cells, Salivary gland mucous cell (polysaccharide-rich secretion), Salivary gland serous cell (glycoprotein enzyme-rich secretion), Von Ebner's gland cell in tongue (washes taste buds), Mammary gland cell (milk secretion), Lacrimal gland cell (tear secretion), Ceruminous gland cell in ear (wax secretion), Eccrine sweat gland dark cell (glycoprotein secretion), Eccrine sweat gland clear cell (small molecule secretion). Apocrine sweat gland cell (odoriferous secretion, sex-hormone sensitive), Gland of Moll cell in eyelid (specialized sweat gland), Sebaceous gland cell (lipid-rich sebum secretion), Bowman's gland cell in nose (washes olfactory epithelium), Brunner's gland cell in duodenum (enzymes and alkaline mucus), Seminal vesicle cell (secretes seminal fluid components, including fructose for swimming sperm), Prostate gland cell (secretes seminal fluid components), Bulbourethral gland cell (mucus secretion), Bartholin's gland cell (vaginal lubricant secretion), Gland of Littre cell (mucus secretion), Uterus endometrium cell (carbohydrate secretion), Isolated goblet cell of respiratory and digestive tracts (mucus secretion), Stomach lining mucous cell (mucus secretion), Gastric gland zymogenic cell (pepsinogen secretion), Gastric gland oxyntic cell (hydrochloric acid secretion), Pancreatic acinar cell (bicarbonate and digestive enzyme secretion), Paneth cell of small intestine (lysozyme secretion), Type II pneumocyte of lung (surfactant secretion), Clara cell of lung, Hormone secreting cells, Anterior pituitary cells, Somatotropes, Lactotropes, Thyrotropes, Gonadotropes, Corticotropes, Intermediate pituitary cell, Magnocellular neurosecretory cells, Gut and respiratory tract cells, Thyroid gland cells, thyroid epithelial cell, parafollicular cell, Parathyroid gland cells, Parathyroid chief cell, Oxyphil cell, Adrenal gland cells, chromaffin cells, Ley dig cell of testes, Theca interna cell of ovarian follicle, Corpus luteum cell of ruptured ovarian follicle, Granulosa lutein cells, Theca lutein cells, Juxtaglomerular cell (renin secretion), Macula densa cell of kidney, Metabolism and storage cells, Barrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract), Kidney, Type I pneumocyte (lining air space of lung), Pancreatic duct cell (centroacinar cell), Nonstriated duct cell (of sweat gland, salivary gland, mammary gland, etc.), Duct cell (of seminal vesicle, prostate gland, etc.), Epithelial cells lining closed internal body cavities, Ciliated cells with propulsive function, Extracellular matrix secretion cells, Contractile cells; Skeletal muscle cells, stem cell, Heart muscle cells, Blood and immune system cells, Erythrocyte (red blood cell), Megakaryocyte (platelet precursor), Monocyte, Connective tissue macrophage (various types), Epidermal Langerhans cell, Osteoclast (in bone), Dendritic cell (in lymphoid tissues), Microglial cell (in central nervous system), Neutrophil granulocyte, Eosinophil granulocyte, Basophil granulocyte, Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, Natural Killer T cell, B cell, Natural killer cell, Reticulocyte, Stem cells and committed progenitors for the blood and immune system (various types), Pluripotent stem cells, Totipotent stem cells, Induced pluripotent stem cells, adult stem cells, Sensory transducer cells, Autonomic neuron cells, Sense organ and peripheral neuron supporting cells, Central nervous system neurons and glial cells, Lens cells, Pigment cells, Melanocyte, Retinal pigmented epithelial cell, Germ cells, Oogonium/Oocyte, Spermatid, Spermatocyte, Spermatogonium cell (stem cell for spermatocyte), Spermatozoon, Nurse cells, Ovarian follicle cell, Sertoli cell (in testis), Thymus epithelial cell, Interstitial cells, and Interstitial kidney cells.
[0289] In some embodiments of any one of the methods disclosed herein, the condition can be a cancer or tumor. Non-limiting examples of such condition can include Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor.
[0290] In accordance with various embodiments, numerous types of neoplasms can be detected, including (but not limited to) acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), anal cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, breast cancer, Burkitt's lymphoma, cervical cancer, chronic lymphocytic leukemia (CLL) chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, diffuse large B-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, fallopian tube cancer, follicular lymphoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, hairy cell leukemia, hepatocellular cancer, Hodgkin lymphoma, hypopharyngeal cancer, Kaposi sarcoma, Kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell cancer, mesothelioma, mouth cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors, pharyngeal cancer, pituitary tumor, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, skin cancer, small cell lung cancer, small intestine cancer, squamous neck cancer, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, uterine cancer, vaginal cancer, and vascular tumors.
[0291] Many embodiments are directed to diagnostic or companion diagnostic scans performed during cancer treatment of an individual. When performing diagnostic scans during treatment, the ability of agent to treat the cancer growth can be monitored. Most anti-cancer therapeutic agents result in death and necrosis of neoplastic cells, which should release higher amounts nucleic acids from these cells into the samples being tested. Accordingly, the level of circulating-tumor nucleic acids can be monitored over time, as the level should increase during early treatments and begin to decrease as the number of cancerous cells are decreased. In some embodiments, treatments are adjusted based on the treatment effect on cancer cells. For instance, if the treatment isn't cytotoxic to neoplastic cells, a dosage amount may be increased or an agent with higher cytotoxicity can be administered. In the alternative, if cytotoxicity of cancer cells is good but unwanted side effects are high, a dosage amount can be decreased or an agent with less side effects can be administered.
[0292] Various embodiments are also directed to diagnostic scans performed after treatment of an individual to detect residual disease and/or recurrence of cancer. If a diagnostic scan indicates residual and/or recurrence of cancer, further diagnostic tests and/or treatments may be performed as described herein. If the cancer and/or individual is susceptible to recurrence, diagnostic scans can be performed frequently to monitor any potential relapse.
F. Computer Systems
[0293] In one aspect, the present disclosure provides a computer program product comprising a non-transitory computer-readable medium having computer-executable code encoded therein, the computer-executable code adapted to be executed to implement any one of the preceding methods.
[0294] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. The system can, in some cases, include components such as a processor, an input module for inputting sequencing data or data derived therefrom, a computer-readable medium containing instructions that, when executed by the processor, perform an algorithm on the input regarding one or more cell-free nucleic acids molecules, and an output module providing one or more indicia associated with the condition.
[0295] FIG. 27 shows a computer system 2701 that is programmed or otherwise configured to implement partial or all of the methods disclosed herein. The computer system 2701 can regulate various aspects of the present disclosure, such as, for example, (i) identify, from sequencing data derived from a plurality of cell-free nucleic acid molecules, one or more cell-free nucleic acid molecules comprising the plurality of phased variants, (ii) analyze any of the identified cell-free nucleic acid molecules, (iii) determine a condition of the subject based at least in part on the identified cell-free nucleic acid molecules, (iv) monitor a progress of the condition of the subject based at least in part on the identified cell-free nucleic acid molecules, (v) identify the subject based at least in part on the identified cell-free nucleic acid molecules, or (vi) determine an appropriate treatment of the condition of the subject based at least in part on the identified cell-free nucleic acid molecules. The computer system 2701 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.
[0296] The computer system 2701 includes a central processing unit (CPU, also "processor" and "computer processor" herein) 2705, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 2701 also includes memory or memory location 2710 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 2715 (e.g., hard disk), communication interface 2720 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 2725, such as cache, other memory, data storage and/or electronic display adapters. The memory 2710, storage unit 2715, interface 2720 and peripheral devices 2725 are in communication with the CPU 2705 through a communication bus (solid lines), such as a motherboard. The storage unit 2715 can be a data storage unit (or data repository) for storing data. The computer system 2701 can be operatively coupled to a computer network ("network") 2730 with the aid of the communication interface 2720. The network 2730 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 2730 in some cases is a telecommunication and/or data network. The network 2730 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 2730, in some cases with the aid of the computer system 2701, can implement a peer-to-peer network, which may enable devices coupled to the computer system 2701 to behave as a client or a server.
[0297] The CPU 2705 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 2710. The instructions can be directed to the CPU 2705, which can subsequently program or otherwise configure the CPU 2705 to implement methods of the present disclosure. Examples of operations performed by the CPU 2705 can include fetch, decode, execute, and writeback.
[0298] The CPU 2705 can be part of a circuit, such as an integrated circuit. One or more other components of the system 2701 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).
[0299] The storage unit 2715 can store files, such as drivers, libraries and saved programs. The storage unit 2715 can store user data, e.g., user preferences and user programs. The computer system 2701 in some cases can include one or more additional data storage units that are external to the computer system 2701, such as located on a remote server that is in communication with the computer system 2701 through an intranet or the Internet.
[0300] The computer system 2701 can communicate with one or more remote computer systems through the network 2730. For instance, the computer system 2701 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple.RTM. iPad, Samsung.RTM. Galaxy Tab), telephones, Smart phones (e.g., Apple.RTM. iPhone, Android-enabled device, Blackberry.RTM.), or personal digital assistants. The user can access the computer system 2701 via the network 2730.
[0301] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 2701, such as, for example, on the memory 2710 or electronic storage unit 2715. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 2705. In some cases, the code can be retrieved from the storage unit 2715 and stored on the memory 2710 for ready access by the processor 2705. In some situations, the electronic storage unit 2715 can be precluded, and machine-executable instructions are stored on memory 2710.
[0302] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
[0303] Aspects of the systems and methods provided herein, such as the computer system 2701, can be embodied in programming. Various aspects of the technology may be thought of as "products" or "articles of manufacture" typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. "Storage" type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.
[0304] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
[0305] The computer system 2701 can include or be in communication with an electronic display 2735 that comprises a user interface (UI) 2740 for providing, for example, (i) analysis of any of the identified cell-free nucleic acid molecules, (ii) a determined condition of the subject based at least in part on the identified cell-free nucleic acid molecules, (iii) a determined progress of the condition of the subject based at least in part on the identified cell-free nucleic acid molecules, (iv) the identified subject suspected of having the condition based at least in part on the identified cell-free nucleic acid molecules, or (v) a determined treatment of the condition of the subject based at least in part on the identified cell-free nucleic acid molecules. Examples of UI's include, without limitation, a graphical user interface (GUI) and web-based user interface.
[0306] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 2705. The algorithm can, for example, (i) identify, from sequencing data derived from a plurality of cell-free nucleic acid molecules, one or more cell-free nucleic acid molecules comprising the plurality of phased variants, (ii) analyze any of the identified cell-free nucleic acid molecules, (iii) determine a condition of the subject based at least in part on the identified cell-free nucleic acid molecules, (iv) monitor a progress of the condition of the subject based at least in part on the identified cell-free nucleic acid molecules, (v) identify the subject based at least in part on the identified cell-free nucleic acid molecules, or (vi) determine an appropriate treatment of the condition of the subject based at least in part on the identified cell-free nucleic acid molecules.
EXAMPLES
[0307] The following illustrative examples are representative of embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way.
Example 1: Genomic Distribution of Phased Variants
[0308] Described is an alternative to duplex sequencing for reducing the background error rate that involves detection of `phased variants` (PVs), where two or more mutations occur in cis (i.e., on the same strand of DNA FIG. 1A and FIG. 1E). Similar to duplex sequencing, this method provides lower error profiles due to the concordant detection of two separate non-reference events in individual molecules. However, unlike duplex sequencing, both events occur on the same sequencing read-pair, thereby increasing the efficiency of genome recovery. Phased mutations are present in diverse cancer types, but occur in stereotyped portions of the genome in B-cell malignancies, likely due to on-target and aberrant somatic hypermutation (aSHM) driven by activation-induced deaminase (AID). The most common regions of aSHM in B-cell non-Hodgkin lymphomas (NHL) are identified. Described herein is phased variant Enrichment and Detection Sequencing (PhasED-Seq), a novel method to detect ctDNA through phased variants to tumor fractions on the order of parts per million. Described herein is demonstration that PhasED-Seq can meaningfully improve detection of ctDNA in clinical samples both during therapy and prior to disease relapse.
[0309] To identify malignancies where PVs may potentially improve disease detection, the frequency of PVs across cancer types were assessed. Publicly available whole-genome sequencing data was analyzed to identify sets of variants occurring at a distance of <170 bp apart, which represents the typical length of a single cfDNA fragment consisting of a single core nucleosome and associated linker. The frequency of these `putative phased variants," (Example 10) controlling for the total number of SNVs, from 2538 tumors across 24 cancer histologies including solid tumors and hematological malignancies (FIG. 1B, FIG. 5, and Table 1) was identified and summarized. PVs were most significantly enriched in two B-cell lymphomas (DLBCL and follicular lymphoma, FL, P<0.05 vs all other histologies), a group of diseases with hypermutation driven by AID/AICDA.
Example 2: Mutational Mechanisms Underlying PVs
[0310] To investigate the origin of PVs, the single base substitution (SBS) mutational signatures contributing to SNVs occurring within 170 bp of another SNV, and SNVs occurring in isolation (e.g., not having another SNV within 170 bp) (Example 10) were compared. As expected, PVs were highly enriched in several mutational signatures associated with clustered mutations. Signatures of clustered mutations associated with activity of AID (SBS84 and SBS85) were significantly enriched in PVs from B-cell lymphomas and CLL, while signatures associated with activity of APOBEC3B (SBS2 and SBS13)--another mechanism of kataegis hypermutation--were significantly enriched in PVs from multiple solid cancer histologies, including ovarian, pancreatic, prostate, and breast adenocarcinomas (FIG. 1C and FIGS. 6A-6WW). Signatures of clustered mutations associated with activity of AID (SBS84 and SBS85) were enriched in PVs found in lymphomas and CLL, while signatures associated with activity of APOBEC3B (SBS2 and SBS13) were significantly enriched in breast cancer (FIG. 1C and FIGS. 6A-6WW). PVs from multiple tumor types were also associated with SBS4, a signature associated with tobacco use. Furthermore, among PVs across multiple tumor histologies, it was observed that novel enrichments in several other signatures without clearly associated mechanisms (e.g., SB S24, SBS37, SBS38, and SB S39). In contrast, aging-associated mutational signatures such as SBS1 and SBS5 were significantly enriched in isolated SNVs.
Example 3: PVs Occur in Stereotyped Genomic Regions in Lymphoid Cancers
[0311] To assess the genomic distribution of putative PVs, these events were first binned into 1-kb regions to visualize their frequency across tumor types. It was observed that a strikingly stereotyped distribution of PVs in individual lymphoid neoplasms (e.g., DLBCL, FL, Burkitt lymphoma (BL), and chronic lymphocytic leukemia (CLL); FIG. 1D and FIG. 7). In contrast, non-lymphoid cancers generally did not exhibit substantial recurrence of clustered PVs in stereotyped regions. This lack of stereotype in the position of PVs was true even when considering melanomas and lung cancers, diseases with frequent PVs.
[0312] Notably, the majority of hypermutated regions were shared between all three lymphoma subtypes, with the highest densities seen in known targets of aSHM including BCL2, BCL6, and MYC, as well as the immunoglobulin (Ig) loci encoding the heavy and light chains IGH, IGK, and IGL (Table 2). Strikingly, certain regions within Ig loci were densely mutated in nearly all lymphoma patients as well as in patients with CLL (FIG. 1D). Among lymphoma subtypes, DLBCL tumors harbored the most 1-kb regions recurrently containing PVs (FIG. 8A), consistent with the highest number of recurrently mutated genes being observed in this tumor type. In total, 1639 unique 1-kb regions recurrently containing PVs in B-lymphoid malignancies were identified. Among these lymphoma-associated 1-kb regions, nearly one-third fell into genomic areas previously associated with physiological or aberrant SHM in B-cells. Specifically, 19% (315/1639) were located in Ig regions, while 13% (218/1639) were in portions of 68 previously identified targets of aSHM (Table 2). While most PVs fell into noncoding regions of the genome, additional recurrently affected loci not previously described as targets of aSHM, including XBP1, LPP, and AICDA, among others, were also identified.
[0313] The distribution of PVs within each lymphoid malignancy correlated with oncogenic features associated with the distinct pathophysiology of the corresponding disease. For example, cases of FL--where more than 90% of tumors harbor oncogenic BCL2 fusions--were significantly more likely to contain phased variants in BCL2 than other lymphoid malignancies (FIG. 1D and FIG. 8B). Similarly, significantly more Burkitt lymphomas (BL) harbored PVs in MYC and ID3, two driver genes strongly associated with the BL pathogenesis, than other lymphoid malignancies (FIG. 1D and FIGS. 8C-8D). DLBCL molecular subtypes associated with distinct cell-of-origin also demonstrated distinct distributions of PVs (Table 2). Specifically, while germinal center B-cell like (GCB) and activated B-cell like (ABC) DLBCLs harbored similar frequencies of PVs overall (median 798 vs 516, P=0.37), significant enrichment for PVs in the telomeric IGH class-switch regions (S.gamma.1, and S.gamma.3) in ABC-DLBCLs, consistent with previous reports41 (FIG. 8E), was found. Conversely, GCB-DLBCLs harbored more phased haplotypes in centromeric IGH class switch regions (S.alpha.2 and S.epsilon.) and in BCL2.
Example 4: Design and Validation of PhasED-Seq Panel for Lymphoma
[0314] To validate these PV-rich regions and assess their utility for disease detection from ctDNA, a sequencing panel targeting putative PVs identified within WGS from three independent cohorts of patients with DLBCL, as well as in patients with CLL (FIG. 2A and Example 10) was designed. This final Phased variant Enrichment and Detection Sequencing (PhasED-Seq) panel targeted .about.115 kb of genomic space focused on PVs, along with an additional .about.200 kb targeting genes that are recurrently mutated in B-NHLs (Table 3). While the 115 kb of space dedicated to PV-capture targets only 0.0035% of the human genome, it captures 26% of phased variants observed in mature B-cell neoplasms profiled by WGS (FIG. 9A), thus yielding a .about.7500-fold PV enrichment by PhasED-Seq over WGS.
[0315] Expected SNV and PV recovery was compared to previously reported CAPP-Seq selector designed to maximize SNVs per patient in B-cell lymphomas (FIG. 9A-C). When considering diverse B-NHLs with available WGS data, PhasED-Seq recovered 3.0.times. more SNVs (81 vs. 27) and 2.9.times. more PVs (50 vs. 17) in the median case than previous CAPP-Seq panel. This observation highlights the importance of including non-coding portions of the genome for maximal mutation recovery. To validate these yield improvements experimentally, 16 pretreatment tumor or plasma DNA samples from patients with DLBCL (Table 4) were profiled. Both CAPP-Seq and PhasED-Seq panels were applied to each specimen in parallel and then sequenced them to high unique molecular depths (FIG. 2B). Compared to the expected enrichment established from WGS, similar improvements in yield of SNVs by PhasED-Seq compared to CAPP-Seq (2.7.times.; median 304.5 vs. 114) were observed. However, when enumerating PVs observed in individual sequenced DNA fragments, an improvement in favor of PhasED-Seq beyond the expected improvement from WGS (7.7.times.; median 5554 vs 719.5 PVs/case) was found. This improvement is potentially due to either 1) the higher sequencing depth in targeted sequencing which leads to improvement in rare allele detection, or 2) enumeration of higher order PVs in targeted sequencing with PhasED-Seq or CAPP-Seq, which was not accounted for in the WGS design (i.e., >2 SNVs per fragment; FIGS. 9D-9F). Furthermore, across 1-kb windows in the panel, robust correlation between the frequency of putative PVs in WGS data and PVs from targeted sequencing by PhasED-Seq across 101 DLBCL samples (FIG. 2C) was observed, further validating the frequency and distribution of PVs in B-cell malignancies.
Example 5: Differences in Phased Variants Between Lymphoma Subtypes
[0316] Having validated the PhasED-Seq panel, the biological differences in PVs between various B-cell malignancies, including DLBCL (n=101), primary mediastinal B-cell lymphoma (PMBCL) (n=16), and classical Hodgkin lymphoma (cHL) (n=23) were examined. The number of SNVs identified per case was not significantly different between lymphoma subtypes (FIGS. 9G-9K). However, when considering mutational haplotypes, cHL had a significantly lower burden of PVs than either DLBCL or PMBCL. In addition to this quantitative disparity, differences in the genomic locations of PVs between different B-cell lymphoma subtypes were also observed (FIGS. 2D-2E and FIGS. 10-12). This included previously established biological associations in DLBCL subtypes, including more frequent PVs in BCL2 in GCB-type than ABC-type DLBCL, with the opposite association seen for PIM1. More frequent PVs in CIITA in PMBCL compared with DLBCL, a gene in which breakpoints are common in PMBCL, was also observed. Relative enrichments were also observed throughout the IGH locus, with more frequent PVs seen in S.gamma.3 and S.gamma.1 regions in ABC-DLBCL (compared with GCB-DLBCL) and interestingly, more frequent PVs in the Sc locus in cHL compared with DLBCL (FIG. 2E and FIG. 13). In total, after correcting for testing multiple hypotheses, significant relative enrichments in 25 genetic loci between ABC- and GCB-DLBCL, 24 between DLBCL and PMBCL, and 40 between DLBCL and cHL were found (FIG. 10-12).
Example 6: Recovery of Phased Variants Through PhasED-Seq
[0317] To facilitate detection of ctDNA using PVs, efficient recovery of DNA molecules is desired. Hybrid-capture sequencing is potentially sensitive to DNA mismatches, with increasing mutations decreasing hybridization efficiency. Indeed, AID hotspots can contain a 5-10% local mutation rate, with even higher rates in certain regions of IGH. To empirically assess the effect of mutation rate on capture efficiency, DNA hybridization of 150-mers with varying mutation rates in silico was simulated. As expected, predicted binding energy decreased with an increasing number of mutations (FIG. 14A). Notably, randomly distributed mutations had a greater effect on binding energy than clustered mutations. To assess the effect of this decreased binding affinity, 150-mer DNA oligonucleotides with 0 to 10% difference from the reference sequence in MYC and BCL6, two loci that are targets of aSHM were synthesized. To assess the worst-case scenario for hybridization, non-reference bases were randomly distributed rather than in clusters (Example 10). An equimolar mixture of these oligonucleotides were then captured with PhasED-Seq panel. Concordant with the in silico predictions, increased mutational rates resulted in decreased capture efficiency (FIG. 3A). Molecules with a 5% mutation rate were captured with 85% efficiency relative to fully-wildtype counterparts, while molecules with 10% mutation were captured with only 27% relative efficiency. To assess the prevalence of this degree of mutation in human tumors, the distribution of variants in panel in 140 patients with B-cell lymphomas, calculating the fraction of mutated bases in overlapping 151-bp windows (Example 10) was examined. Only 7% (10/140) of patients had any 151-bp window exceeding 10% mutation rate (FIG. 14B-C). Indeed, in the experiment with synthetic oligonucleotides, a 5% mutation rate was recovered nearly as efficiently as the wild-type sequence. In over half of all cases considered, no locus had >5% mutation rate at any window, while in all cases >90% of windows had <5% mutations. Overall, these observations indicate that the majority of phased mutations are recoverable by efficient hybrid capture, despite hybridization biases.
Example 7: Error Profile and Limit of Detection for Phased Variant Sequencing
[0318] Previous methods for highly error-suppressed sequencing applied to cfDNA have utilized either a combination of molecular and in silico methods for error suppression (e.g., integrated digital error suppression, iDES) or duplex molecular recovery. However, each of these has limitations, either for detecting events at ultra-low tumor fractions or for efficient recovery of original DNA molecules, which are important considerations for cfDNA analysis where input DNA is limited. The error profile and recovery of input genomes from plasma cfDNA samples form 12 heathy adults by PhasED-Seq were compared with both iDES-CAPP-Seq and duplex sequencing. While iDES-enhanced CAPP-Seq had a lower background error profile than barcode-deduplication alone, duplex sequencing offered the lowest background error rate for non-reference single nucleotide substitutions (FIG. 3B, 3.3.times.10.sup.-5 vs. 1.2.times.10.sup.-5, P<0.0001). However, the rate of phased errors--e.g., multiple non-reference bases occurring on the same sequencing fragment--was significantly lower than the rate of single errors in either iDES-enhanced CAPP-Seq or duplex sequencing data. This was true for the incidence of both two (2.times. or `doublet` PVs) or three (3.times. or `triplet` PVs) substitutions on the same DNA molecule (FIG. 3B, 8.0.times.10' and 3.4.times.10.sup.-8 respectively, P<0.0001). Phased errors containing C to T or T to C transition substitutions were more common than other types of PVs (FIG. 14D). Notably, the rate doublet PVs errors in cfDNA was also correlated with distance between positions, with the highest PV error-rate consisting of neighboring SNVs (e.g., DNVs) and decreasing error rate with increasing distance between constituent variants (FIG. 14E). When considering unique molecular depth, duplex sequencing recovered only 19% of all unique cfDNA fragments (FIG. 3C). In contrast, the unique depth of PVs within a genomic distance of <20 bp was nearly identical to the depth of individual positions (e.g., molecules covering individual SNVs). Similarly, PVs up to 80 bps in size had depth greater than 50% of the median unique molecular depth for a sample. Importantly, almost half (48%) of all PVs were within 80 bp of each other, demonstrating their utility for disease detection from input-limited cfDNA samples (FIG. 3D).
[0319] To quantitatively compare the performance of PhasED-Seq to alternative methods for ctDNA detection, limiting dilutions of ctDNA from 3 lymphoma patients into healthy control cfDNA were generated, resulting in expected tumor fractions between 0.1% and 0.00005% (1 part in 2,000,000; (Example 10). The expected tumor fraction was compared to the estimated tumor content in each of these dilutions using PhasED-Seq to track tumor-derived PVs, as well as to error-suppressed detection methods depending on individual SNVs (e.g. iDES-enhanced CAPP-Seq or duplex sequencing; FIG. 3E). All methods performed equally well down to tumor fractions of 0.01% (1 part in 10,000). However, below this level (e.g., 0.001%, 0.0002%, 0.0001%, and 0.00005%), both PhasED-Seq and duplex sequencing significantly outperformed iDES-enhanced CAPP-Seq (P<0.0001 for duplex, `2.times.` PhasED-Seq, and `3.times.` PhasED-Seq; FIG. 3E). In addition, when compared to duplex-sequencing, tracking either 2 or 3 variants in-phase (e.g., 2.times. and 3.times. PhasED-Seq) more accurately identified expected tumor content, with superior linearity down to 1 part in 2,000,000 (P=0.005 for duplex vs 2.times.PhasED-Seq, P=0.002 for 3.times.PhasED-Seq) (Example 10). Specificity of PVs by looking for evidence of tumor-derived SNVs or PVs in cfDNA samples from 12 unrelated healthy control subjects and the healthy control used for the limiting dilution was assessed. Here again, both 2.times.- or 3.times.-PhasED-Seq showed significantly lower background signal levels than did CAPP-Seq and duplex sequencing (FIG. 3F). This lower error rate and background from PVs improves the detection limit for ctDNA disease detection. In some instances, the method of sequencing-based cfDNA assays described herein (e.g. the method depicted in FIG. 3E and FIG. 3F) does not require molecular barcodes to achieve exquisite error-suppression and low limits of detection. Signal assessed by the method without barcode used limiting dilution series from 1:1,000 to 5:10,000,000, and `blank` controls (FIGS. 23A-23B).
[0320] This dilution series was used to assess the limit of detection for a given number of PVs (FIGS. 3G-3I). When considering a set of PVs within 150 base pair (bp) regions, the probability of detection for a given sample may be accurately modelled by binomial sampling, considering both the depth of sequencing and the number of 150 bp regions with PVs (Example 10).
Example 8: Improvements in Detection of Low-Burden Minimal Residual Disease
[0321] To test the utility of the lower LOD afforded by PhasED-Seq for detection of ultra-low burden MRD from cfDNA, Serial cell-free DNA samples were sequenced from a patient undergoing front-line therapy for DLBCL (FIG. 4A). Using CAPP-Seq, this patient had undetectable ctDNA after only one cycle of therapy, with multiple subsequent samples during and after treatment also remaining undetectable. This patient had subsequent re-emergence of detectable ctDNA >250 days after the start of therapy, with eventual clinical and radiographic disease progression 5 months later, indicating falsely negative serial measurements with CAPP-Seq. Strikingly, all four of the plasma samples that were undetectable by CAPP-Seq during and after treatment had detectable ctDNA levels by PhasED-Seq, with mean allelic fractions as low as 6 parts in 1,000,000. This increased sensitivity improved the lead-time of disease detection by ctDNA compared to radiographic surveillance from 5 with CAPP-Seq to 10 months with PhasED-Seq.
[0322] Next, the performance of PhasED-Seq ctDNA detection in a cohort of 107 patients with large B-cell lymphomas and blood samples available after 1 or 2 cycles of standard immuno-chemotherapy was next assessed. Importantly, ctDNA levels measured by PhasED-Seq were highly correlated with those measured by CAPP-Seq. In total, 443 tumor, germ-line, and cell-free DNA samples, including cfDNA prior to therapy (n=107) and after 1 or 2 cycles of treatment (n=82 and 89), were assessed. Prior to therapy, patient-specific PVs were detectable by PhaseED-Seq in 98% of samples, with 95% specificity in cfDNA from healthy controls (FIGS. 15 and 16A). Importantly, ctDNA levels measured by PhasED-Seq were highly correlated with those measured by CAPP-Seq, considering both pretreatment and post treatment samples (Spearman rho=0.91, FIG. 16B). Next, quantitative levels of ctDNA measured by PhasED-Seq and CAPP-Seq from cfDNA samples after initiation of therapy were compared. In total, 72% (78/108) of samples with detectable ctDNA by PhasED-Seq after 1 or 2 cycles were also detected by conventional CAPP-Seq (FIG. 4B). Among 108 samples detected by PhasED-Seq, disease burden was significantly lower for those with undetectable (28%) vs. detectable (72%) ctDNA levels using conventional CAPP-Seq, with a >10.times. difference in median ctDNA levels (tumor fraction 2.2.times.10.sup.-4 vs 1.2.times.10.sup.-5, P<0.001, FIG. 4B). In total, an additional 16% (13/82) of samples after 1 cycle of therapy and 19% (17/89) of samples after 2 cycles of therapy had detectable ctDNA when comparing PhasED-Seq with CAPP-Seq (FIG. 4C).
[0323] ctDNA molecular response criteria was previously described for DLBCL patients using CAPP-Seq, including Major Molecular Response (MMR), defined as a 2.5-log reduction in ctDNA after 2 cycles of therapy22. While MMR at this time-point is prognostic for outcomes, many patients have undetectable ctDNA by CAPP-Seq at this landmark (FIGS. 4D-4E). Importantly, even in patients with undetectable ctDNA by CAPP-Seq, detection of occult ultra-low ctDNA levels by PhasED-Seq was prognostic for outcomes including event-free and overall survival (FIG. 4D). Indeed, in the 89 patients with a sample available from this time-point, 58% (52/89) had undetectable ctDNA by CAPP-Seq at their interim MMR assessment, after completing 2 of 6 planned cycles of therapy. Using PhasED-Seq, 33% (17/52) of samples not detected by CAPP-Seq had evidence of ctDNA as evidenced by PVs, with levels as low as .about.3:1,000,000 (FIGS. 17A-17D)--these 17 cases additionally detected by PhasED-Seq represent potential false negative tests by CAPP-Seq. Similar results were seen at the Early Molecular Response (EMR) time-point (i.e., after 1 cycle of therapy, FIGS. 18A-18H).
[0324] While detection of ctDNA in DLBCL after 1 or 2 cycles of therapy is a known adverse prognostic marker outcomes for patients with undetectable ctDNA at these time-points are heterogeneous (FIG. 4E and FIG. 18F). Importantly, even in patients with undetectable ctDNA by CAPP-Seq after 1 or 2 cycles of therapy, detection of ultra-low ctDNA levels by PhasED-Seq was strongly prognostic for outcomes including event-free survival (FIG. 4F, FIG. 17C-D, FIG. 18C-D, and FIG. 18G). When combining detection by PhasED-Seq with previously described MMR threshold, patients could be stratified into three groups--patients not achieving MMR, patients achieving MMR but with persistent ctDNA, and patients with undetectable ctDNA (FIG. 4G). Interestingly, while patients not achieving MMR were at especially high risk for early events despite additional planned first line therapy (e.g., within the first year of treatment), patients with persistent low levels of ctDNA appeared to have a higher risk of later relapse or progression events. In contrast, patients with undetectable ctDNA after 2 cycles of therapy by PhasED-Seq had overwhelmingly favorable outcomes, with 95% being event-free and 97% overall survival at 5 years. Similar results were seen at the EMR time-point after 1 cycle of therapy (FIG. 18H).
Example 9: Exemplary Embodiments of Mutation Detection Using Next Generation Sequencing (NGS) when the Mutation is not a Single Base Substation, but Rather a Pair of Mutations
[0325] In many instances, a limitation of cfDNA tracking may be the limitation on the number of molecules available for detection. Additionally, there are multiple potential limitations on tracking tumor molecules from cell-free DNA, including not only the sequencing error profile, but also the number of molecules available for detection. The number of molecules available for detection--here termed the number of "evaluable fragments"--can be thought of as both a function of the number of recovered unique genomes (e.g., unique depth of sequencing) and the number of somatic mutations being tracked. More specifically, the number of evaluable fragments is equal to: EF=d*n.
[0326] Where d=the unique molecular depth considered and n=the number of somatic alterations tracked. For the typical cell-free DNA samples, less than 10,000 unique genomes are often recovered (d), requiring any sensitive method to track multiple alterations (n). Furthermore, as stated above, the major limitation for duplex sequencing is difficulty recovering sufficient unique molecular depth (d); thus, from a typical plasma sample with duplex depth of .about.1,500.times., even if following 100 somatic alterations, there are only 150,000 evaluable fragments. Thus, in this scenario, sensitivity is limited by the number of molecules available for detection. In contrast, other methods such as iDES-enhanced CAPP-Seq consider all molecules recovered. Here, as many as 5,000-6,000.times. unique haploid genomes can be recovered. Therefore, the number of evaluable fragments, tracking the same 100 somatic alterations, may be 500,000-600,000.times.. However, the error profile of single-stranded sequencing, even with error suppression, allows detection to levels of at best 1 part in 50,000. Therefore, methods aiming to improve on the detection limits for ctDNA must overcome both the error-profile of sequencing and the recovery of sufficient evaluable fragments to utilize said lower error-profiles.
[0327] To remedy this apparent deficiency, the method of PhasED-Seq, as described in the instant disclosure, allows for lymphoid malignancies and was applicable to other cancer histologies, (e.g., using a "personalized" approach). For a personalized approach, customized hybrid-capture oligonucleotides (or primers for PCR amplicons) were used to capture personalized somatic mutations identified from whole exome or genome sequencing. The PCAWG dataset assessed for SNVs occurring within 170 bp of each other in genomic space was re-analyzed. It was found that in 14 of 24 cancer histologies considered, the median case contained >100 possible phased variants, including in several solid tumors such as Melanoma (median 2072), lung squamous cell carcinoma (1268), lung adenocarcinoma (644.5), and colorectal adenocarcinoma (216.5).
[0328] Next, the expected limit of detection in all cases in the PCAWG dataset using either duplex sequencing or PhasED-Seq was assessed. Again, the limit of detection was defined by the expected number of evaluable fragments, and thus depends on both the number of variants tracked and the expected depth of sequencing. Utilizing the data from optimized hybrid capture conditions, a model to predict the expected deduplicated (single-stranded) and duplex (double-stranded) molecular depth with a given DNA input and number of sequencing reads was constructed. Using this, along with the number of SNVs or possible PVs from the PCAWG dataset, for each case, which method would lead to a greater number of evaluable fragments, and therefore a superior limit of detection was assessed. The results of this exercise, assuming 64 nanograms (ng) of total cfDNA input and a total of 20 million sequencing reads are shown in FIG. 19. Notably, in the majority of cancer types (18/24 histologies), PhasED-Seq had a lower limit of detection than duplex sequencing. This importantly included not only B-cell lymphomas, but common solid tumors, including lung squamous cell carcinoma and adenocarcinoma, colorectal adenocarcinoma, esophageal and gastric adenocarcinoma, and breast adenocarcinoma, among others. Indeed, taking lung cancers as a specific example, an almost 10-fold lower limit of detection was found for the median squamous cell and adenocarcinoma lung cancer case using PhasED-Seq compared to duplex sequencing (FIG. 20). Both PhasED-Seq and duplex sequencing using a personalized approach had a lower limit of detection than non-personalized approaches (e.g., iDES-enhanced CAPP-Seq).
[0329] To further confirm the applicability of phased variants and PhasED-Seq in diverse solid tumors, WGS (20-30.times.) was performed on paired tumor and normal DNA to identify PVs from five solid tumor patients predicted to have low ctDNA burden prior to treatment (lung cancer (n=5)). After identifying putative PVs in each case, a set of personalized hybrid capture oligonucleotides was subsequently designed to performed targeted resequencing of tumor and normal DNA to validate candidate PVs. Finally, plasma samples were sequenced from all 5 patients to high unique molecular depth using personalized PhasED-Seq to detect ctDNA. Considering these five lung cancer cases the PhasED-Seq approach achieved a .about.10-fold improvement in analytical sensitivity, achieving a median LOD of 0.00018% compared to 0.0019% using customized CAPP-Seq (FIG. 21).
[0330] To demonstrate the clinical significance of this improved limit of detection for ctDNA from PhasED-Seq in solid tumors, serial plasma samples from a patient with stage 3 adenocarcinoma of the lung treated with chemoradiotherapy with curative intent (LUP814) were analyzed using both CAPP-Seq and PhasED-Seq. As outlined above, both CAPP-Seq and PhasED-Seq quantified a similar level of ctDNA prior to therapy (.about.1% tumor fraction). However, 3 subsequent samples after beginning therapy had undetectable ctDNA by standard CAPP-Seq, including samples during and after chemoradiation and during adjuvant immunotherapy with Durvalumab. Despite the lack of detectable disease by CAPP-Seq, the patient had biopsy-confirmed recurrent disease after an initial radiographic response. However, when analyzing these same samples with PhasED-Seq, molecular residual disease in 3/3 (100%) of samples was detected, with mean tumor fraction as low as 0.00016% (1.6 parts per million). Furthermore, the trend in ctDNA quantitation mirrored the patient's disease course, with an initial response to chemoradiotherapy but disease progression during immunotherapy. Importantly, this patient's disease remained detectable at all timepoints, with detectable disease at the completion of chemoradiotherapy 8 months prior to the patient's biopsy-confirmed disease progression (FIG. 22).
Example 10: Methods of Phased Variant Enrichment for Enhanced Disease Detection from Cell-Free DNA
10(a): Whole-Genome Sequencing Analysis
10(a)(1): Whole-Genome Sequencing Data Putative Phased Variant Identification
[0331] Whole-genome sequencing data were obtained from two sources. Data for lymphoid malignancies (diffuse large B-cell lymphoma, DLBCL; follicular lymphoma, FL; Burkitt lymphoma, BL; chronic lymphocytic leukemia, CLL) were downloaded from the International Cancer Genome Consortium (ICGC) data portal on May 7, 2018. Data from all other histologies were part of the pan-Cancer analysis of whole genomes (PCAWG) and downloaded on Nov. 11, 2019. Only cancer histologies with at least 35 available cases were considered; details of the dataset considered are provided in Table 1. All samples had somatic mutations called from WGS using matched tumor and normal genotyping. Queries were limited to base substitutions obtained from WGS (single, double, triple, and oligo nucleotide variants; SNVs, DNVs, TNVs, and ONVs). Having thus identified the cases and variants of interest, the number of putative phased variants (PVs) in each tumor was next identified. To function as a PV on a single cell-free DNA (cfDNA) molecule, two variants, such as two single nucleotide variants (SNVs) generally must occur within a genomic distance less than the length of a typical cfDNA molecule (.about.170 bp). Therefore, putative PVs were defined as two variants occurring on the same chromosome within a genomic distance of <170 bp. DNVs, TNVs, and ONVs were considered as the set of their respective component SNVs. The number of SNVs as well as the identity of putative PVs for each case are detailed in Table 1. The raw number of SNVs and putative PVs, as well as the number of putative PVs controlling for the number of SNVs, is shown in FIG. 5A-C.
10(a)(2): Mutational Signatures of Phased Variants from WGS
[0332] To assess the mutational processes associated with phased and non-phased mutations across different cancer types/subtypes, the mutational signatures of single base substitutions (SBS) were enumerated for each WGS case described above using the R package `deconstructSigs`. The list of SNVs for each patient was first divided into two groups: 1) SNVs contained within a possible PV; that is, with an adjacent or `nearest neighbor` SNV <170 bp away, and 2) isolated SNVs (i.e., non-phased), defined as those occurring .gtoreq.170 bp in distance from the closest adjacent SNV. `DeconstructSigs` was then applied using the 49 SBS signatures described in COSMIC (excluding signatures linked to possible sequencing artefacts) to assess the contribution of each SBS signature to both candidate phased SNVs and un-phased SNVs for each patient. To compare the contribution of each SBS signature to phased and isolated SNVs, a Wilcoxon signed rank test was performed to compare the relative contribution of each SBS signature between these two categories for each cancer type (FIGS. 6A-6WW). To account for multiple hypotheses, Bonferroni's correction was applied, by considering any SBS signature that differed in contribution to phased vs. un-phased SNVs to be significant if the Wilcoxon signed rank test resulted in a P-value of <0.05/49 or 0.001. The distributions of these comparisons, along with significance testing, are depicted in FIGS. 6A-6WW. A summary of this analysis is also shown in FIG. 1C using a heat-map display, where the `heat` represents the difference between the mean contribution of the SBS signature to phased variants to the mean contribution to isolated/un-phased variants.
10(a)(3): Genomic Distribution of Phased Variants from WGS
[0333] The recurrence frequency for PVs was assessed in each cancer type across the genome within each tumor type. Specifically, the human genome (build GRCh37/hg19) was first divided into 1-kb bins (3,095,689 total bins); then, for each sample, the number of PVs (as defined above) contained in each 1-kb bin was counted. For this analysis, any PV with at least one of its constituent SNVs falling within the 1-kb bin of interest was included. The fraction of patients whose tumors harbored a PV for each cancer type within each genomic bin was then calculated. To identify 1-kb bins recurrently harboring PVs across patients, the fraction of patients containing PVs in each 1-kb bin vs. genomic coordinates (FIG. 1D and FIG. 7) was plotted; for this analysis, only bins where at least 2% of samples contained a PV in at least one cancer subtype were plotted.
10(a)(4): Identification of Recurrent 1-Kb Bins with Phased Variants
[0334] To identify 1-kb bins that recurrently contain PVs in B-lymphoid malignancies, WGS data was utilized from the following diseases: DLBCL, FL, BL, and CLL. Any 1-kb bin where >1 sample from these tumor types was considered to recurrently contain PVs from B-lymphoid malignancies. The genomic coordinates of 1-kb bins containing recurrent PVs in lymphoid malignancies are enumerated in Table 2, and are plotted in FIG. 8A.
10(b): Design of PhasED-Seq Panel for B-Lymphoid Malignancies
10(b)(1): Identification of Recurrent PVs from WGS Data at Higher Resolution
[0335] Given the prevalence of recurrent putative PVs from WGS data in B-cell malignancies, a targeted sequencing approach was designed for their hybridization-mediated capture--Phased variant Enrichment Sequencing (PhasED-Seq)--to enrich these specific PV events from tumor or cell-free DNA. In addition to the ICGC data described above, WGS data was also utilized from other sources in this design, including both B-cell NHLs as well as CLL.
[0336] Previous experience with targeted sequencing from cfDNA in NHLs was also examined. Pairs of SNVs occurring at a distance of <170 bp apart in each B-cell tumor sample were identified. Then, genomic "windows" that contained PVs was identified as follows: for each chromosome, the PVs were sorted by genomic coordinates relative to reference genome. Then, the lowest (i.e., left-most) position was identified for any PV in any patient; this defined the left-hand (5') coordinate seeding a desired window of interest, to be captured from the genome. This window was then extended by growing its 3' end to capture successive PVs until a gap of .gtoreq.340 bp was reached, with 340-bp chosen as capturing two successive chromatosomal sized fragments of .about.170-bp. When such a gap was reached, a new window was started, and this iterative process of adding neighboring PVs was repeated again until the next gap of .gtoreq.340 bp was reached. This resulted in a BED file of genomic windows containing all possible PVs from all samples considered. Finally, each window was additionally padded by 50 bp on each side, to enable efficient capture from flanking sequences in rare scenarios when repetitive or poorly mapping intervening sequences might preclude their direct targeting for enrichment.
[0337] Having identified the regions of interest containing putative PVs, each window was then into 170 bp segments (e.g., the approximate size of a chromatosomal cfDNA molecule). Then, the number of cases containing a PV was enumerated in each case. For each 170 bp region, the region in final sequencing panel design was included if one or more of the following criteria was met: 1) at least one patient contained a PV in the 170 bp region in 3 of 5 independent data-sets, 2) at least one patient contained a PV in the region in 2 of 5 independent data-sets if one dataset was prior CAPP-Seq experience, or 3) at least one patient contained a PV in the region in 2 of 5 independent data-sets, with a total of at least 3 patients containing a PV in the region. This resulted in 691 `tiles`, with each tile representing a 170 bp genomic region. These tiles, along with an additional .about.200 kb of genomic space targeting driver genes recurrently mutated in B-NHL, were combined into a unified targeted sequencing panel as previously described for both tumor and cfDNA genotyping using NimbleDesign (Roche NimbleGen). The final coordinates of this panel are provided in Table 3.
10(b)(2): Comparison of PhasED-Seq and CAPP-Seq Performance in PV Yield
[0338] To evaluate the performance of PhasED-Seq for capturing both SNVs and PVs compared to previously reported CAPP-Seq selector for B-cell lymphomas, the predicted number of both SNVs and PVs that may be recovered with each panel by limiting WGS in silico to the capture targets of each approach (FIG. 9A-C) was quantified. The predicted number of variants was then compared using the Wilcoxon signed rank test. Both CAPP-Seq and PhasED-Seq were also performed on 16 samples from patients with DLBCL. In these samples, tumor or plasma DNA, along with matched germ-line DNA, was sequenced. The resulting number of variants were again compared by the Wilcoxon signed rank text (FIG. 2B, and FIGS. 9D-9E). The sequencing depth for the samples included in this analysis are provided in Tables 4.
10(c): Identification of Phased Variants from Targeted Sequencing Data
10(c)(1): Patient Enrollment and Clinical Sample Collection
[0339] Patients with B-cell lymphomas undergoing front-line therapy were enrolled on this study from six centers across North America and Europe, including Stanford University, MD Anderson Cancer Center, the National Cancer Institute, University of Eastern Piedmont (Italy), Essen University Hospital (Germany), and CHU Dijon (France). In total, 343 cell-free DNA, 73 tumor, and 183 germ-line samples from 183 patients were included in this study. All patient samples were collected with written informed consent for research use and were approved by the corresponding Institutional Review Boards in accordance with the Declaration of Helsinki. Cell-free, tumor, and germ-line DNA were isolated as previously described. All radiographic imaging was performed as part of standard clinical care.
10(c)(2): Library Preparation and Sequencing
[0340] To generate sequencing libraries and targeted sequencing data, CAPP-Seq was applied as previously described. Briefly, cell-free, tumor, and germ-line DNA were used to construct sequencing libraries through end repair, A-tailing, and adapter ligation following the KAPA Hyper Prep Kit manufacturer's instructions with ligation performed overnight at 4.degree. C. CAPP-Seq adapters with unique molecular identifiers (UMIDs) were used for barcoding of unique DNA duplexes and subsequent deduplication of sequencing read pairs. Hybrid capture was then performed (SeqCap EZ Choice; NimbleGen) using the PhasED-Seq panel described above. Affinity capture was performed according to the manufacturer's protocol, with all 47.degree. C. hybridizations conducted on an Eppendorf thermal cycler. Following enrichment, libraries were sequenced using an Illumina HiSeq4000 instrument with 2.times.150 bp paired-end (PE) reads.
10(c)(3): Pre-Processing and Alignment
[0341] FASTQ files were de-multiplexed and UMIDs were extracted using a custom pipeline as previously described. Following demultiplexing, reads were aligned to the human genome (build GRCh37/hg19) using BWA ALN. Molecular barcode-mediated error suppression and background polishing (i.e., integrated digital error suppression; iDES) were then performed as previously described.
10(c)(4): Identification of Phased Variants and Allelic Quantitation
[0342] After generating TIMID error-suppressed alignment files (e.g., BAM files), PVs were identified from each sample as follows. First, matched germ-line sequencing of uninvolved peripheral blood mononuclear cells (PBMCs) was performed to identify patient-specific constitutional single nucleotide polymorphisms (SNPs). These were defined as non-reference positions with a variant allele fraction (VAF) above 40% with a depth of at least 10, or a VAF of above 0.25% with a depth of at least 100. Next, PVs were identified from read-level data for a sample of interest. Following UMID-mediated error suppression, each individual paired-end (PE) read and identified all non-reference positions were using `samtools calmd`. PE data was used rather than single reads to identify variants occurring on the same template DNA molecule, which may subsequently fall into either read 1 or read 2. Any read-pair containing .gtoreq.2 non-reference positions was considered to represent a possible somatic PV. For reads with .gtoreq.2 non-reference positions, each permutation of size .gtoreq.2 was considered independently: i.e., if 4 non-reference positions were identified in a read-pair, all combinations of 2 SNVs (i.e., `doublet` phased variants) and all combinations of 3 SNVs (i.e., `triplet` phased variants) were independently considered. PVs containing putative germ-line SNPs were also removed as follows: if in a given n-mer (i.e., n SNVs in phase on a given molecule) .gtoreq.n-1 of the component variants were identified as germ-line SNPs, the PV was redacted. This filtering strategy ensures that for any remaining PV, at least 2 of the component SNVs were not seen in the germ-line, as relevant for both sensitivity and specificity.
[0343] Putative somatic PVs were filtered using a heuristic blacklisting approach in considering sequencing data from 170 germ-line DNA samples serving as controls. In each of these samples, PVs were identified on read-pairs as described above, but without filtering for matched germ-line. Any PV that occurred in one or greater paired-end read, in one or more of these control samples, was included in the blacklist and removed from patient-specific somatic PV lists.
[0344] To calculate the VAF of each PV, a numerator representing the number of DNA molecules containing a PV of interest was calculated over a denominator representing the total number of DNA molecules that covered the genomic region of interest. That is, the numerator is simply the total number of deduplicated read-pairs that contain a given PV while the denominator is the number of read-pairs that span the genomic locus of a given PV.
10(c)(5): Genotyping Phased Variants from Pretreatment Samples
[0345] The above strategy resulted in a list of PVs of .gtoreq.1 read-depth in each sample. To identify PVs serving as tumor-specific somatic reporters for disease monitoring, for each case a `best genotyping` specimen--either DNA from a tumor tissue biopsy (preferred), or pretreatment cell-free DNA was identified. After identifying all possible PVs in the `best genotyping sample`, the list for specificity was further filtered as follows. For any n-mer PV set, if .gtoreq.n-1 of the constituent SNVs were present as germ-line SNPs in the 170 control samples described above, the PV was removed. Furthermore, only PVs that meet the following criteria were considered: 1) AF >1%; 2) depth of the PV locus of .gtoreq.100 read-pairs, and 3) at least one component SNV must be in the on-target space. Finally, 4) any PV meeting these criteria was assessed for read-support in a cohort of 12 healthy control cfDNA samples. If any read-support was present in >1 of these 12 samples, the PV was removed. For genotyping from cell-free DNA samples identified as low tumor fraction by SNVs (i.e., <1% mean AF across all SNVs), the AF threshold for determining PVs was relaxed to >0.2%. This filtering resulted in the PV lists used for disease monitoring and MRD detection.
10(c)(6): Determination of Tumor Fraction in a Sample from Phased Variants
[0346] For evaluation of a sample for minimal residual disease (MRD) detection with prior knowledge of the tumor genotype, the presence of any PV identified in the best pretreatment genotyping sample in the MRD sample of interest can be assessed. Given a list of k possible tumor-derived PVs observed in the best genotyping sample, all read-pairs covering at least 1 of the k possible PVs were determined. This value, d, can be thought of as the aggregated `informative depth` across all PVs spanned by cfDNA molecules in a PhasED-Seq experiment. It was then assessed how many of these d read-pairs actually contained 1 or more of the k possible PVs--this value, x, represents the number of tumor-derived molecules containing somatic PVs in a given sample. The number of tumor-derived molecules containing PVs divided by the informative depth--x/d--is therefore the phased-variant tumor fraction (PVAF) in a given sample. For detection of MRD in each sample, PVAF was calculated independently for doublet, triplet, and quadruplet PVs.
10(c) (7): Monte Carlo Simulation for Empirical Significance of PV Detection within a Specimen
[0347] To assess the statistical significance of the detection of tumor-derived PVs in any sample, an empiric significance testing approach was implemented. A test statistic f was first defined as follows--from a given list of k possible tumor-derived PVs observed in the best genotyping sample, the arithmetic mean of allele fractions was calculated across all k PVs (allele fraction defined as the number of read-pairs containing an individual PV (x.sub.i) over the number of read-pairs spanning the PV positions (d.sub.i)):
f = i = 1 k x i d i k ( 1 ) ##EQU00001##
to assess the hypothesis that f is not significantly different from the background error-rate of similar PVs assessed from the same sample. A Monte Carlo approach was used to develop a null distribution and perform statistical testing as follows:
[0348] 1. Given a set of k PVs, {pv.sub.1 . . . pv.sub.i . . . pv.sub.k}, an `alternate` list of PVs, {pv'.sub.1 . . . pv'.sub.i . . . pv'.sub.k}, was generated such that for each alternate PV had the same type of base change and distance between SNVs as the test PV. For example, if a doublet PV, chr14:106329929 C>T and chr14:106329977 G>A, was identified in the genotyping sample and searched for an alternate two positions at the same genomic distance (here, 48 bp) with reference bases C and G, and assessed for read-pairs with the same types of base changes (i.e., C>T and G>A), using the heuristic search scheme below.
[0349] 2. For each tumor pv.sub.i in the set of k, 50 such alternates were identified. This was performed with a random search algorithm to scan the genomic space and identify alternates. To find these 50 alternates, a random position on the same chromosome as the test pv.sub.i was identified and then searched for the same types of reference bases at the same genomic distance as described above. Synteny of observed/alternate PVs was used to control for regional variation in SHM/aSHM as well as copy number variation, as potential confounders of the null distribution. Alternate positions that were identified as a germ-line SNP, defined as having AF >5%, were excluded.
[0350] 3. After identifying 50 such alternates for each pv.sub.i, 10,000 random permutations of 1 alternate were generated for each of the k original PVs and calculated the phased-variant fraction f' for these alternate lists in the sample of interest being evaluated for presence of MRD, as described above.
[0351] 4. An empiric P-value was calculated, defined as the fraction of times the true phased-variant fraction f is observed to be less than or equal to the alternate f' across the 10,000 random PV lists as an empirical measure of significance of MRD significance in the blood sample of interest.
[0352] While this resulting comparison is a measure of the significance for PV detection of tumor-reporter list compared to the empirically defined background PV error-rate within the sample of interest, its relationship to specificity of detection across cases and control samples was also evaluated, as described below.
10(c)(8): Assessment of Specificity of PhasED-Seq
[0353] To determine the specificity of disease and MRD detection through PhasED-Seq, patient-specific PVs from 107 patients with DLBCL were first identified using pretreatment tumor or plasma DNA along with paired germ-line samples. 40 independent plasma DNA samples were then assessed from healthy individuals for presence of these patient-specific PVs, using the Monte Carlo approach outlined above. A threshold for P-values was empirically determined from Monte Carlo such that 95% specificity was achieved for disease detection from doublet, triplet, and quadruplet PVs. The P-value threshold yielding 95% specificity for each size of PV was as follows: <0.041 for doublets, <1 for triplets, and <1 for quadruplets. The results of this specificity in control cfDNA analysis is shown in FIGS. 15 and 16.
10(c)(9): Calculation of Error Rates
[0354] To assess the error profile of both isolated SNVs and PVs, the non-reference base observation rate of each type of variant was examined across all reads. For isolated SNVs, the error-rate for each possible base change e.sub.n1<n1' was calculated as the fraction of on-target bases with reference allele n1 that are mutated to alternate allele n1', when considering all possible base-changes of the reference allele. Positions with a non-reference allele rate exceeding 5% were classified as probable germ-line events, and excluded from the error-rate analysis. A global error rate, defined as the rate of mutation from the hg19 reference allele to any alternate allele, was also calculated.
[0355] For phased variants, a similar calculation was performed. For the error-rate of a given type of phased variant composed of k constituent base-changes {e.sub.n1>n1' . . . e.sub.nk>nk'}, the error-rate was calculated by determining both the number of instances of the type of base change (i.e., the numerator), as well as the number of possible instances for the base change (i.e., the denominator). To calculate the numerator, N, the number of occurrences of the PV of interest over all read-pairs was counted in a given sample. For example, to calculate the error-rate of C>T and G>A phased doublets, the number of read-pairs that include both a reference C mutated to a T as well as a reference G mutated to an A was first counted.
[0356] To calculate the denominator, D, the number of possible instances of this type of phased variant was also calculated; this was performed first for each read-pair i, and then summed over all read pairs. A PV with k components can be summarized as having certain set of reference bases p.sub.A,p.sub.C,p.sub.G,p.sub.T, where P.sub.N is the number of each reference base in the PV. Similarly, a given read pair contains a certain set of reference bases b.sub.A, b.sub.C,b.sub.G,b.sub.T, where b.sub.N is the number of each reference base in the read pair. Therefore, for each read pair in a given sample, the number of possible occurrences of PV type of interest can be calculated combinatorically as:
D i = ( b A p A ) ( b C p C ) ( b G p G ) ( b T p T ) ( 2 ) ##EQU00002##
For example, consider a read-pair with 40 reference As, 50 reference Cs, 45 reference Gs, and 35 reference Ts. The number of positions for a C>T and G>A PV is:
D i = ( 4 0 0 ) ( 5 0 1 ) ( 4 5 1 ) ( 3 5 0 ) = 2 2 5 0 ( 3 ) ##EQU00003##
The aggregated denominator, D, for error rate calculation is then simply the sum of this value over all read pairs. The error rate for this type of PV is then simply N/D.
10(d): Differences in Phased Variants Between Lymphoma Subtypes
[0357] To compare the distribution of phased variants in different types of lymphomas, tumor-specific PVs were identified in 101 DLBCL, 16 PMBCL, and 23 cHL patients via sequencing of tumor biopsy specimens and/or pre-treatment cell-free DNA and paired germ-line specimens. After identifying these tumor-specific PVs, their distribution was the assessed across the targeted sequencing panel. The panel was first divided into 50 bp bins; for each patient, it was then determined if each patient had evidence of a PV within the 50 bp bin, defined as having at least one component of the PV within the bin. The nearest gene to each 50 bp bin was further determined, based on GENCODEv19 annotation of the reference genome.
[0358] To assess how the distribution of PVs between subtypes of lymphoma varies at the level of specific genes, the distribution of PVs was examined across the 50 bp bins spanning each gene (or nearest gene). For example, consider a given gene with n such 50 bp bins represented in targeted sequencing panel. For each bin, it was first determined the fraction of patients, f in each type of lymphoma with a PV falling within the 50 bp bin--i.e., determining {f.sub.type1,1, . . . f.sub.type1,n} and if {f.sub.type2,1, . . . f.sub.type2,n}. Then, any two histologies were then compared for the fraction of cases harboring PVs in the set of 50 bp bins assigned to each gene. These comparisons are depicted for individual genes on gene-specific plots in FIG. 2D and FIGS. 10-12.
[0359] The enrichment in PVs was statistically compared in a specific lymphoma type or subtype vs. another by calculating the difference in the fraction of patients which contain a PV in each 50 bp bin across all bins assigned to a gene (i.e., overlapping a given gene or with a given nearest gene). Specifically, for any comparison between two lymphoma types (type.sub.1 and type.sub.2), this set of differences in PV-rate was first identified between histologies if {.sub.type1,1-f.sub.type2,1, . . . f.sub.type1,n-f.sub.type2,n}. This set of gene-specific differences in frequency of PVs was the compared between types of lymphoma against the distribution of all other 50 bp bins in the sequencing panel by the Wilcoxon rank sum test. For this test, the set of n 50 bp bins assigned to a given gene was compared to all other 50 bp bins (i.e., 6755-n, since there are 6755 50 bp bins in sequencing panel). This P-value, along with the mean difference in fraction of patients with a PV in each bin for each gene between histologies, is depicted as a volcano plot in FIG. 2E. To account for the global difference in rate of PVs between different histologies, the mean difference in fraction of patients with a PV between histologies was centered on 0 by subtracting the mean difference across all genes.
10(e): Hybridization Bias
[0360] To assess the effect of mutations on hybridization efficiency, the affinity of mutated molecules to wildtype capture baits in silico was first estimated by considering DNA fragments harboring 0-30% mutations across the entire fragment For each mutation condition across this range, 10,000 regions were first randomly sampled, each 150 bp in length, from across the whole genome. These 150-mers were then mutated in silico to simulate the desired mutation rate in 3 different ways: 1) mutating `clustered` or contiguous bases starting from the ends of a sequence, 2) mutating clustered bases started from the middle of the sequence, or 3) mutating bases selected at random positions throughout the sequence. The energy.c package was then used to calculate the theoretical binding energy (kcal/mol) between the mutated and wild-type sequences, in relying on a nearest-neighbor model employing established thermodynamic parameters (FIG. 14A).
[0361] This in silico experiment was then replicated by testing the effects of same mutation rates in vitro. Specifically, oligonucleotides (IDT) were synthesized and annealed to form DNA duplexes harboring 0-10% mutations at defined positions relative to the human reference genome sequence. These synthetic DNA molecules were then captured together at equimolar concentrations and quantified the relative capture efficiency of mutated duplexes compared to the wild-type, unmutated species (FIG. 3A). Two sets of oligonucleotide sequences were selected from coding regions of BCL6 and MYC to capture AID-mediated aberrant somatic hypermutations associated with each gene (Table 5); the preserved mappability of the mutated species was ensured by BWA ALN. These synthetic oligonucleotide duplexes were then subjected to library preparation, then captured and sequenced using PhasED-Seq, performed in triplicate using distinct samples. This allowed assessment of the relative efficiency of hybrid capture and molecular recovery as directly compared to wildtype molecules identical to the reference genome.
10(f): Assessment of Limit of Detection with Limiting Dilution Series
[0362] To empirically define the analytical sensitivity of PhasED-Seq, a limited dilution series of cell-free DNA from 3 patients that were spiked into healthy control cell-free DNA at defined concentrations was utilized. The dilution series contained samples with an expected mean tumor fraction of 0.1%, 0.01%, 0.001%, 0.0002%, 0.0001%, and 0.00005% or ranging from 1 part in 1,000 to 1 part in 2,000,000. The sequencing characteristics and ctDNA quantification via CAPP-Seq, duplex sequencing, and PhasED-Seq are provided. To compare the performance of each method, the difference was calculated, .delta., between the observed and expected tumor fraction for each patient i at each dilution concentration j:
.delta..sub.i,j=tumac.sub.i,j-tumor frac.sub.i,j (4)
This value was calculated for patients i={1,2,3} and concentrations j={0.001%, 0.0002%, 0.0001%, 0.00005%} for each ctDNA detection method (CAPP-Seq, duplex, doublet PhasED-Seq, and triplet PhasED-Seq). The performance of each method was then compared to each other by paired t-test across this set of patients and concentrations.
10(g): Model to Predict the Probability of Detection for a Given Set of Phased Variants
[0363] To build a mathematical model to predict the probability of detection for a given sample of interest, it began with the common assumption that cfDNA detection can be considered a random process based on binomial sampling. However, unlike SNVs occurring at large genomic distances apart from one another, detection of PVs can be highly inter-dependent, especially when PVs are degenerate (i.e., when two PVs share component SNVs) or occur in close proximity. To account for this, only PVs occurring >150 bp apart from each other was considered as independent `tumor reporters`. The number of `tumor reporters` to allow for disease detection in a given sample can thus be determined as follows. The PhasED-Seq panel was broken apart into 150 bp bins. Each PV in a given patient's reporter list was then turned into a BED coordinate, consisting of the start position (defined as the left-most component SNV) and end position (defined as the right-most component SNV). For each PV, the 150 bp bin from the PhasED-Seq selector panel containing the PV was determined; if a PV spanned two or more 150 bp bins, it was assigned to both bins. The number of independent tumor reporters was then defined as the number of separate 150 bp bins containing a tumor-specific PV.
[0364] A mathematical model was then developed comparing the expected probability of detection for a given sample at a given tumor fraction with a given number of independent tumor reporters (e.g., 150 bp bins). With a given number of tumor reporters r, at a given tumor fraction f, with a given sequencing depth d, the probability of detecting 1 or more cell-free DNA molecule containing a tumor-specific PV containing can be defined as:
Pr ( detection ) = 1 - Pr ( nondetection ) ( 5 ) = 1 - ( d * r 0 ) f 0 ( 1 - f ) d * r ( 6 ) ##EQU00004##
based on simple binomial sampling. However, as ctDNA detection method was trained to have a 5% false positive rate, this false positive rate term was added to the model as well:
Pr ( detection ) = 1 - Pr ( nondetection ) + 0.05 * Pr ( nondetection ) ( 7 ) Pr ( detection ) = 1 - 0 . 9 5 * Pr ( nondetection ) ( 8 ) = 1 - 0 . 9 5 * ( d * r 0 ) f 0 ( 1 - f ) d * r ( 9 ) ##EQU00005##
FIG. 3G shows the results of this model for a range of tumor reporters r from 3 to 67 at depth d of 5000. The confidence envelope on this plot shows solutions for a range of depth d from 4000 to 6000.
[0365] To empirically validate this model assessing the probability of disease detection, samples from limiting dilution series were utilized. In this dilution series, 3 patient cfDNA samples, each containing patient-specific PVs, were spiked into healthy control cfDNA. For each list of patient specific PVs, 25 random subsamplings of the 150 bp bins containing patient-specific PVs were performed to generate reporter lists containing variable numbers of tumor-specific reporters. A maximum bin number of 67 was selected to allow sampling from all 3 patient-specific PV lists, followed by scaling down the number of bins by 2.times. or 3.times. per operation. This resulted in reporter lists containing patient-specific PVs from 3, 6, 17, 34, or 67 independent 150 bp bins. Disease detection was then assessed using each of these patient-specific PV lists of increasing size in each of `wet` limiting dilution samples from 1:1,000 to 1:1,000,000 (FIG. 3H, closed circles). In silico mixtures was further created using sequencing reads from limiting dilution samples with varying expected tumor-content, and again assessed for the probability of disease detection using patient-specific subsampled PV reporter lists of varying lengths (open circles). For this experiment, both the `wet` and `in-silico` dilution bam files were down-sampled to achieve a depth of .about.4000-6000.times. to correspond with modeled depth. The final mean and standard deviation of depth across all down-sampled bam files was 4214x .+-.789. The probability of detection was summarized across all tests at a given expected tumor fraction, for a given patient-specific PV list. For each given dilution, multiple independently sampled sets of reads were considered to allow superior estimation of the true probability of detection. Specifically, the following number of replicates at each dilution indicated was considered in Table 7.
TABLE-US-00001 TABLE 7 Replicates at each dilution for predicting the probability of detection for a given set of phased variants. Number of Tests Dilution Replicates (Replicates * 25) Wet or In silico 1:1,000 1 25 Wet 5:10,000 3 75 In silico 3.5:10,000 3 75 In silico 2:10,000 3 75 In silico 1:10,000 3 75 Wet 5:100,000 3 75 In silico 3.5:100,000.sup. 3 75 In silico 2:100,000 3 75 In silico 1:100,000 3 75 Wet .sup. 5:1,000,000 8 200 In silico 3.5:1,000,000 8 200 In silico .sup. 2:1,000,000 8 200 Wet .sup. 1:1,000,000 8 200 Wet
[0366] The total number of tests, for each patient-specific PV list, is therefore the number of randomly subsampled PV lists (e.g., 25) times the number of independently downsampled bam files; this number is provided in the table above. In FIG. 3H, the points and error-bars represent the mean, minimum, and maximum across all three patients. The concordance between the predicted probability of disease detection from theoretical mathematical model and wet and in silico samples validating this model, is shown in FIG. 3I.
10(h): Statistical Analyses & Software Availability
[0367] All P-values reported in this manuscript are 2-sided unless otherwise noted. Comparisons of matched samples and populations were performed using the Wilcoxon signed rank test; comparisons of samples drawn from unrelated populations were performed using the Wilcoxon rank-sum test. Comparisons of paired samples were performed by paired t-test. Survival probabilities were estimated using the Kaplan-Meier method; survival of groups of patients based on ctDNA levels were compared using the log-rank test. Other statistical tests are noted in the manuscript text where utilized. All analyses were performed with the use of MATLAB, version 2018b, R Statistical Software version 3.4.1, and GraphPad Prism, version 8.0.2. The contribution of known mutational processes to phased and isolated SNVs from WGS was assessed with the deconstruct Sigs R package using the COSMIC signature set (v2) as described. Calculation of AUC accounting for survival and censorship was performed using the R `survivalROC` package version 1.0.3 with default settings. An executable version of the PhasED-Seq software, developed in C++ 17, is available at phasedseq(dot)stanford(dot)edu.
Example 11
[0368] Additional details of the tables described throughout the present disclosure are provided herein:
[0369] TABLE 1: 1000 bp regions of interest throughout the genome containing putative phased variants (PV) in various lymphoid neoplasms. Only regions containing >1 subject with a PV are shown. Coordinates are in hg19. Regions from genes that were previously identified as targets of activation-induced deaminase (AID) are labeled. Regions that contain PVs in >5% of subjects in any histology (BL, CLL, DLBCL, FL) are also labeled. BL, Burkitt lymphoma; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphoma; FL, follcicular lymphoma.
[0370] TABLE 2: 1000 bp regions of interest throughout the genome containing putative phased variants (PV) in the ABC and GCB subtypes of DLBCL. Only regions containing >1 subject with a PV are shown. Coordinates are in hg19. Regions from genes that were previously identified as targets of AID are labeled. ABC, activated B-cell subtype; GCB, germinal center B-cell subtype.
[0371] TABLE 3: Regions used for the PhasED-Seq capture reagent described in this paper focused on lymphoid malignancies. Coordinates are in hg19. The closest gene and the reason for inclusion (Phased Variants vs general DLBCL genotyping) is also shown.
[0372] TABLE 4: Enrichment of PVs at genetic loci throughout the PhasED-Seq targeted sequencing panel for different types of B-cell lymphomas (DLBCL including ABC and GCB subtypes, PMBCL, and cHL). The PhasED-Seq selector was binned into 50 bp bins in hg19 coordinates, and each bin was labelled by gene or nearest gene. The mean of the fraction of cases of a given histology with a PV across all 50 bp bins is shown. Significance was determined by rank-sum (Mann-Whitney U) test of 50 bp bins for a given gene against the remainder of the sequencing panel. Uncorrected P-values are shown; multiple-hypothesis testing correction was performed by Bonferroni method. DLBCL, diffuse large B-cell lymphoma; PMBCL, primary mediastinal B-cell lymphoma; cHL, classical Hodgkin lymphoma; ABC, activated B-cell DLBCL; GCB, germinal center B-cell DLBCL.
[0373] TABLE 5: Sequences of oligonucleotides synthesized to assess hybridization and molecular recovery bias with increasing mutational burden (SEQ ID NOs. 1331-1358).
[0374] TABLE 6: Nucleic acid probes for Capture Sequencing of B-cell Cancers (SEQ ID NOs. 0001-1330).
[0375] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Sequence CWU
1
1
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1002100DNAHomo sapiens 2gagcctcctg gagactgggg
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ctcctctccg tctgctcgca 1003100DNAHomo sapiens
3gatcccagtt ctgaccccag ggcctcccac agatctcttc cccatgcccc tgtcctggcc
60gttgctggct ccggcgtcca gcccgtcccc tgctgcctgg
1004100DNAHomo sapiens 4ccatgttgct ggcttacttg gcatttccca tgatctcaca
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ttgctggctt acttggcatt 1006100DNAHomo sapiens
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60tacaagtcct caaaactttg attatataga gagctaaact
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tatactatga tgaaaaaata 60aatgaataat tgtgaaatag ccaaaaatac taaaatacag
1008100DNAHomo sapiens 8aatgaataat tgtgaaatag
ccaaaaatac taaaatacag ctataaggtt aaaaataaat 60ctgaataaaa aatgtaggag
ggaaaagtga ttaccttacc 1009100DNAHomo sapiens
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10011100DNAHomo sapiens 11gaggagcccc tgggtcccca
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ggcaaattca gagaagaatt 10012100DNAHomo sapiens
12agtagaaaaa aagggcgtcg tgctggattc tccttctgga tggtacatga cagtggatgc
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10014100DNAHomo sapiens 14acccgagacc tctcactgag
cccgagccgc gcgcgacatg agccacggga agggaaccga 60catgctcccg gagatcgccg
ccgccgtggg cttcctctcc 10015100DNAHomo sapiens
15agcctcctga ggacccgggg ctgcgtgagc gagcagaggc ttaaggtctt cagcggggcg
60ctccaggagg cactcacagg tgagcgcatg ccgaggggcc
10016100DNAHomo sapiens 16tggcgccacc gggggtcggc cccatccctg ccagggccgt
ctttcttcta ctcctgcggc 60agggtgaccc acgggagcag ctttgggact cggtggccct
10017100DNAHomo sapiens 17cctccgaccc ccggggcggc
ccgcagtccc cagtttcctg ggtcctcctc cccagccctg 60tgctcgggtc tcggccgtgg
cggttctgat ggggcgcgcc 10018100DNAHomo sapiens
18cctctacgct ctcggaggcg cagaccctgg tcctggagtg ccagcccgag tccccagctt
60atgcccctgt ctcattacgg gctcgtctcc ctcgctggac
10019100DNAHomo sapiens 19cctcgagatc ttaagaccct cgatggatgt tgttgcgggc
cgcccggtcg gccgaggggt 60cccgatgagg gaagaaggtg cagtcgagcc ttttcaacaa
10020100DNAHomo sapiens 20tttggagtcc cagtgcggtt
cttcctgccg gtcggggtgc gctgtgcctg gggtagtcca 60ctggttgctg actggcttca
agttggaatt tgggccccct 10021100DNAHomo sapiens
21ttgtgttatc tttggttccc cttagccatc tgccacctat tgtggtaggg aggagagcct
60cgtagctcgt gaccctgccg tgcgggcctt caagttggga
10022100DNAHomo sapiens 22ggtgaagaga taagcagccc gctcgctggc tggggagaga
cctctctccc agctgtttct 60agctggttac tgtcagtttt gggaagcgat agccatctcg
10023100DNAHomo sapiens 23gaacgcaccc acacagaccc
tgccttctga ggaaaacaga tgtttcatca aaacaaccca 60gttttcactc ccttaggcac
tgctaaggaa ggttctctga 10024100DNAHomo sapiens
24ctcttctgaa ggaagcagag ggaacacagg gtgggaggtc cagtgacttg ctgtggaccc
60aacaatgttg gcagccttcc tggccctgaa acttcagctc
10025100DNAHomo sapiens 25acaggtctcc agaggccctg cctggacatg ccagtcccag
tcacaccctt cccttgcttt 60gggggtgtgc caaaagcaat acactggcca ctagagagta
10026100DNAHomo sapiens 26ccctagagct ctagaatccc
ctcccaacac gcacacacac acacacacac actctctctc 60tcacacacac acactcagtc
acacacacac acacacacac 10027100DNAHomo sapiens
27ctttcagatc tttcgcagcg tcccaacagg gcaaaggctc cagcattctg ccagaaggaa
60ttcccgcctc cacattcccg gtccccggct gtgctgaggg
10028100DNAHomo sapiens 28gctgccccca agcaagccca gcgttgggga ccctccctcc
actctgtcgg agagctgcca 60acgccccccg cccacggggg ccccacttcg ggcctcctca
10029100DNAHomo sapiens 29gggcctacgg aggccagggc
cctgggcagc ctggaccagc tcagggaatc agaggactct 60gcgctttgca cgctcacagt
cgtctcctct ggccttttgc 10030100DNAHomo sapiens
30ccacttcagg ctccccagag cccggcatgc cacagggcag atatcctttc cccatcttcc
60cagggggttc tccatcgcgg ggcccgcccc tttctggggc
10031100DNAHomo sapiens 31tgggcttgtc tcactgccca gaaactgccc ctgcctctcc
accagggcct ctgggggctg 60caggtcctca agctcacggg ctctcccaga cggctcagtg
10032100DNAHomo sapiens 32agggcaagat cctgtggacg
gtgtggccca gtggatgtaa ctctcgctgc cacttccgtg 60gccatcgtta agctagctcc
gaacagcccc aatgagggag 10033100DNAHomo sapiens
33ctaggcagct ccgagttccc ggggtaggag agcccctttt gtcaatttcc atagctgtgg
60gtgagccaca gcggggactg gcagggatac ccttctccat
10034100DNAHomo sapiens 34ccttacaaaa gcggatggac cctgagcctc tgatcctgta
ggggcagccc ggccgggaag 60aggtggcatt cctttcttca cctgcgagga gcataggctg
10035100DNAHomo sapiens 35ggccctcctt tcctcccgga
gtcggttcct gaagtctctg gacattgctc cccccaggac 60tttgtcctcc gttcctcgct
ccgggcgccc tgaaccagga 10036100DNAHomo sapiens
36cccttccagg gggctgactg ctgctgcgga aggggcacgg ggagggcgag cgagccctgc
60ccaaacgcgg gctgcggggc gcttgaatgg cggagctctg
10037100DNAHomo sapiens 37tgcctggatg tgcgcctcaa acatgcccac tttctggttc
acctgcacgt tctgcaactc 60gcgctgcaag atccgcagct tcctcttggc ctcctccggc
10038100DNAHomo sapiens 38cctggcgggg agagggtacc
ggctgccacc acctgctgcc ggtcccctcg caggcgacca 60gcccaacttg ggctgctcac
gctactgccg ctgctgccgc 10039100DNAHomo sapiens
39tgccactgcc gctgctacta ttcagcctgc gccggccgct ccgccagccc ccggggctcc
60ggggctcctc gggggacagc gactcggctg gggggaagag
10040100DNAHomo sapiens 40gaaagaggcg cctctcccgg ggctgaaaac gctgccgggg
ctcagcactg ccctcctcgg 60gggcgggggc gtctcgctgc cactgggccc cgggccgccg
10041100DNAHomo sapiens 41ccgctcttca tctcgttggc
gctattcatg atcaccaggc tattgagcgc atagcagtac 60acagccatag tactgggtcc
cgcgctgccc gccgccgcgg 10042100DNAHomo sapiens
42ctcccgctcc tgctccgccg ccggcgcctc ctcctcccgg cgctcccggc tcagccccgg
60aggcccggca gccgcggctc cgcgcgcaga tggggcggca
10043100DNAHomo sapiens 43aagtgcgaag gaagtgtcag gctggatgtc aaaatgaaca
ccttggagaa ctggatgatg 60gaacagacgg taaaaatcag ctaaacatca gagaaaatgg
10044100DNAHomo sapiens 44aggaagaggt caaaactgtg
aacaggaact agaagaaagt gtagcagaaa aagacttgtc 60acaaacttcg agagatttgg
agaaaatgat gtcaaaacac 10045100DNAHomo sapiens
45atcttcctca agcccatgct gagtatctct gatttggtta atttcttggt aagtgttcca
60agtacagaca acaaagcaga aaagcactga ttacagggaa
10046100DNAHomo sapiens 46tatgcagaat gatccttcag atcatgtgaa cgctataatt
aaatgttgct accaaatccc 60cactaccctt tctcccacct agaaaaagtt aatgcatgaa
10047100DNAHomo sapiens 47ttcagtatga gcaaattgtg
atttataaaa acaaacaaac aaacaaacaa acaaaaccca 60ccctattcac tccgtagggg
aataaagctt tcttgcatta 10048100DNAHomo sapiens
48aacaaacaaa acccacccta ttcactccgt aggggaataa agctttcttg cattaagtca
60cgcatcatgg gggtaggaaa aaagcacagt actgaaagaa
10049100DNAHomo sapiens 49gtgaagtgat ccaaatgtag cccagagatc ctaaagaaaa
aacgatgctc atgtgttaca 60aaacaaaatt ttaaggcaat cagtgaggaa tcacagacaa
10050100DNAHomo sapiens 50atttccttag tgcttttatc
aaggttgaat ctgaatataa attactagag gaaagcaaat 60cagatttcac atctgaaaat
taaaaacaaa attcttagct 10051100DNAHomo sapiens
51aggcaacaaa atgagatcct gtccctagaa aacatttcaa aaaattaaca gcatggtgac
60gcacacttgt agccctagct acttgggagg ctgagtggga
10052100DNAHomo sapiens 52aagaacttaa gcagactagg atataaagta taggagcgta
ttgtgtacag gaacgggaaa 60tactgtttcc tggatctttt gtttcactta cgcacacacc
10053100DNAHomo sapiens 53cacacccgcc agtagtgtac
caggttgcga tggaaatctc tctctttctg tggatgagtt 60tgtggaagcc cttgctccag
catgccctcc ttcctgccca 10054100DNAHomo sapiens
54cccctggacc attccttccc ttcacagcac tgtcccatgg gtaggccaca gcccagcaca
60ggccccagcc tggcggctgc agcaggagcc ccatcccagg
10055100DNAHomo sapiens 55gcctgagggg ccatgcgggg gtctgggtgg gagtgggaac
cgctgaggaa ggtgaaggga 60aatatggtga gatgacaggc ccgctgtcag ggagagtggg
10056100DNAHomo sapiens 56aggagccctg gagtgcccta
cctctgtggg gctggaactc cctgtatccg agctagggtc 60ttccacacgc atgctactac
cccaagtgcc acagctggag 10057100DNAHomo sapiens
57tcatctccca ctggataaca gtgttgtcgg gaacttccat ccagcactgg cggacactcc
60cgtcgcagct gctcctgact gagcaagtca tttaaggggg
10058100DNAHomo sapiens 58tccttggcac tcataagcac tcacagaatg gggctggcag
tgcgcccggc ctccctggga 60tgggtccaga atggtaggaa gcgcagtccg ggagggaccc
10059100DNAHomo sapiens 59actgcttaga gctctcagcc
ctagatggcg tatcacagtt aatgctctat aaaacccatc 60atggcttttc cctagtaagc
ctcaaatcgc tgcaagcaag 10060100DNAHomo sapiens
60gcttcatata tgagagtttc tgctgtctcc tggagccatc tcacccaaag ccactgactc
60tgggagacca gcccaggcca caaaccagca aagcaccagt
10061100DNAHomo sapiens 61tatagttaga gctgcattat aaagtggcca gaggacattt
ctttgcagtg agatgtgtat 60cgtgaacgtt tggggcctgt gctcgcctag tcctcatctt
10062100DNAHomo sapiens 62tgcttttcta ggtacacaaa
gccatcccat ggctgcaaat gttagctggg ctgggctccc 60tacttgcctc aagccccttc
atagaccctt caggcacatg 10063100DNAHomo sapiens
63cttttctctg gacgtttaca gacaggtcct cagaggtcag agcaggttgt cctagggagc
60agggaggctt cctagggagg tcagactcca aatagtggat
10064100DNAHomo sapiens 64atggcaaaaa tgcagctgca gactcatgag gagtcgccct
gggctgccac tagggctccc 60acagtgtgcg ctgccaacct gctgcccgtg cagaaactct
10065100DNAHomo sapiens 65caactgtgcc ctgcactgtt
agggcccttg tcaaaacaac acatttctca gtgattctga 60gactctttct cttatctata
gaagtcataa ctcaagagta 10066100DNAHomo sapiens
66aaatcatacc aatattttac ataaacccta gaatttttat agatctatta tttcttttta
60gagtacatat tggaagtaac ttcacaagga acattttctt
10067100DNAHomo sapiens 67tctggtcaaa ccactccaca aataaagtgg actgatcctc
ttgactctat gtgtaagtgc 60ccattgtgtg tgcacagagc tggtgagaac ggccatggtg
10068100DNAHomo sapiens 68ctaggtgggg gtggtgttgg
tggagttgga ctagattatc tgggatcatg cgaaatggaa 60attcatttct agctggctgg
cttcagaagg tgccatctcc 10069100DNAHomo sapiens
69tatttttata tgaagcgtgc tttggaactc agggcaacga agggtgggtg tgctgcacaa
60ggacagcaga agagtgagct gactggtccc tgaaatcgca
10070100DNAHomo sapiens 70gttggaaagt ggattaccag tgcagtagaa ctcttcacgg
aggcctggac catcaggtct 60aatggtgttg ttccaggtgg gtggtcatgt ggagcaaaaa
10071100DNAHomo sapiens 71tatttgaaat cagcgagcac
gtacctgaga gatgactttt ccacttgggc tagtctcttg 60atatttctgg tcctgtttct
tcatctgtaa actgggttag 10072100DNAHomo sapiens
72aaggagacca agaagcgtat ttaaaatctt gatgttttga gtttcttcct agcttccccc
60tattccttaa taaagttcta aattgttttg ttggagctct
10073100DNAHomo sapiens 73ttgcagccat tctgagggct ttgcatgctt ttctgacctt
gcagtaaact caatgcttta 60ggcaaagaat ggccacgtca tccgaccccc tcagagttta
10074100DNAHomo sapiens 74gaattcagaa caggtctgaa
gaagaccagg cagcggctga gtcaaggaaa gcctccgtcc 60gcttttattt cccctgtgcc
tcttccagga ctgtgctggg 10075100DNAHomo sapiens
75ataacaggct cccgggggtt actttggctg ggctgggcta aaacctccct gcagagcagg
60ccctgagccc tgcctctgcg cctgggtggt gtcagcccct
10076100DNAHomo sapiens 76ccaccttctg actgttccag caactctcta agccctccca
aaggcctcaa ggcctgtaac 60catatgcagc aattttcagc cataccagga gaggtcaact
10077100DNAHomo sapiens 77gtaatcttgg ccacctgcct
aagaggaagt ggctagcttc acttctgacc ctcagcaact 60gccaggtggc ctcttggaaa
tccccctctg ggggattcca 10078100DNAHomo sapiens
78cccgttgggt gggagagcag tagttaaaat gtaaaataag aatcttttgc tgggagaagt
60caacagatag ggagaagtca gctgataaca gaaatagttt
10079100DNAHomo sapiens 79taaaactaac ttcactgtta accaagcagt tcaacatgaa
agactgaatc tcttatgttt 60aatattttct tctcttttaa tcttcataac taattttttt
10080100DNAHomo sapiens 80cagataattg tataaaataa
ccatggtagc aaaataatgt gatcactgga aaataagcag 60ggaaaaacat gctatgaaga
tactcctatc tgggtgaatt 10081100DNAHomo sapiens
81cttgatagct ttacattttt catctggcat ttaaacatta aacagttaat gtatttgaca
60tgaaaattat ttcaagttat cttattagtt ttaatagagt
10082100DNAHomo sapiens 82ttaaaaagtg tttaaaagag ttttcaaaag gctctaaaat
cattttgaaa tagtttaaaa 60cagttttgaa tcgttgtaag ttagttttaa tagagcttta
10083100DNAHomo sapiens 83aaaaggccct aaaatagtcc
tatcaagttg ttgcagacca aaataatctc cttaaatatc 60acttttgaga tcagctgggg
taaacgacag caacacaatg 10084100DNAHomo sapiens
84acaaatcatt aaactatttt agagattatg aaattaaaat actcagatta aaattttcct
60atcacagaat taaggtactg gaaaatatgt ttaagttttt
10085100DNAHomo sapiens 85attaatcaca ttgctatagg tttagatatt ttgtacaact
gaaataaaat cacacactgg 60cagctacatt tttgaaagtt aaaaacatgg tcacgaatat
10086100DNAHomo sapiens 86atcttatttt aaaatcagtt
aatatacctt aatggtattt aatgccaaat tcaaagtgaa 60ttgatcaagc cctcagtggc
caggtcatgg gtgtgatttt 10087100DNAHomo sapiens
87tactctgaaa gaattacata tttctttctt tttggttgag cttttgttat ttaaatacat
60ttgatgagag gatattgaaa taattaaata gcactgaaaa
10088100DNAHomo sapiens 88aaaaaaagct ttaaattatt tacaatcccc taatggaaat
tttcactaat gagatatcat 60aatgaatgtg aattttattt ctgaaatctc taataaatca
10089100DNAHomo sapiens 89aagctttaaa ttatttacaa
tcccctaatg gaaattttca ctaatgagat atcataatga 60atgtgaattt tatttctgaa
atctctaata aatcagtctt 10090100DNAHomo sapiens
90ctccctggtt ttcccagctc agcgcccatt acgtttctgt tctctttccc ttagtggcat
60tatttgtatc actgtgcatc aggaaagctg gctacggcag
10091100DNAHomo sapiens 91catcaatcgg gcagacacag ggtggccacg gccactagcg
gcaaggcggc tgccccaaga 60gcgcggtggc atggccacca aagccactca atcgagaaag
10092100DNAHomo sapiens 92accgcggctc tgtctacagc
tcgcggtgcc acggccttct tggcagaata aaaatgtaga 60caagtaataa cagaggataa
tgaaagaaca tactctttaa 10093100DNAHomo sapiens
93aatatttcct atttttttca cagacccacg gtcattaaaa aatgcaatta tttacttttt
60ttcatttaaa cacatttctt tgagattgag cttttgggaa
10094100DNAHomo sapiens 94taaccacctt tccaccatta caataagaga taatttcacg
tttagtctaa tgtacaaatt 60ggatttttaa aaaatgagct ctatctgtga agcccttatt
10095100DNAHomo sapiens 95aaaatgagct ctatctgtga
agcccttatt cctatagaat gtgtcttttt gagtttatta 60cttattacag actctaaaaa
caacattgct gctgattttc 10096100DNAHomo sapiens
96aagtaagctg cctcttctac atagcaaata ggtacacttc acttttccct gatttttctt
60agggcgtgct attgattttt attgttgtct gacaaaataa
10097100DNAHomo sapiens 97tttatcaaac aaaagggaga aagactaaaa aatgtatttt
tccacttttc tgtatcatgc 60ataatcagca acaaccaata caatatttgg caagagtgaa
10098100DNAHomo sapiens 98caaaaataaa tttacttttg
ctccttagaa atacaagggt tcctttttag ttacactttt 60tttttttact ttgtgtcatt
cagtttagag caatttaatc 10099100DNAHomo sapiens
99tttttttctc caaatccatt tttgaagctg agtttaactt ttgcaaccca tggcaaatct
60taaatgccct catttaccaa tctttaccaa actcctattt
100100100DNAHomo sapiens 100aagcctctaa aagtcaatac tggccatcag acccaaattt
cagaagacaa tagtgaaaaa 60ttacttacgt ttaatctcca gtcgtgtccc ttggccgaag
100101100DNAHomo sapiens 101gtgatccaca gtgttaactt
aattactttc cccttaacaa aaatctcttt tcgctgttaa 60tatcactaac ctgaccgatg
cagagaaaat cttgcaattg 100102100DNAHomo sapiens
102agatgcctca cttaactggc tagcgcttgg ctgttcctta agatgaacta attttctatc
60ccttactcat ctgacttttt gaaagaatct ggtactcttt
100103100DNAHomo sapiens 103ggaattgacc tgagctaata tctcaaacac aaaaacgctc
caaatttaaa accttataag 60aaaaagcatt aggaaagtgc acttacgttt gatctccacc
100104100DNAHomo sapiens 104ttggtccctc cgccgaaagt
gagccacagt gagggatctc accctttccc ctcaacaaaa 60acctctcttg aagccaatca
tatgagatag gctgcttgtt 100105100DNAHomo sapiens
105cagagaaaaa tctagctatt tcttccccat ttcccccatg aatcctattc tcctctcaaa
60cccaatgatt cgtctatttg ctcagctttt taagttcatt
100106100DNAHomo sapiens 106ttctggtgtc ctgctattta cttctgggtc accaggttta
ttcaaccaaa atatcacaaa 60acttgcacaa atgatacaat ggcactaaaa tctcacgaat
100107100DNAHomo sapiens 107aattgagaca gatgtactta
cgtttgatat ccactttggt cccagggccg aaagtgaatc 60acagtgattc gtcttaactt
ttccctttac aaaaacctcc 100108100DNAHomo sapiens
108ctgaaagctc agcaagcctc tttcccccaa tgaagttatt ttgatttaga aatcttaaaa
60attagccaca agctagcgtc ctgtggaaca atttcccctc
100109100DNAHomo sapiens 109ctctgtacct aacctgggaa tgaagtttgt tagatccctg
gcatccgact aatgaaaatc 60cacacaaagg aacacaaagt aaactaatta gcaacagtga
100110100DNAHomo sapiens 110agaatcagtg gaaaaaagta
cttacgtttg atctccagct tggtcccctg gccaaaagtg 60tacacacaat ggttcctctt
aacttccctc ctatacaaaa 100111100DNAHomo sapiens
111actccctttc tgacaattga ccaaggctct gtccagaaca tgttatgttc cccaggacat
60ttctgaagct attacttaga caagttattc tcacccaatg
100112100DNAHomo sapiens 112actgaatctt gcttgctctt caaagaaaat gtgcaatcaa
ttctcgagtt tgactacaga 60cttatcttta tcttttccct gaaggatatc agaggctgat
100113100DNAHomo sapiens 113tgcagagtca ccttatagat
cacttcatag acacagggaa cagaagacac agacaactga 60ggaagcaaag tttaaattct
actcacgttt gatttccacc 100114100DNAHomo sapiens
114ttggtccctt ggccgaacgt ccaccacagt gagagctctc cattgtcttg ctgaacaaaa
60acccttctca ccaaagggga acagagtcct gggtcagctg
100115100DNAHomo sapiens 115atcaacttaa ggctcataac tttgaaatgc attttgaaat
gtagctccag atggtatacg 60aaaccaaagt gaagactaat agagtagaaa agtagacttt
100116100DNAHomo sapiens 116acttggttgg tttgtctgtt
ttcacagcac aggaagagct cagctcttac tgagctggac 60caggcgcatg ccatctttgg
agctgccatg gagtcccagt 100117100DNAHomo sapiens
117gttccatagt gtttccatag taatctcatc aacaacactg aagacctttt cagtattttc
60ttttgagtcc agctccattt ttgcagcctt gtatctctct
100118100DNAHomo sapiens 118ccgcgcccag ccgagtgcct gtttattttt acctgctttc
agattctctt ctacccttct 60aaattataag ctgtttgatg ttttatttgc cctgtatttg
100119100DNAHomo sapiens 119ggaggctccg tccagtatct
ttacttagca aatgcttaac aaacattttc agaataaata 60aaaaaaaata cctaattgaa
agtcaataat agatcagaga 100120100DNAHomo sapiens
120tgctatcata gaccaaagac taatactgac tgccacaaca gtaactttta caacagaaat
60cataactaca attctaaaga ttaggggtag gtttatttga
100121100DNAHomo sapiens 121ttctgtcact ggcagctttg ctagttgcct tgaatagcag
aattagcatt tggtctcacc 60agaagatgag gaaggagagg gatcaagtta gaggtggaga
100122100DNAHomo sapiens 122gttaacattg gcaagtgaaa
tttaatgtgc aaaatagctg accaagggca tagtcctttt 60ttaaagggga cacaaagtga
ttttctctgc agacatacac 100123100DNAHomo sapiens
123gcaataccaa tcataaaggg tgacatttat tgagcactta ctaagtgcca gacattgtac
60atggatcatc acatttaatt attcccaaga ctctatgaac
100124100DNAHomo sapiens 124tgagcactta ctaagtgcca gacattgtac atggatcatc
acatttaatt attcccaaga 60ctctatgaac taggaactaa tattatcccc tactttgtag
100125100DNAHomo sapiens 125gtgcaaaaac ttgagggcag
agaggtcaag gaactggctt atggcagtaa gtggcagagc 60tgtgacctaa actcagatcc
catgttttta actgaactat 100126100DNAHomo sapiens
126atgcagatta tactccagga gtaaagtcac tcaacggaag caacaagcgt gacagggaat
60gctgggatgg gggaaggtaa aaggaactcc ttagactggg
100127100DNAHomo sapiens 127ataagtgtgt acagacgtat gtataagact acacatggaa
atattgttta aagagtgaaa 60aataactaaa atcctcatta ataggagttt ggttaaactg
100128100DNAHomo sapiens 128tgctagagct ttacaatgta
gcacaaagca gacattaagg ggaagacgta gacttctata 60tagttacgtg gaaggtgttt
gtgaaaatgc aggtcactga 100129100DNAHomo sapiens
129agagtatgtg tggtgagata tcatgatccc atctacattg aatatatatg tatataaata
60cgggctgaat tttaaaagac ataaattgtg cttggtagtt
100130100DNAHomo sapiens 130aaatacgggc tgaattttaa aagacataaa ttgtgcttgg
tagttatctc ctgggattgc 60agaggaggaa caatgacact ttatgccatc tcctcctact
100131100DNAHomo sapiens 131cttctgtatg gtgatgtgaa
tatattcatt ttatagtttt tagaaataat aaaactgtac 60taattttgaa aaacagtaaa
ctctgacatt gcctattagc 100132100DNAHomo sapiens
132attctcgata ttcctgtgca atgcataaac ataacttttt aaaagatatg tacacacatg
60tgtgagtttt ctttgtcaaa tacttttcta taatctttaa
100133100DNAHomo sapiens 133atcaagcatg ccaaaaaggt aaaagctttc ctgtttcagt
gtaggagata gtcgtctgca 60aaggaaagag atgtagggga tagaaacagg aatgaaaaag
100134100DNAHomo sapiens 134atgactgagc tgttcgaggg
acttatgttc ctaagtgagc taattggaaa tctaatatga 60acagtgcaac cgaataacta
ttgtaaagca gtatttgtaa 100135100DNAHomo sapiens
135acaataaaag atgattatca taagtaccat tgttgcaaaa actattttat tgatcacatg
60cagtggtgat ctgtaggaat gattgttgtg atgtttgctg
100136100DNAHomo sapiens 136taacataaaa tgaaacatgg gaagtggctg agatctttag
gatgtgtgtg gttcattttt 60tgaaagcaaa tgttgtctca gaagcatctg tgagactctg
100137100DNAHomo sapiens 137ccaggatcca ccgttctaca
aaatatctgt gatggacatt gataagattg atctgttgag 60gaaaggcaag gtgtcagtaa
gatagtctga gagcttcttg 100138100DNAHomo sapiens
138gatttcatgt aaaagagtgc tggaaataga atttcttggg gaacattcca actaactcat
60cactgaaggt gctttacatt gaaccctcag caaagttaga
100139100DNAHomo sapiens 139ttatcagaaa aaaaatataa actgctgtgg aggggacagg
aaggaaagtc agggagggag 60gggggcaagg agagaaagag cgagagagag gagagaaaga
100140100DNAHomo sapiens 140agagaggaga gagagagcac
aagtacacac ttcaatgcac atctataaat catcctgaaa 60actactgata aattatttta
gcaatgttcc tcagatgtaa 100141100DNAHomo sapiens
141catttcaaga aatatcattt ttgcttttta tttggcataa tttactagcc aatttaggaa
60gttcccctca catcagtaac atacagtaca tcacccagta
100142100DNAHomo sapiens 142tgtcagagga cacaatggca taagtttgcc ttttgcaagg
tttgagggat ggccatttcc 60ctacctgact caggaaagtc tgtagctgat atccatcttc
100143100DNAHomo sapiens 143aagtttgtgg ttctttctct
ctatatatat atttgagctc agcagtcatg ctggagtcca 60gagtaggtga ttctttctgc
tttagcttga ctcctcctta 100144100DNAHomo sapiens
144tatatatttg agctcagcag tcatgctgga gtccagagta ggtgattctt tctgctttag
60cttgactcct ccttaagatt gtaactctct cagttttaca
100145100DNAHomo sapiens 145ttttttgtca gacgtaagct gacattccac aaggagagga
ggaaattctg tggttcacat 60ccagtggtgc ttggaacctg attggttgtc attcttccag
100146100DNAHomo sapiens 146ctagtttgtc acgagtggat
atctgtcctg gattcccaag gatcaaggct gccccattag 60ccaggaagta gggagataga
ggaggtcact tgagaaagag 100147100DNAHomo sapiens
147ctgcttcttt gccgcctcca ggttgtgtct gtttcctctc atatctgaag acagatgtgc
60tggcagaagc aaagtccttt gtccggccac gtgcaaatgc
100148100DNAHomo sapiens 148atgggacata aatatgaaca gagattcttg tcccactcta
gaaaatgtag atgttcatct 60tgtttccaag gggacagtaa ggctgcaggt gttttttgac
100149100DNAHomo sapiens 149cttttgtact cactggttgt
ttttgcatag gcccctccag gccacgacca gctgtttgga 60ttttataaac gggccgtttg
cattgtgaac tgagctacaa 100150100DNAHomo sapiens
150caggcaggca ggggcagcaa gatggtgttg cagacccagg tcttcatttc tctgttgctc
60tggatctctg gtgaggaatt aaaaagtgcc acagtctttt
100151100DNAHomo sapiens 151cagagtaata tctgtgtaga aataaaaaaa attaagatat
agttggaaat aatgactatt 60tccaatatgg atccaattat ctgctgactt ataatactac
100152100DNAHomo sapiens 152attaagatat agttggaaat
aatgactatt tccaatatgg atccaattat ctgctgactt 60ataatactac tagaaagcaa
atttaaatga catatttcaa 100153100DNAHomo sapiens
153ttatatctga gacagcgtgt ataagtttat gtataatcat tgtccattac tgactacagg
60tgcctacggg gacatcgtga tgacccagtc tccagactcc
100154100DNAHomo sapiens 154ctggctgtgt ctctgggcga gagggccacc atcaactgca
agtccagcca gagtgtttta 60tacagctcca acaataagaa ctacttagct tggtaccagc
100155100DNAHomo sapiens 155agaaaccagg acagcctcct
aagctgctca tttactgggc atctacccgg gaatccgggg 60tccctgaccg attcagtggc
agcgggtctg ggacagattt 100156100DNAHomo sapiens
156cactctcacc atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata
60ttatagtact cctcccacag tgcttcagcc tcgaacacaa
100157100DNAHomo sapiens 157acctcctccc catacgctgg gccagtaggt ctttgctgca
gcagctgctt cctctgcaca 60cagcccccaa catgcatgct tcctctgtgt gttggggagg
100158100DNAHomo sapiens 158aatacatgaa aacaactacc
gaaatgttat gaaattatag tttagtagaa ctaacaagtg 60cattaatgca aaagaaaagt
agggctcagt aatcagggaa 100159100DNAHomo sapiens
159ccaagtgtgc attgtaaaag tgcagcctct ctaacactgg gtttcatcac aagtaacaga
60acaggatgcc tgatgcaggg aaaaaagaaa ggcaattgtt
100160100DNAHomo sapiens 160gatctctggt aagagaaaca cttcctctcc tctgtgccac
caagtcccct gcatatccac 60aaaaataata tattttcata aggaattgat tttcctcatt
100161100DNAHomo sapiens 161ctctgcaaat atgatgcatt
tgatttatgt tttttacttt gctccataat cagataccag 60ggcagaaacg acactcacgc
agtctccagc attcatgtca 100162100DNAHomo sapiens
162gcgactccag gagacaaagt caacatctcc tgcaaagcca gccaagacat tgatgatgat
60atgaactggt accaacagaa accaggagaa gctgctattt
100163100DNAHomo sapiens 163tcattattca agaagctact actctcgttc ctggaatccc
acctcgattc agtggcagcg 60ggtatggaac agattttacc ctcacaatta ataacataga
100164100DNAHomo sapiens 164atctgaggat gctgcatatt
acttctgtct acaacatgat aatttccctc tcacagtgat 60acaccctgtt acaaaaacct
ccaagttctc tcagtgggat 100165100DNAHomo sapiens
165gccctctgtc ctggagacac ggccaaggag gctggagact gggtcagcac aatgtcccca
60ttgcagcctg aaatgataaa gacagataaa ttatatcaga
100166100DNAHomo sapiens 166tatactgaga ctgtccccat gtaggccatg cattggtgac
acttgtaacc acagtcatat 60gcaacatctt gagtaaccag aaaacaaaag ataactgggg
100167100DNAHomo sapiens 167aacttacaac ctacaatgag
tgccctaaat ccaacaacca agaatccaga gacacaaaaa 60acaatgatgg ccacatgagt
ttgcccgatg tttccctata 100168100DNAHomo sapiens
168taccaacacc atcagagtgt ggctgcatct gaggaccact ctcagctgat agaggcatca
60ggaggagcag ctggggcagc cctgcctcac acatctgctt
100169100DNAHomo sapiens 169ggggtttatg ttcgggtgtg taacactgtg ggagaataac
tattatactg ttggcagtaa 60taagttgcaa aatcatcagg ctgcaggctg ctgatggtga
100170100DNAHomo sapiens 170gccgctgaac cttgatggga
ccccactttc taaactagac gccttataga tcaggagctt 60aggggctttc cctggtttct
gctgatacca ggccaaccag 100171100DNAHomo sapiens
171ctactaatac tctgactggc ccggcaagtg atggtgactc tgtctcctac agatgcagac
60agggtggaag gagactgggt catctggatg tcacatttgg
100172100DNAHomo sapiens 172ggatgtcaca tttggcacct gagattggaa atagaaacac
aaatattcat actattgatc 60atattatagg aagacttccc tgaataacca ggcagtactg
100173100DNAHomo sapiens 173agcacactgg gctgagtaaa
ttcctagtgt tctccttcct tacctgggag ccagagcagc 60aggagcccca ggagctgagc
ggggaccctc atgtccatgc 100174100DNAHomo sapiens
174gggactattt tattatgaga aacaattttt aggtattttt ttgagaattt taaatattcc
60tcaggagccg atagagtaat gtatttcatt ggtgtatcag
100175100DNAHomo sapiens 175gattatttag gagaatattc ttgtttgtag gaaacacata
gtaaaatgtt agatggtagg 60attctcaagt cttcaaaaga ctctcataag attccgggta
100176100DNAHomo sapiens 176tattcttgtt tgtaggaaac
acatagtaaa atgttagatg gtaggattct caagtcttca 60aaagactctc ataagattcc
gggtagggaa gggggtaatt 100177100DNAHomo sapiens
177tgtaagtatt aggtaatggt gttatgcctt tgttcttact agtattagat caagcaattt
60attacagata tacaaagatg ataccgtgtt gtctccatgc
100178100DNAHomo sapiens 178atgcagcact cacagatcca ccactatcaa gaactgcagg
tctctttaat acccagagac 60taaatgaggt gcaccttatt cttgttttgg gtaccttcat
100179100DNAHomo sapiens 179ttgggtgtgt aacactgtgg
gagggtaact ataatactgt tgacagtaat aagttgcaaa 60atcttcagac tgcaggcagc
tgatggtgag agtgaaatct 100180100DNAHomo sapiens
180ctgactcgcc cgacaagtga tggtgactct gtctcctgta gatgcagaga atgaggatgg
60agactgggtc atccggatgg cacatctggc acctgagatt
100181100DNAHomo sapiens 181ctttcccctg gagacaaaga cagggtgcct ggagactgcg
tcaacacaat ttctccggtg 60gtatctgaga ttggaaataa aacagaaaag tcacccatgt
100182100DNAHomo sapiens 182aatctaaatc aaacccattg
tcttcccaga agagccagaa ttattgcttt atattgagct 60ttaattattg tattgactga
gcagagttgc caggtaacag 100183100DNAHomo sapiens
183gacttgagag ggttttcact gacatgcaaa accatcccat gttcccctca cctgggagcc
60agagtagcag gaggaagaga agctgcgctg gggtttccat
100184100DNAHomo sapiens 184agctcttctc cagagctctg acccaggcat tgatatgggc
tctggactgc agggcggctg 60ggagggacat gcaaagcagc tggggcgggt gctgggcttg
100185100DNAHomo sapiens 185cagctgcaga gacaatctgc
ctcccctttc tgctctcagc agcccatgcc caggtgatca 60ggccagaaaa ggccgttggc
tcagtctgag ggtagaactt 100186100DNAHomo sapiens
186ctcccctgcg gccacagaat ttaacccctg tgtcctcttg tctcaccatc acctagattg
60agccacagaa tgtttggtac aagtctgtta gaaacaaaat
100187100DNAHomo sapiens 187agaaggctgt ggtttcattt ttctctttct gctccaactt
gtgcccagtc agctccctaa 60atgcatgatg gatcaggttg aaaggaagag tctattacaa
100188100DNAHomo sapiens 188ctttatcttc cggatatact
tgtatttact tgttagtgat ctttcctgag ggtccagaag 60ctgtctcatt ctttgcagaa
attaaaagag taacattcaa 100189100DNAHomo sapiens
189ttaacctcag cactgtgggt gtgaggactt tcacaactgc acagataagt gagacctggg
60ctccaaatcc tcagggtagt gataccattt ccctaaagac
100190100DNAHomo sapiens 190agaagatggt tttgtccatg caggcaaaga actatttctt
gggtgatcct ctaaactatc 60cagtcttttt attctgtata gctggtatag tttaccctta
100191100DNAHomo sapiens 191ggctatatat gtatttgttc
atatttcaaa aatacacagt ttcaaaatgg aactcaaggg 60atccaaggct caaaggggtc
tccagaagac cccacaccat 100192100DNAHomo sapiens
192cccctttctg tgtcagtctt ccccagagca cagatccttg tttctgcttg aatcttcctc
60actctcacag atctgatcat cacatgcccc actctggagg
100193100DNAHomo sapiens 193acaacatgtg catgtccaat acaggaaagg aacacacata
ggagtgtagt gagaccccca 60gagatcactg ttgttagagg cagtggggcc ccagaactca
100194100DNAHomo sapiens 194ggagcagcag cgggtggaga
ccccatgggc tggccgagac aagaggactc ctcagccagt 60cctcctgacc tgagacaggt
ctcaggaatg tgcggaggac 100195100DNAHomo sapiens
195acaccgggac atacatttcc cttcatgctc ccaacataca catgcaaaca tacacagacc
60catacaggca cgcgcgagca gccatgcccc accccctccc
100196100DNAHomo sapiens 196ccaacacaca cacgtataaa agtgtgtgta tatgggcaaa
ctgctcgcat ccccaaatgg 60caggctcttt ccctagaggc gcccagtccg cggcggggag
100197100DNAHomo sapiens 197aagctcactc actggggcca
ttgactggga tccagtctgt ggccatgtca tggtttctat 60ttttgaggtt atagctaatg
agcaacatga ggttaagaca 100198100DNAHomo sapiens
198cacttttcat aaggccccag ccagcatcat aaatatgtgt gtgagcatgt tcacactcag
60gttatgtctt ctttatgtgc accctctacc acacacacac
100199100DNAHomo sapiens 199gccaagaacc acgactctct aattttactt cccagcaggt
attcagtgca taatagttcc 60tacttagaag tatcatattt gcccaaacac aaggtgatac
100200100DNAHomo sapiens 200ccaaaatgag gtaagtttcc
tgttttctca gtgagatctt ttgttgttgt tgttgttgtt 60gttgttttgt tgtcgatgtt
gttgtttttg gttttggtct 100201100DNAHomo sapiens
201ccgggtcgtc cagccccggg ccgccgcggc tgcccactac acccacgcca accgcccgca
60agcagcgctg caggggctcc gctgggcgac acgccaggct
100202100DNAHomo sapiens 202ctgtcccaca gggtgctggg gagcgactgg gcggctccgc
cgcgagcgtc tttgaattgc 60gcgccgctgc aggaaaccaa aaactcccta gcaagagggt
100203100DNAHomo sapiens 203ttcaaaaggt ttctggaaac
caccgacggt taaacatcac aactggactc ggagagagcc 60aaacggtttc cccacttgca
cctgccagtc ttcgcggcgg 100204100DNAHomo sapiens
204cgacctggca gcccaggtgc ggtcttaacc gcccccgccc ctcaccccgt acccgctcct
60atccccggag cgcaaatctc agggctggca gctgcgcggt
100205100DNAHomo sapiens 205ggaaggtttt ccccctcaaa cccaaagcgc gcgggcggat
caactcctag ctgctgccac 60cactcgatcc cctcagagga tcggcgcggt gggtccaccc
100206100DNAHomo sapiens 206gcctctcccg ccctctgcct
actgtgctgg gagactggca cagctccgtc ggccgcacag 60agtttaacaa acacgcaccc
agtgtcaaga acagtcacca 100207100DNAHomo sapiens
207ggcgcttaac cccgaagtta aagcgggcgc aatctcctcc tgggaactca gcccaggcac
60gccgccctcc gcctctaaat tcagacaatg taactcgctc
100208100DNAHomo sapiens 208caagacatcc ccgcttcccc aaggaagaga ccggtggtct
gagtcccgag gcagcgcgca 60cgccttctct gcacttgtgc acagaatgtt cttacgtttg
100209100DNAHomo sapiens 209caaacagcgt gcaagccgcc
gcgcgcggcg ggactcaagg gggagacaca tgcagccact 60ggaacgctct ttccagtcgt
ttctcctcga ctcacagaga 100210100DNAHomo sapiens
210aaaagattcc aatcctgctc cccccccacc cacccgcact atataggcat ggtcaagaaa
60actcctttcg gtgacccttt tttggagtac gggtacctcc
100211100DNAHomo sapiens 211aatgtcctgg ccgcttctgc ccgctcggag aggggctgcg
ctctaagttc aaacgtttgt 60acatttatga caaagcaggt tgaaactgga cttacactga
100212100DNAHomo sapiens 212tcccctccat ggtaaccgct
ggttctccag atgcggtggc tactggagca ctcaggccct 60cggcgtcact ttgctacctg
ctgccgcagc caacaaactg 100213100DNAHomo sapiens
213cccattgctg acatacttac tccctgagag tggctcttca tgcacctcca aggggttgct
60ctccggtcca tccagtgtct tgctcacccc ctgtggtgaa
100214100DNAHomo sapiens 214agttctccac catctccctc tccggagggt gagctgggct
gcttggcgag gggcacctcc 60cctctggggc ctgagctggg ctctgggctt tggtttctcc
100215100DNAHomo sapiens 215cagccggagc actgcacaca
tccccagtcc ccggtttctc attctccagt gacgcgtgat 60ccccacgtgc gttttttgca
tctctggcat cctcggtgct 100216100DNAHomo sapiens
216atttgcaggt tatatcctgg atggtggcac gacagcgcct ggaacacaga aggttgggag
60gcgtgacgct catcaggaag gctcttttgg ggagccagga
100217100DNAHomo sapiens 217agagtccccc agaagcccac ttggcaccct atctataaca
agttgctctt taagaatcat 60gggaactcca gaatcatttt cacaaatacc ttccactcat
100218100DNAHomo sapiens 218gattcaatta aatggcagaa
aacacaaacc ttccgttccc actggcaaac tgggtctagc 60taactgagca cagctagcac
aaggcaggcc ccctgctagc 100219100DNAHomo sapiens
219agggcaagtg gcggcccggt ccccaaggcc caggggagcc tctgcagctc cctggaagga
60cggtcaagtg aacagagagc tggctgccat ctgggttctt
100220100DNAHomo sapiens 220atgagatcac cagtttatcg taactagagg cctctcccat
ctaaagcatc tttgtaactg 60ctttcccttt ccccacactg cctacacata aagaagcccc
100221100DNAHomo sapiens 221taatttgtaa caagtcattt
gacaactcca gaagaggggc cacatccttt ttctctatgt 60ctgttgatta acaaagacaa
cattatgttt ccaacaccag 100222100DNAHomo sapiens
222tcagaccaag ggggaaaaaa gtccccatga cttcagtaat tttccatcct ttggaacaag
60gaaatataca caaaaggttt actatagaat gtaagcattg
100223100DNAHomo sapiens 223aactgttcaa gattgggctc tcacactaac acacctcttc
cttgcaactt gcacccaatt 60tgactctggt cctaggcatg ctgacctgaa atagttgctg
100224100DNAHomo sapiens 224gctgcggcaa gcaccacgcg
gtggcaggag aattcctgaa tgtccacaca caagatgaca 60tctgtcagag cgttttccat
tcgcagggtt tccaggccat 100225100DNAHomo sapiens
225tctgaagaat taaggagagt cccgcgtcgt caaatttgac cttttcccca tttaagatct
60cgaccaagtc tcctgttttc tgggagggct catctgtaga
100226100DNAHomo sapiens 226aggtgccagg ggcccttcca aactcttctc gaccacatca
cccatggtcc aggcgcccct 60ttgtcctgcc atcaacatcg agactgaagg agcgcccaag
100227100DNAHomo sapiens 227ccttcctgtt ggccactaca
tacgtgtccc ccgcttcttg cccctctctg cttgggtccc 60tgctacactg gtatcctgca
ctttccacct tgtattgcca 100228100DNAHomo sapiens
228gtttgtttcc aaggccatct ccactttgag cttgttcatg accacctcac acagcacact
60tggtctgtgt ggtggtttga ggggttctgt ctgtacactg
100229100DNAHomo sapiens 229tgctttggct gtgttggagg cgggcaggtg ggaaggaaga
aatgtattct tggggagatt 60tgtttttaga gacatgagac atggaaaata gttaagtaat
100230100DNAHomo sapiens 230aatataatat gggaggcatg
gactatcaga ggaggcaggc aggactgccc aacctcctca 60ctgggcacgt tacgctactt
cctcctgacc tctatagtcc 100231100DNAHomo sapiens
231ctatcattgc cctttcttac cttgatatcc taaaaagctg gtggtctgtc ttctctatct
60tttgtcctgg tcagttatcc taactatttt gtgtctgttt
100232100DNAHomo sapiens 232ctgtggatta gtaaacgggg tccccacccc cactccacaa
ggagaacatc tggcacccag 60aagtcactga gagaatagct gttgctttgg tagaattctg
100233100DNAHomo sapiens 233cctctgagtg gcttgttctt
ttcccagacg gagaggtctc ctgacagcag ctctcttctt 60tttctttttt tttttttttg
agacagagtt ttgctcttgc 100234100DNAHomo sapiens
234ctcctgtacc ctgtgggcct gagagaggag acaatgggac aagaagaccc agtggcttcc
60ttggaagctt ttgtgctagc tggagagaga agacctactt
100235100DNAHomo sapiens 235cctatatgcc tagcaacagt ccacactgac tggactgcaa
ccaggacatt tccagattac 60tcagtggggc ttatcttgaa ataatagttg atgccatttg
100236100DNAHomo sapiens 236ttaaatatat tatatatacc
atctaagggt cttacatgcc ttctctcatt tgatcttcat 60ggcaaaccct gtgaggtatg
accaccaacc accattttac 100237100DNAHomo sapiens
237ctcagaactc aggctcccag agtttaagtt gctcacagga gcccagaaag taagcgacag
60aggtgggatt tggttctagg tgtttgccac cagcacttta
100238100DNAHomo sapiens 238aatcaccaaa gctttctgga agctccaact tttcttctca
agatactgaa agacaggtat 60ctggatgggt tggcagggcg ggtgggaggt gggcgagatt
100239100DNAHomo sapiens 239tccatcaaca acgggtctaa
aaccagcgat ggtgagctgg gtgattttga tggaacccct 60gccatacagt ctattaatat
cataattgga gctaaaattt 100240100DNAHomo sapiens
240aatcatgatg gcaatcatga gttctggggc ttcttgattt gggccagcag acacagtctc
60agtcactagt tctccgaatc agagaaagga tgccttcagg
100241100DNAHomo sapiens 241ctgtgtcttc acatggcttt tcctctgtgc gtggtggaaa
gagagagctc tgcgggtctc 60ttcttgttgt aaggacactg gccccattgg attagggccc
100242100DNAHomo sapiens 242caccacatga cacatttaat
cctaattacc tccctcacag ccctatttcc aaacagggta 60ttagtcacat tagggattag
ggcttcaaca taggaattct 100243100DNAHomo sapiens
243gggggcacac aattcagtct ataacagagg gaaaacagat ttgagaagaa aaaagtccaa
60aatatgcaca gtggtaatat ctgaagatgt gcgtgcgtgc
100244100DNAHomo sapiens 244tcaagggctc agcaaacgac aacttaagca tttagagtcc
catccctatc caccaaaccc 60agaataagtt agtcttttca agaaagcatt ggtataaaac
100245100DNAHomo sapiens 245ccttcaaaac tgaaaagaag
aaaggggcaa ttggagaatt cccacttttt ctggctgtct 60ccttcaagtc gcccagtttt
tatgaacagc atctagcctt 100246100DNAHomo sapiens
246actgtcacta tcaacaaccc ttaaaactag ccaatgcttc ggcctctagt attggaaagt
60cttccaaata ggatactgga aacttctatt tataagcttg
100247100DNAHomo sapiens 247gggtggcggg cggggcgggg aggtggagag agagttgcca
tctacaggtt tctattttgg 60cctgaagact caactgcagt cattagagta agggaatgcc
100248100DNAHomo sapiens 248ttatttatta aaaccacaca
caccttgcaa agaaaaaggg aaactggcag tctctgtaga 60ggaagccggt ggcatcgctc
agagccacaa actgtatttc 100249100DNAHomo sapiens
249taaacagccc tttccctggt tccctctctc ctgccccact ttttttaaaa tccagactgt
60aaaaaacaca tctactgaca ctcactttac tttaaaaaaa
100250100DNAHomo sapiens 250gaagagaaaa agtaaagcgt tacaagactt tcctcctgga
aactataaac tgaaaaaaaa 60atccataaaa gattaaatcc tggcgggttg tggggtggcg
100251100DNAHomo sapiens 251ggggccggcg gggagggggc
gcggagtgga gattggctct ctgaggtggt caggggccct 60gtgacagctt gggactttca
gcacctggtt tggggtcatt 100252100DNAHomo sapiens
252tatctgctca actgtcagga ccccccaccc ccaaacccca gccaccaaca caaccatcgt
60agaagggaac acaacacaga gggtcttttt tcattttttt
100253100DNAHomo sapiens 253tttttaaaaa atcggtttgg ttgtgttttt gttttccatg
ggggagcttt aaaactcatt 60attgcaacac tagttccatt tttcgccagg gttccaataa
100254100DNAHomo sapiens 254caagacattt accacggtca
ctacatccgg cagcggggtg gcccctagct cctgctgccc 60ccccgccctt tctccccgcc
cgcccccgga gctcagccga 100255100DNAHomo sapiens
255tttctgaggc tccaactcta cccactccct ccccgggccg ccgccgccgc gccttccccc
60attcttactc cctcgaggag agccacaggt tgcaaatcca
100256100DNAHomo sapiens 256accaacctcg caatctattt ttgcaaaatc actcacaaag
atctcccttt cgcgcccgcg 60cccgctcctc ccgcgccggg tcccctcagc cacggccaca
100257100DNAHomo sapiens 257aagtgccctt ctctcctcct
gagtcttgca cataaggaac gcgggctggg gctctgttcg 60tctttctcct cgcccaaggt
aaggacctcg ggaatctgaa 100258100DNAHomo sapiens
258gcctggcgtc cactacgctc aggcccgcag ttcccttttt acagagcttg caccatggga
60aaaaataaaa taaaatttag gaaagggagg caacagccat
100259100DNAHomo sapiens 259taaaatttag gaaagggagg caacagccat tgggagccaa
cacagagtca cgcagcgccc 60aaaatacaaa caccgcagcg gccagaaatc ccgccacctt
100260100DNAHomo sapiens 260tctcgttctc ccaggctgtc
ctgtcgaggt tccctgagtc cccccgcaca ctgaaaggca 60tcgcaggtgc agtgcgcacc
cctttcccac ccaccccaag 100261100DNAHomo sapiens
261aagccctgtc ccgccatcag tctctctcct cgggatgagc agggagagcg cgcggaggtt
60cccgactccc tcgactacaa ccaagaaaga ataattttca
100262100DNAHomo sapiens 262aagtgttcaa catccccgcc cccaagctcc ccaaaacaca
ggggcaggga acaccaaaac 60actcggctct cattaggaag atcacggctc tgaaaggaaa
100263100DNAHomo sapiens 263tagtagacac gatacttcat
ctcatctgga tttatgacca aaaaaacaaa aacaaaaacc 60caaagagttc gcttgcattt
tttccttcca aatctcggtt 100264100DNAHomo sapiens
264aacaaaaacc caaagagttc gcttgcattt tttccttcca aatctcggtt cggctcgaag
60gcagggaatc taaaagaccg aggccgatgg aagagagcca
100265100DNAHomo sapiens 265gcggggcgag cgagcgggca gcctcccttt ttgcctcccg
gagttaccca gaaggacagg 60ggaagggaag gaagaagagg cgaggaaaaa gaggagggag
100266100DNAHomo sapiens 266ggaagcggag gccaggagcg
acggagcaag gaaagcagtt tgcaagcgag aaaagaggga 60aaaaacacag ccgcacgaat
ccagagagat cacaagccgt 100267100DNAHomo sapiens
267acgcaagcag cagcagaaag agcgagagcg cgagcgcgcg tcctctccgc ggtctggggc
60cagacagccc ccagactagc ccgaatcacc ccccaagcac
100268100DNAHomo sapiens 268tgtctcgtcc tctctgctcc ggccgccccc taattcccct
ccttcctctc ctccacctcc 60tttccaaaaa ccaaaacaac acaagggagg gtggcaaaag
100269100DNAHomo sapiens 269cctccccaaa ccggccgatt
cactcaaaga caacaataat aataataaat acataacaat 60ctatatccta tggtgggaga
gacgtgggac taatcttcgg 100270100DNAHomo sapiens
270acataacaat ctatatccta tggtgggaga gacgtgggac taatcttcgg catttatttt
60aacacctgac agctagaata aataaatata tacatttata
100271100DNAHomo sapiens 271aataaatata tacatttata tcaatagata cacatagaaa
acttggagcc aaagcatttg 60gcaagagcgg aaaaaaaaag aattaaaagg taaaataatg
100272100DNAHomo sapiens 272atcatgagca gcggcggcgg
cagcggcacc agcggcaaca gcggcggcgg cggcagtagc 60agcagcagcg gcggcagcaa
cagcaataat cacctggtgt 100273100DNAHomo sapiens
273ccggcctttc ctagaaactt cttgcatcac cacttctaag aaccccagtt ctaagaatca
60acagagctca attctcggaa tttgagcttc ggactttacc
100274100DNAHomo sapiens 274actgctacgt ggcaggggag gacttggtgt cagctctccg
agatttttac tgcccctggc 60caaccaaaag ccctcaaagc cacaagattt tttcactggc
100275100DNAHomo sapiens 275cggcatattt cgaggtcctc
ataagcagag cgtctcggat ttggaggttc cggttcgagg 60ctcgaggggc ctgaaggtgg
ctctccctcc ccgggcccaa 100276100DNAHomo sapiens
276gacgatggta tggcctgctc cgccaccatc acgtgggctc ctcctctgtg acgtcggcgc
60cttcgctgta gcaaagctcg gcctctggaa ttctgagaac
100277100DNAHomo sapiens 277gcacaaaagg gagcgagagg tttgaaccac tgggaaaagt
atgttatata tatagtaggg 60ttagagaggc gagtaagaga aaaataaaat aaaataaaca
100278100DNAHomo sapiens 278aaaataaaat aaacatcaca
gctctttcca actagaatat taggcaccac gagaaaaata 60tttgccaagc agttttcggt
gggttcattt gctttatttt 100279100DNAHomo sapiens
279tatttaggac aggggttttt gctgttgttc tgggtttttt tctttctggt gtggtggctt
60gggatttttg gtttctgtat tttgatggtt tatggatttt
100280100DNAHomo sapiens 280tgcttctgat tttttgcctt ttgcaagttt gtggtgttac
gtaaatcaca ggatcggcat 60cggttggatt tttttgtacg tgccttttct ttccctatct
100281100DNAHomo sapiens 281aatccctcaa gcgttttaaa
gatgtattat ttcaatacta atactattga aagaagctta 60aatttttggc catatgtaac
aatcccagcc cccacttttt 100282100DNAHomo sapiens
282attatcatca tcaccaccaa catcctctgc cctggagacc aagagaattc aaacaggtca
60gcacctctaa ttgctgtata gaacattgac cctactgtct
100283100DNAHomo sapiens 283cccagttcct gaggatggtg tgataataat acatctcaga
gttctgtagt ttcttcacca 60ctgtgcaggt gtggttggtg ggagcaatgc cctggatgga
100284100DNAHomo sapiens 284taagccaagc tcttgtgtcc
tggcagataa acaaggtgaa ccctcaatcc gtgtagcagg 60agtttccaga caaactcact
ttgcatggaa ggacactaac 100285100DNAHomo sapiens
285ccttccaggt gcatggaaat attttgtagt ttttactgtc tcccccttcc tccactgcct
60catctttttt gttttttccc ctgtgagact atttgctctg
100286100DNAHomo sapiens 286cctttccaac actggcctgc cttagggact caccgtctgc
actccgcctg cacaggtgga 60actgagttca gatgagggag aattgctttc cattgttcag
100287100DNAHomo sapiens 287taggcttttt gtaatttcta
gttttgctta cctttcctac tcaccacaca cacaaaacag 60tgtgagcttt ctcattctag
tgcataaaca caggtcggtc 100288100DNAHomo sapiens
288aatacccaca agtgttccaa aaggtgagct ggcattgctg cccaactggg cattatagtc
60ccttctgtcc ctgcccatca ggcttgcctt cctcggcaac
100289100DNAHomo sapiens 289ctttctagct tgaattgtac tgtgactcct tctcacggac
cactcccgga gactggtgaa 60agttgggccc attcttgaag cctctgcttc taaatcatgt
100290100DNAHomo sapiens 290tttccataaa gtctccctca
tcgtgcttgc ttccaccttc tcctatttgg aattactggt 60gggctcttcc actgtcccat
agcaagtgtt ctatacattc 100291100DNAHomo sapiens
291tgaaggcaca tttgaatata tactttgtca tggttgcttg gaaccatgtc gtcttttcca
60agtaggctgt gaacattcag tggcatggat cataccgtgc
100292100DNAHomo sapiens 292cccattgttc aaagaaaggc attatggagt ctccaaaagc
cattggcagg tggtgtctgt 60gacttcctta gcctggaaat aaacaaataa acaagcacaa
100293100DNAHomo sapiens 293aaacaaataa acaagcacaa
attagaagtc tttgccctat tactgcacta ttagtattga 60ttgcgcaaca tcatgcaaaa
agtcacttta atttatctgg 100294100DNAHomo sapiens
294caggtcctat gtaaacacca atacagtcaa gagggcttgg atgggtattt gctttcattt
60ctaatgaaat ttcaggcctc tagggtagga tatcaaaatt
100295100DNAHomo sapiens 295ggtagatcat ttgcaattta ttttatccca aacacctcac
tttacagtca gagaaactga 60ggcccagaga agtaaaatga gttgctcaag gtctcagaga
100296100DNAHomo sapiens 296actgaggccc agagaagtaa
aatgagttgc tcaaggtctc agagagcaag aaatagagat 60gggacttgag cacctagatc
tctggtattg ctgtcctgta 100297100DNAHomo sapiens
297gttcatggag ctggcagatg gatacatctg tgacctggga tgatggagag actgctggac
60ccttcagagg atctcatctc aaggtggggt ttatgtgtaa
100298100DNAHomo sapiens 298atgatatctg tgtgtttcat tttcctttca taaactaatt
taaaaatcct tttggtatca 60aattttaagc caaaaagtag tgagggggaa catgggtagg
100299100DNAHomo sapiens 299aatagcttac agcttgccta
acaaggttgt tgactgcata agagtcagga gttttgggta 60agagtgtgtg tgtgtgtgtg
tgtgtgtgtg tgtgtgtgag 100300100DNAHomo sapiens
300cgtactgaat ttgactgctt tattttgtag ggaaggaaac tgatgtgcct agagtagttg
60agagctttat tcaaactcat tccactgtta ttgagtagtt
100301100DNAHomo sapiens 301aggatattag accagcaaca tatttgggta gaaactttca
tataaaaaag cgtaatcata 60actatccaat catgtcaact agtaaggctg ctcaggtggg
100302100DNAHomo sapiens 302ataacacatc aaccttcttt
gggattcttc cctcagacat ggttttggtg ggaggagcat 60ggcaagggag gggcgagctc
caaatgcagg gctgctctgt 100303100DNAHomo sapiens
303cctcggcgac ctgagcagac acacgagcag agatcagaga cactcttagt gaatgaacct
60ccctattggc tatattaaag taatgctctg aaaaagttcc
100304100DNAHomo sapiens 304tatgtatgca tagtctaaag tgatgatttt agaggtagca
agacagtgag aatgtcccta 60catgtgaaat gggcacagtt ttatcaggga agtgtcaata
100305100DNAHomo sapiens 305gagggttaat gttccacgta
gtggctgcaa gaatgataag tggtcatggg gatagcctga 60cactctagga gcagaaggtg
gtgggtatgg atagaactac 100306100DNAHomo sapiens
306tgatatagca tgaatccaac ctgctgttat ctgcgcaggc ctctctgcag ctgtttgccc
60tgaagtacat gctgtacgtt tctccagctg atcctgcatg
100307100DNAHomo sapiens 307actgggtata aacgcctgtc cgctgtgtgc tggacagccc
cagacaccct cggcagcctg 60ctgtgtttgt gtgagacatg ctgtgttagg gatttaagca
100308100DNAHomo sapiens 308acagctttct catctacatg
gacaacctat ttttaaagaa tcttcagaga gtcgttgact 60ttgttataac tactactata
tacgtaattt cagatgatag 100309100DNAHomo sapiens
309aattgaaaat ttaacttgtt tttctagaaa gagtttattt tccctataac ttcaaagagt
60aatggtgggg agtaggacat tctgaaaata agaagaaaca
100310100DNAHomo sapiens 310tgtcaaatga atttctgact tccagctagg catatggaat
aaaggtcttt attccagtga 60cctctgctca ttggaaaact ttgggctggt agatttcatg
100311100DNAHomo sapiens 311tctcttgcat tcttaacttg
caatttagta ctgtttatat tctgcttgaa ggttagagac 60attcgactaa atggtctttt
ctccacattg ctgtcattca 100312100DNAHomo sapiens
312ttaatgtcct ggtcctggac tttactcatt gaccacagga caagtggctc aactctctcc
60tgccactacc caggctgtta gtcctgttgg gaggctcagg
100313100DNAHomo sapiens 313gcccaactca ctcatctgta actctcatct ccattcagct
gcagcctcta cagcccctgg 60ttataccctg gatcttatca ttgcttcgct ctattttacc
100314100DNAHomo sapiens 314tcctaaatcg taaaaattaa
aaccagcctc ggaacacaac ccctcattct tccagcactc 60tctctcattc aggtaactcc
tattctactt ttcttcagca 100315100DNAHomo sapiens
315ttgttttttt ttactttacc ttaatttctc tttttggact aagatgttaa aatgtttctt
60aatgtgactg tctccgaaac tgttttgtgt ctaccactca
100316100DNAHomo sapiens 316tcctagtggc agtcattgat ccttttcttg ttgcgagtgt
ttgagtgtgg gtgtgtgtga 60gtgtgtatat gtatttgtag agggaaaaac aagagagagg
100317100DNAHomo sapiens 317tgtgagtgtg tatatgtatt
tgtagaggga aaaacaagag agagggaaac agacattgga 60gccacctttc ccccactagc
cacgtacctg ttgaaccttc 100318100DNAHomo sapiens
318aagcctctct atagaatcag atatacacaa gcacagtgac agaactacat gtgtcctaca
60gtccagcttt taagatatga taaaaactct tgtattcaca
100319100DNAHomo sapiens 319gagctaaatg gcaataacca taggagattg catattgcta
cattatgtaa agacagagtc 60ccaagaaaat agtgagaact cagtttgatg tatgatgtga
100320100DNAHomo sapiens 320tatgtgatat cttactttac
atggctaaca gttgacattc tttgtggatt ctatattgtc 60taaggctaca gaagagccat
atgataaatt catcggcaac 100321100DNAHomo sapiens
321cagtgaaaag gcttgggccg cttttgtttt cacctgcttt tgttgaacaa atttgatttc
60cggagtcagt cattttactg tcaagacatt tcttcggcat
100322100DNAHomo sapiens 322tctgcaacag gtaaggattt tgcttcctta aaagtatttc
tttggtgtca aaagaaattt 60ttctaatttt atttagcttt tactctaggc caaacatcgt
100323100DNAHomo sapiens 323aatgactctg agctacctgc
tgtaaggtgt agaatcaatt tacaggggga cgggggtcgg 60gggggtgagt gttgctttga
tattcactgc ccctcaccac 100324100DNAHomo sapiens
324agtcctaaca agatttttga aacatgaaaa gttacaatag ttggcttttt ggttttccag
60atattctaga gaatgcatat gcttgtgact gtggctgagc
100325100DNAHomo sapiens 325tcaactgtat gggtagttta aatactaccc aaggtttgat
gaagtaaatc taaagatgct 60ctaagttgtg caaatatgaa ttttaaagtt gtctagttca
100326100DNAHomo sapiens 326gaaaagaaac agaaccgaag
tctaaatgat gtagatttca atctggaatt tctagcttgt 60gtttttcacc tattgccaat
gttaatgacc atttcccaaa 100327100DNAHomo sapiens
327agtgctctat gatgtataac atgtattttt taattaaatt taatctttct tctgaggtgg
60tttgatttgg agatatgcta cgaggtacca gtcagtagcc
100328100DNAHomo sapiens 328tgagttgtaa ctaaacaaag tttgggaaat caccggtttt
aggtgcttta ctaaatgaaa 60gttgccattg acgtattcaa gcaggcaaca agtagttggt
100329100DNAHomo sapiens 329gtccccttat tggttctaag
ctggtgccgt ggaggatata agagaaatat tttaaaaatc 60tctactttga aggaccctat
aatctggtag ttgtgataag 100330100DNAHomo sapiens
330tttaaaaatc tctactttga aggaccctat aatctggtag ttgtgataag aagtaaaatt
60taggaagcaa tgcaagatga gaattcagtg atgagtgggg
100331100DNAHomo sapiens 331cagcacaggc ttgaagagtt ctgtgaattc catggagggg
gcctgggggc aaactggagt 60tgtcaggaag atctgggctt tggaagaatg cgaagtgtcg
100332100DNAHomo sapiens 332gtagaaggag aaggggcagg
tgatttcaga ctgggaggac cttgtgggca aaggcacaaa 60ggcgagactg acctggagat
gataaggcca gttgaagaga 100333100DNAHomo sapiens
333acattgcagg aaatcagatt agacagttag ggtgtggaca caaaagcgag gaccttgcag
60gcactgggga gaagtgaccc cattcaatag tccttggtct
100334100DNAHomo sapiens 334ccttctgccc tgcggctgcg cttcctcggc tctcacggca
ccagcagaat tccatgtgag 60agggagcttg tcgagcgtgg cctcttccca cttggggctg
100335100DNAHomo sapiens 335ctttctgcat ccctgtgcct
ggctgtgggc ctccatttgc cctctactgt cttcccttag 60gacatcattt atgcagagaa
aggttcgtgt ggctcggggt 100336100DNAHomo sapiens
336ggacgttgtt tagagagtca gtagatcata ataattcaga cacttttttt ctggaccata
60aaatatctga acccatataa taacaaacat acagcacggt
100337100DNAHomo sapiens 337gaataagaac ccaacttttg agccagatca ctttgcatgg
aatccccatt ctatcattct 60atcatttctg ggctgtggga acctcagaca agttacttaa
100338100DNAHomo sapiens 338cttcttcaat gctcagatta
aaaaaaaaat tcacaaaata tctctaataa cagtaataat 60aactgaaaat acctacctca
gagggttgtc gtagagatca 100339100DNAHomo sapiens
339aaaattcaca aaatatctct aataacagta ataataactg aaaataccta cctcagaggg
60ttgtcgtaga gatcaaatga gataaaaata tgtaaagcat
100340100DNAHomo sapiens 340gtagcctagt gcctgactga aaaaaaaatc tctcaataga
tgcaactctt atgattctta 60ttaaggactt ggctattgcc acaaatgaag gtgttatgag
100341100DNAHomo sapiens 341ccctggctta agagcaagaa
gcctgcaaag ctaactctcc taatcccaac attcctttcc 60agggaaagta gggtgacagg
tggaggctgg gaattaacgt 100342100DNAHomo sapiens
342tttttgagca ccaaatatgg acaaggcaca ggggttgggt gtttttctag tgagaataca
60tatgaaagaa ggaaaacaaa cttggaaacc gctattttaa
100343100DNAHomo sapiens 343gccatttggt aacagtttct ctagcttatg agatgagaga
ggtcctctca gtatccgctg 60cattacttgt gggcctcctt ggttgacgtc gctctctgaa
100344100DNAHomo sapiens 344cgcttggggt ggaattctag
aggtgctttt cattagaggc agagagcatg acctttcttc 60cttgcccagt ttaaattaaa
ttattttatc ttacaatgtg 100345100DNAHomo sapiens
345ttaattttag tgctagcaag gcacagctaa aattccattt ctacttagga gtggggatca
60ttgtggcagt gagtgcttat ttgggtttgg gatgcttgga
100346100DNAHomo sapiens 346tctgggtgaa agccaggatt aaaaagcatc ctccttcccc
attccactct ctaggttata 60aatatttttt tggattaaaa gcctccttta aaaaaatgca
100347100DNAHomo sapiens 347aatccacctg gcatgttaat
tgtgcagggg attcctaatt atgtgtgcag atgacgtgag 60tcacacggtg atagtgttcc
ttctagagtc ccactggtgt 100348100DNAHomo sapiens
348actaggcgtt catcctgtgt aatttgaaaa tatgtcacac gtggtgatga gaatctattt
60gaggaacatg ggcagtttga aataatatat gcaatgtatg
100349100DNAHomo sapiens 349actagtttat ataatgaaag gaagtattta aaaagataga
atgacataga ctaatctaat 60tgagaaatat gaaagtctaa cagaaatgat tgcttgtgaa
100350100DNAHomo sapiens 350attttatgaa gaaatccaca
gataaattct ccaccttgat ctatgtaatc cgaaatttag 60atgttaaaaa tatgttgatt
ctgaaaattt atatttattc 100351100DNAHomo sapiens
351tttggtatga ataggtcaaa acaagtcacc attaactgac aggaagcaca gaattctcaa
60tttagttttg gcaaagacat tattttataa atatgagttt
100352100DNAHomo sapiens 352ttaaatgatt cttatgaaga aactagcacc aaagtgaatg
cactctgcaa ataactccca 60gcttctctga atttcaaaag cagccactaa atattattag
100353100DNAHomo sapiens 353caaatcaatt tagctgaaag
cgatgaatta cagaagtaaa tctttaggta caaagtagac 60agctgacaca catgtagcat
atacacacta gtgatctgcc 100354100DNAHomo sapiens
354ttccttcttt accaacatag agtttcccat gagccctgaa tccggggcac ttttgctaac
60ttcccctgca gcggcgacgc tgccactccc agtgcccccg
100355100DNAHomo sapiens 355cagtggaagg ggctcgcgcc acctccattg ctcttggccc
caaagccata gaggtgcccc 60ccggaagggg cctggctgcc actgccattc tggtggccct
100356100DNAHomo sapiens 356gaagcaggtc gtgcttgtcc
ttcctggatt tccccgcatc cttatcccgc ttggcgcctc 60ggctgctctg gcttttacct
ggcttctcct ctttgctttt 100357100DNAHomo sapiens
357cccacaggag cctgcccccg cggtggcggc agaggtgctg gtgctggtac tattgctgtt
60tgggttgccg ctgccgccgc tgctcacact ttgacccagc
100358100DNAHomo sapiens 358gctgaattca tgccagttgc ctctccaggg cgcccttgga
cttcctgcct cttgccagtg 60ctgctgatct cgggaatccc atacaaggca gcagaaggca
100359100DNAHomo sapiens 359gagatttatt agcatcctta
gaagttttac tccttttcac ttttgatttg ctggtctctt 60tgtgtgaatt cccctgggga
gcagaggcct gaacagaagc 100360100DNAHomo sapiens
360aaattttagg ccatcagcta aggctgcggt agcaccagcc ccactggagg ccggacctcc
60acaatccttg gagttgctgc tactagtggt ggtggtggaa
100361100DNAHomo sapiens 361ttattcatct caaatttctg tctgtccttc tccaaatcag
cgtccaaatc aattattaaa 60tttccaaccc cgatttccca atcatcgcca ctgtcataag
100362100DNAHomo sapiens 362tatcaactgt atttggatcc
acaccttttc ctgcagtaga aatgttcact gacatcctga 60agatgagctc tctagaataa
aaatccgatg aacttttctt 100363100DNAHomo sapiens
363ttcctcagga atttgagctg gggatctgca tcctggccat tgcagtcctt tagcatcctc
60gccgcgccct gagcgcgctg gaggctcgca ggctgcgccc
100364100DNAHomo sapiens 364tcccagggct gatgccgcgt cctgctccgc cgttctggga
cgtcggggac aaaagtggag 60gagacgggag agcccgggca gaaaaagcag gacgcgcgtc
100365100DNAHomo sapiens 365ccaggtgccc acctcttcgc
tttgaggcgg gggcggtggg atggaatatg ggtgcgcgag 60gtcggggctg gtaactctcg
gaggggcacg gcctccacgc 100366100DNAHomo sapiens
366tgggagggat gaatggacgc tgggccccgg caaatgaggc gctgtgggtc cccaggaagt
60ggggtaccag gctctactcc caccccggcc tctgaaacgc
100367100DNAHomo sapiens 367ggccaggagg ggtggcggct gggtggggag agagggtgca
agacgagcgg cgcgtgtcgg 60gagcctttgg gctgcgggtg cgttacagga gagcaggcgg
100368100DNAHomo sapiens 368gtaggagcct tcgcgggggc
cgagctcgga aggcggacgg ctgtgcccgc ccaggggatg 60cgcccgggcc ggccgcgaag
gtgccttctt ccgggggccc 100369100DNAHomo sapiens
369ggacgaccct gacacggcac gcgcgcgctt cgcagcctca aagactccgg ggcctcgtgg
60tcactggcgc aggggatcgg ggcggggtgc ccggagtgcg
100370100DNAHomo sapiens 370cccgcagtgc agagcagagc gggcggagga ccccgggcgc
gggcgcggac ggcacgcggg 60gcatgaacct ggagggcggc ggccgaggcg gagagttcgg
100371100DNAHomo sapiens 371catgagcgcg gtgagctgcg
gcaacgggaa gctccgccag tggctgatcg accagatcga 60cagcggcaag taccccgggc
tggtgtggga gaacgaggag 100372100DNAHomo sapiens
372aagagcatct tccgcatccc ctggaagcac gcgggcaagc aggactacaa ccgcgaggag
60gacgccgcgc tcttcaaggt ctccggcctc gggagccggc
100373100DNAHomo sapiens 373cccgcgcgcc acagctctgc agctcgtggc agcggcgcag
cgctccagcc atgtcgcgcg 60gcctccagct tctgctcctg agctgcggta gggctcgcga
100374100DNAHomo sapiens 374gcgcctgtct cgcctgtcgc
cccccgcccc tccacgacac cccctcccgt cggtcgcttg 60ctcacgacgc gctctctctt
tcttgtagcc tacagcctgg 100375100DNAHomo sapiens
375ctcccgcgac gccggaggtg aaggtggctt gctccgaaga tgtggacttg ccctgcaccg
60ccccctggga tccgcaggtt ccctacacgg tctcctgggt
100376100DNAHomo sapiens 376caaggtaggt gctgcgatac ccacgggctg gggtttggtg
ggctcatttg aagacagcag 60gaaccatctc ccctaggctg gcgaccctct gtggctgcca
100377100DNAHomo sapiens 377ggtgggggcg aggggcgtct
cccgcagctg aacttggagt acccagcctc ccgtcgcgcc 60tcccccaccc catccgcatc
caggtacagg gccgaattag 100378100DNAHomo sapiens
378gttttgctct ccgcagacct caatcccctt cctgtcactg aaggtggcct gagatgaatg
60atccacttaa gatgttttgg aagggcagag actctcattt
100379100DNAHomo sapiens 379ggattaattc tggaggccac ctgtggttgt gggccagcag
gtcaggaaga aagcaacagg 60gacctagatt tgggcattgg acagggggaa tgtctccaga
100380100DNAHomo sapiens 380ctctccagtt cctatattct
aatacccctc cgccgccaaa taaaatttgg cgtctggcca 60cagctctttt agtgggtatc
tgggtggctc ttaaaagagc 100381100DNAHomo sapiens
381ctttggggtt aggtgttaag acgcttactt ggaatgttta cttggagctg gtgtacttgg
60tgacggcctt ggtgccctcc gacacggcgt gcttggccag
100382100DNAHomo sapiens 382ctccggcccc tgccgagaag actcccgtga agaagaaggc
ccgcaagtct gcaggtgcgg 60ccaagcgcaa agcgtctggg cccccggtgt ccgagctcat
100383100DNAHomo sapiens 383tactaaagct gttgccgcct
ccaaggagcg cagcggcgta tctttggccg ctctcaagaa 60agcgctggca gccgctggct
atgacgtgga gaagaacaac 100384100DNAHomo sapiens
384agccgcatca agctgggtct caagagcctg gtgagcaagg gcaccctggt gcagaccaag
60ggcaccggcg cgtcgggttc cttcaaactc aacaagaagg
100385100DNAHomo sapiens 385cggcctctgg ggaagccaag cctaaggcta aaaaggcagg
cgcggccaag gccaagaagc 60cagcaggagc ggcgaagaag cccaagaagg cgacgggggc
100386100DNAHomo sapiens 386ggccaccccc aagaagagcg
ccaagaagac cccaaagaag gcgaagaagc cggctgcagc 60tgctggagcc aaaaaagcga
aaagcccgaa aaaggcgaaa 100387100DNAHomo sapiens
387gcagccaagc caaaaaaggc gcccaagagc ccagcgaagg ccaaagcagt taaacccaag
60gcggctaaac caaagaccgc caagcccaag gcagccaagc
100388100DNAHomo sapiens 388caaagaaggc ggcagccaag aaaaagtaga aagttccttt
ggccaactgc ttagaagccc 60aacacaaccc aaaggctctt ttcagagcca cccaccgctc
100389100DNAHomo sapiens 389tcagtaaaag agctgttgca
ctattagggg gcgtggctcg ggaaaacgct gctaagcagg 60ggcgggtctc ccgggaacaa
agtcggggag aggagtggga 100390100DNAHomo sapiens
390ctccttagcc agactcgatt acaagcactg catgcattac tcagtgtgat aagatcatga
60taatcccttt aaaaagatcg cccgaattta agcctggatt
100391100DNAHomo sapiens 391aggaacacgt gtttacagct ctaatatcga taatttaagt
ggctcttaaa agagcctttg 60gggttgggct ttaagacgct tacttggcaa gtttacttag
100392100DNAHomo sapiens 392cgctggtgta cttggtgacg
gccttggtgc cctcggacac ggcgtgcttg gccaactccc 60cgggcagcag caggcgcacg
gccgtctgga tctccctgga 100393100DNAHomo sapiens
393ccccggctcc ggctcctgcg gcagctcctc tgggcaccgt ccctgcgccg acatcctgga
60ggttgggatg ctcttgtcca aaatcaactc gcttgcccac
100394100DNAHomo sapiens 394ctgcgcgccg cgccctgcaa cgacctgcac gccaccaagc
tggcgcccgg tgagagcacc 60ccccgcctcc ggcccgggga tgcggggcgg cggcgggatc
100395100DNAHomo sapiens 395tcctgggtgg ggagctggcg
gctcgcgggc cggcactgag tccccgtgct tccccctttc 60ctaggcaagg agaaggagcc
cctggagtcg cagtaccagg 100396100DNAHomo sapiens
396tgggcccgct actgggcagc ggcggcttcg gctcggtcta ctcaggcatc cgcgtctccg
60acaacttgcc ggtgagtggg cgccccgcgg tggggagggc
100397100DNAHomo sapiens 397gcgccgggcg gggggcgcac gggcgtgctt tagcccggac
gagggaacct gacggagacc 60ctgggcttcc aggtggccat caaacacgtg gagaaggacc
100398100DNAHomo sapiens 398ggatttccga ctggggagag
ctggtgagtg ccctgcagga gcgaccccca ggatgagtgg 60gtggggtgag gggcgccccc
gactcccgcc ctaacgcggc 100399100DNAHomo sapiens
399cccctcgccc ctgcagccta atggcactcg agtgcccatg gaagtggtcc tgctgaagaa
60ggtgagctcg ggtttctccg gcgtcattag gctcctggac
100400100DNAHomo sapiens 400tggttcgaga ggcccgacag tttcgtcctg atcctggaga
ggcccgagcc ggtgcaagat 60ctcttcgact tcatcacgga aaggggagcc ctgcaagagg
100401100DNAHomo sapiens 401agctggcccg cagcttcttc
tggcaggtgc tggaggccgt gcggcactgc cacaactgcg 60gggtgctcca ccgcgacatc
aaggacgaaa acatccttat 100402100DNAHomo sapiens
402cgacctcaat cgcggcgagc tcaagctcat cgacttcggg tcgggggcgc tgctcaagga
60caccgtctac acggacttcg atggtgagcc aggcccggga
100403100DNAHomo sapiens 403gggagctgcc caggtgactc ggcccggccc ggcccagtcc
ggaggcctcg gccagtctcc 60cgcgccagcc ttttgtaaag gtcattgggc cgcctggctc
100404100DNAHomo sapiens 404gatgctagcc ggggtgggac
gcaggagagc ctcccagcgt agtaaagccg gggattttca 60gccagctgaa cctgtaatgt
ttctggcatg attttattct 100405100DNAHomo sapiens
405tcaagtggaa ttcagttagt tccaggcttt cccgatgaat aagaggttgt gggcaaccgg
60cggtagccca gatttttcta aagtctgacc cagtttcccc
100406100DNAHomo sapiens 406ctctaaacag acaaaagcaa aatatctcat taggcatcat
ctccgccaag gttcccacta 60ggcaggaaag gatttttatc taaagtaatt acccttttta
100407100DNAHomo sapiens 407gttaaataca ctcaacagat
gaaatttaca gagagtgaga gactgcagca ctagacagcg 60aaggtgaaaa ccaggaacgc
cgcgtctcgc cgcccgcggg 100408100DNAHomo sapiens
408cccgccggga gactgcgggt ccgtctcgcg ggtggggcgc cccggtccct ctcgtttcct
60ggaggccaca ggtcacggcg acggcggtga ccgggagagc
100409100DNAHomo sapiens 409cgggtctgac agctgctgcg gctcgcgcgg acgcgcgcct
cctgcagccc gccctcccca 60tgcctgactt attactctct gctcctcctc cctctgctgt
100410100DNAHomo sapiens 410tccaaaacac ccttcgacgc
cagcaaaata caatgcgcct cggccgccgt aaacagccgg 60gagggagagc acacattcgg
cgcggcgcgg ccgccggctc 100411100DNAHomo sapiens
411ggctcccacc cccttcccgt tcctagaaaa tgccataaaa gcgggcaggg cgcggggagg
60gcggctgcgc gcccggcggc cggggctccc ttcccgcgcc
100412100DNAHomo sapiens 412tatgaaacag ccagtgctac gtctccttta taccaaaact
ggtagcctga agagctctca 60ggcttaccta taaacgatgt tcagtgaatg caggtagccc
100413100DNAHomo sapiens 413aaggcactgg ctatttcagc
agcatagaaa cgagcccgtg gttccaggaa gcagcgttcc 60ctctggagat ggtagaacaa
ctgcaggaga cagaacaaag 100414100DNAHomo sapiens
414tcattctggg ttgcaaatga atttaattag ttttgacata cacagcaaaa gaacaactgc
60aggaagtggc cccaagtaat ctattaacta taaacctgac
100415100DNAHomo sapiens 415aggttgaagg aaatgctaat tctggtaaca ttctccccac
caaaaatctt tgaaaacttt 60tttctcaaac taaaacaaag caggctgtgc agagacacta
100416100DNAHomo sapiens 416agagttgact tctatccccc
ctgctcacct ctccaccatt aatgtagtct aggacaaagt 60acaatttgtc agcagtctgg
aaagagaagt gaaggcccac 100417100DNAHomo sapiens
417caggaaaggg tgcttcacat tcttcaacag aacattccgc tccgacataa tatgcttctc
60ctaggaaaat gacgattcag atttagtggc atgtttcaac
100418100DNAHomo sapiens 418gaggacatga aggaagtgta ccaaaagatc ttcagatttg
aaattacctt tccaaaactg 60ccctttccga tcactttcaa gaagtgaaag tcagatggtt
100419100DNAHomo sapiens 419tagcatgagg attggacgac
gggccaaggt tgatttgctg agaaggactt ggctagaaaa 60aaaaaaaaag aatttctttt
aataccattg cttcaaagga 100420100DNAHomo sapiens
420aatttctttt aataccattg cttcaaagga agacatctat aacataaacg atgtagaaaa
60tgttacatct acaaatgact gatgcaaatg accatacatc
100421100DNAHomo sapiens 421aataaaataa tactctgact caatacttaa atatttatat
cacttgttat gccataatga 60agcattcctg ccttgatact aatttctaga aatgctattt
100422100DNAHomo sapiens 422taatccatta atgtaggaat
actaactgac tcccttacag ttctccacag atgcacggca 60catacaaaaa cttactggag
gagaagggtt ggcattcata 100423100DNAHomo sapiens
423agctcaggct cctgaggttg ggagatcttc aagatggact gaacttcagg gctgcaggga
60ataaagggca cgatttagaa tccagctcgc cactaggggg
100424100DNAHomo sapiens 424cacaccaaca tcaaaagtga gtttctggct ctaccgactt
ctacccggat aattcactgt 60ttaaactgaa aataccccaa tacattagtc agttaaagaa
100425100DNAHomo sapiens 425aataataaac cccattaaat
acagaaataa ggattgttgc tcatggagaa aggccgtgaa 60ttcggccaac acgaaccatt
tatcttacat ctccagttca 100426100DNAHomo sapiens
426agccaaatca gcaaattaac tttaatgttt aaaatgtgtc aaatatatta gaatttaagg
60agaaatgaga tccccacccc agaagaagtc ttcgccttcc
100427100DNAHomo sapiens 427cgataaacgc cgtgatgaga atgtttaccg ctggcaaatt
caaactatac tagttatttc 60ctcaaatccg gtcaaactta ctgtttgcat gcataggagt
100428100DNAHomo sapiens 428tattggcaat cttctgaata
aagtcgttca gacccatcct cctctgcttc atgaaagctg 60tggatgaagg aggagaaata
aagaaacgtt tagacggctt 100429100DNAHomo sapiens
429cataacgtcc ggcgccacac acactaatct gatccgggac tttcaaaaaa tttccacttt
60gcgtctcctg gagcagaagt cccgcaagat tcctgcactc
100430100DNAHomo sapiens 430accgatgaga attgccacca tgcccctcat cctggagtaa
gtgagggtgc ccttagcagc 60ctcagttttc accgtcatca ccaccgcggg gagacagaaa
100431100DNAHomo sapiens 431gacgttagcg ctcaaagacc
ggctcggcgt atgctgcgcc aggccgcgcg ctcggcctta 60taaaaaaggc accgccgcgg
gggcggggcc tgcgcgacag 100432100DNAHomo sapiens
432agggtgagag gagtcaccag gtaaagatgg gttggaagga cctggcaggc agagcaggga
60gcaggacccc agtccagggc agcagggaag cgggagtctg
100433100DNAHomo sapiens 433ggcagagctg attccaggca gctcagtatt gctggcctgt
gcatcctgag acttatccga 60gtcgcaggtg aagctggtgg gaatcaggca gagtgcagag
100434100DNAHomo sapiens 434ctttagctgg ggcagggtta
gccaagagcc tgtcatggag ctgctctctg ggcactggga 60aacataagtc tggaggcttt
ggctgcagct gcagataaag 100435100DNAHomo sapiens
435atgcaggggc ctctgacgat gggggcctta gtcatctcag aggtggtgca gagggtagaa
60gcctgactgg ggtcagagat gaggaaggag agggtcagaa
100436100DNAHomo sapiens 436acagtgattc taaaccaatt tggttgaggc agaagatact
aatggccgag gggaggagag 60agggagcgta ggctctaaag gggaagcttg ttaggaatga
100437100DNAHomo sapiens 437agacagaggc gcaggcacag
ccctttcatc agctgaccag gagtgctcgg cccggcctgc 60caggaacctc ttatcaaact
ccaccggctg cctgcatcta 100438100DNAHomo sapiens
438caattcaagt ccatggctaa ccttctgtta gagacagaaa ttctgctgca gccagcaagt
60ttgctggtgt acagggcacc gcttcatggg cctagtagga
100439100DNAHomo sapiens 439agcgaagctg aaaggcaact tccgaaagcc agtctcctct
cccaaacgcc ctttaatatc 60tccccagttg gatctggggc gcctgtggtt tcggaccctt
100440100DNAHomo sapiens 440aggagctctg agaactggtg
tgtgtggtcg gaagccatct gagtctccct gtgatttgga 60ctttttaaga aacttctaag
ttgtattact atacccttta 100441100DNAHomo sapiens
441ttcccttgtc atatgacttc catcctcagc actacaatat tatcattaat gtttaaatca
60ttgtcaagtc tgtgattgcc ttagagattt attaagaata
100442100DNAHomo sapiens 442acatgctagg attaggaaag tttaactttt taccatcctt
aaaattagat ttttgaaaac 60tgtcttatcc ccattaaaga aaaaaataaa aaggatgaat
100443100DNAHomo sapiens 443tatacatacc tgcacatata
tacagcatat gtatatgtgt ctgtattata tgtattaaat 60gaaagattat ccacattttg
ttctttagga tcttcagcag 100444100DNAHomo sapiens
444ctctcttccc atcacaatag aaaggcctga gctaacattt ccatttctgc aaaaggcaga
60ttttgttcaa ttaaaaatta taatgcctta aatttccaca
100445100DNAHomo sapiens 445gacatttaag agacttcgtt ttcactgtga taaacaggtt
tgatttggac ttataacttt 60tttctaaaat tatcaaatta ataacgacta taatgaaata
100446100DNAHomo sapiens 446gaggcaaata ttttagagga
ttcattcctt ggggtaacat ttgttctata atttatagtc 60tcataatgtt gagagattaa
agcatttaaa taacattgtc 100447100DNAHomo sapiens
447aactaacttt cagcttacct ttcttaagga aaaaaaacaa aaaaatgtta aaaatagaca
60tgtatttttc aaacatacaa ttcatgtttt tatgtcatta
100448100DNAHomo sapiens 448aagagatgtg agggacttat aaataatatt aagataacag
gaattaaagt ctcggtgtgt 60gaaaatactg tatatctagg atgcacataa aaactgccct
100449100DNAHomo sapiens 449tacagatctt gcagggaaaa
gtacctgact atactgtata agacttctgc tgtaccattt 60aatcatacca aaaaaaatgg
aatcaacaca caaatagatt 100450100DNAHomo sapiens
450tcttttccac tgttctcaat ttaaaaataa ttggagaaat gtgtgctttg tttagaagag
60taaaggaaaa cattcattca atagtaccat gcagaatgat
100451100DNAHomo sapiens 451cagaaaaata gaaagattat catcggattt gggaatcaaa
gacagctcag caaaatacta 60ggacatggct catataagat ggaataagcc tggaaataca
100452100DNAHomo sapiens 452ctttagggga tagctctgca
aggggagagg ttcgggactg tggcgcgcac tgcgcgctgc 60gccaggtttc cgcaccaaga
cccctttaac tcaagactgc 100453100DNAHomo sapiens
453ctcccgcttt gtgtgccccg ctccagcagc ctcccgcgac gatgcccctc aacgttagct
60tcaccaacag gaactatgac ctcgactacg actcggtgca
100454100DNAHomo sapiens 454gccgtatttc tactgcgacg aggaggagaa cttctaccag
cagcagcagc agagcgagct 60gcagcccccg gcgcccagcg aggatatctg gaagaaattc
100455100DNAHomo sapiens 455gagctgctgc ccaccccgcc
cctgtcccct agccgccgct ccgggctctg ctcgccctcc 60tacgttgcgg tcacaccctt
ctcccttcgg ggagacaacg 100456100DNAHomo sapiens
456acggcggtgg cgggagcttc tccacggccg accagctgga gatggtgacc gagctgctgg
60gaggagacat ggtgaaccag agtttcatct gcgacccgga
100457100DNAHomo sapiens 457cgacgagacc ttcatcaaaa acatcatcat ccaggactgt
atgtggagcg gcttctcggc 60cgccgccaag ctcgtctcag agaagctggc ctcctaccag
100458100DNAHomo sapiens 458gctgcgcgca aagacagcgg
cagcccgaac cccgcccgcg gccacagcgt ctgctccacc 60tccagcttgt acctgcagga
tctgagcgcc gccgcctcag 100459100DNAHomo sapiens
459agtgcatcga cccctcggtg gtcttcccct accctctcaa cgacagcagc tcgcccaagt
60cctgcgcctc gcaagactcc agcgccttct ctccgtcctc
100460100DNAHomo sapiens 460ggattctctg ctctcctcga cggagtcctc cccgcagggc
agccccgagc ccctggtgct 60ccatgaggag acaccgccca ccaccagcag cgactctggt
100461100DNAHomo sapiens 461gctccccatc tgtccccaca
gttgctcctt ggctgagcca agggcttgct cacctctcag 60agcattgccc taactggttt
gttttgggct tacattgcaa 100462100DNAHomo sapiens
462gatcaggtcc tccccagagc caggctggag tccgaggcag aaaaggctgt ggagggcact
60ggggtcacca cagactggaa accggttggg cgcaggcccc
100463100DNAHomo sapiens 463aaaccttgag gaatcgtttg ggctgggacc agaacagggg
gctcctctgc acagagctcc 60ccaccgcttt ggtggattac ttcagactca gaaaattgac
100464100DNAHomo sapiens 464acaaagagaa actgacctgc
ccgcagccag ccctggctgc ctacacaagc tttcccctgc 60ttgccaggcc actcagcact
gcgtggcaga cacggacatg 100465100DNAHomo sapiens
465ctcgccccgg gaagctcacc ttcactccag ccgggtctct gctgcctttg ttaaataggg
60gacctgcggc taggaaagct ggatcccagg ctgttgggat
100466100DNAHomo sapiens 466gggggggagc ggggtgggag gaccaggcat ggggacggct
cctagcccgg gagcaactcc 60ctgacctgaa gcccgcagag accccgagcg gcacccgagc
100467100DNAHomo sapiens 467cgaggctgcc gaagcctgtc
accttcctcc agcctggctc tgcagcaaac agaaaggaaa 60cgcgattcgt tccacttgga
atttccttga aatctccgaa 100468100DNAHomo sapiens
468tctaatccgg cgttaactca ccgtgagagg agcgctcatc tcacaggagg ctgtggtaat
60gggtgaattg gcaggatccc tgcgggccag gcagccaggc
100469100DNAHomo sapiens 469ttttcgtttc ttatcctctt tttttaaagg ggagaagcca
tgagaaaagg cgtcctgcag 60agaaggaccc aatggggtct ttaagggtct ctgtatgaac
100470100DNAHomo sapiens 470tggccggctc ctaagcagaa
gctgaactca gaaaccgcta cttccttgat ttttcaaagc 60cccctcctca actccaggac
gcctttggag ccctagcccc 100471100DNAHomo sapiens
471tgtcgccgcc ggagccttga aaggctgcag ctcgctgccc aagctacgcg ttgccggagg
60cgggattccc aggtgcctca gcccgggcgg ccaagtgcgt
100472100DNAHomo sapiens 472tgtttcaggt cccctgcctg ggatccctgc actttgcaaa
gttagctgcg cggctgcaga 60ggtccgagat ccttccggcc ttagtacctg acccacggtc
100473100DNAHomo sapiens 473cggcaccccc aacccggtcc
cggcgggaga gtgagagaag cgagctcgcc gcctacttac 60tatgcatgga tgcaaacggg
tcgtgcttac agtgtatttc 100474100DNAHomo sapiens
474catcggggcg ctccagactg caggccggcc cacgccgccg cctcccggcg ccaaggggct
60gcccagggcg gatagggagc ctcgccacca ggccaggcac
100475100DNAHomo sapiens 475tgtgcgagct gggctcagaa aacactgctg gagcttcggg
gtctctctca gagcctccct 60gctggagacc gcccggagct gcgcggagag gcgggaaatg
100476100DNAHomo sapiens 476gtgctagcgc acccgggcta
ggagcgggtg cccaactccg gctggcttcc ctccctggct 60ggctcaagca gcagctccgg
gcccagcccg gggtagctgc 100477100DNAHomo sapiens
477ggccaaggcg cccgcggctt cgggggcata gcgtaggggc ccgcctccgg gacagccagc
60agcccccggc cccaggaagg agcagctttg aggaggccgc
100478100DNAHomo sapiens 478cggaacaatc ggcccttgac ttcactcagg gggcggagag
acccgggggc tgccaggctg 60gttccgcggc ctcgatgctt ctgaggtccc tcctcgaccc
100479100DNAHomo sapiens 479cacacaggca aacaactttt
ggacacaaac tcatatattt ttacatcttt taaaaataca 60tatactgtaa tgaacacact
gagtccctta tataaacaca 100480100DNAHomo sapiens
480caggccctaa cttgcagacc cccggaagga cgccagcgtg aacattcaga aacagagaaa
60aacacagaca aactcacaga tatttggact gatgcagaag
100481100DNAHomo sapiens 481acagtttgaa gtgtgagcct gaacatgttt gatctaaggt
ctggaggaag atgtgaagca 60aatctgacct aaaaaaaatt ataggaaaaa agcaaattgt
100482100DNAHomo sapiens 482tctggatttg tttcaccaag
gaacaagtaa gcagagaacc agacactgga gaaaaaaagg 60agtcaggaag tagacaagga
aatgttaaaa gaaataatag 100483100DNAHomo sapiens
483gataactgaa agaatgtagc ttccagattg ctagctatca gcagatagat agaaactttt
60atacagcctt taaatcttcc ctagaaacct ttttaaaagt
100484100DNAHomo sapiens 484caagggcctg ccaggatgag aacgggcaaa cctggccaag
gtgaccccat tagggactac 60cctcctaggg acagcactca gggccgttcc caatcacccc
100485100DNAHomo sapiens 485ggatttcctg tcctgctcgt
ctcctgccac acctcctttt gatctacccc caagacaccc 60ctaccttttt attctgtgaa
aatttactca tgctgtgggc 100486100DNAHomo sapiens
486cctgctggaa atgccctcct actgtttccc caaaccccgt cagaaattcc acggggaaac
60tcccttccct tctgctgcag gcaccgtcac tgtgtctctc
100487100DNAHomo sapiens 487agctctgccc cccagcctct gagtaccacc ttatcctagc
ccttagctac tggcttgtca 60ttgtctcttt acgttctcag cctcccacag aagcctggga
100488100DNAHomo sapiens 488aggcacactc gcccctggtc
tccaaggctc tgggtcctca gactggctga gtactgggga 60ccaaggtcac ccaagaagcc
ctgagtggcc ctcttgaggg 100489100DNAHomo sapiens
489ttagcagagc ttctctctgt ccaagacagg tcaggctctc tcccctggcc ccagctccac
60cgtcactcag aggagtggcc taaacaaacg ctgcaggtga
100490100DNAHomo sapiens 490ggctcccgag cccctgacat ggatgtttat ggaagaggac
tcttggcatc agcacctggg 60caaggtgggt agaggcagga gtgggcaaat gggaaagtct
100491100DNAHomo sapiens 491ggagagccgt ttgagattca
ccaggtgaat gaaccccggt ttttttctgg gtaacaggtc 60gaatgtgaat tacttatttt
cacaagctct tgacatgttc 100492100DNAHomo sapiens
492cgtcaaattg ctgttcccca aagagtggac tctggtgaca tataagtgtg tgggaccatt
60gcatcttacc ccagagatcc actcctgatc tggcattatt
100493100DNAHomo sapiens 493caaaatctgc tgaattcaaa acgatcctgt acttcctgct
caccaggtct gaaaagaaaa 60aagaaaaaag aagaaggaaa gactacacct gacaaaagac
100494100DNAHomo sapiens 494ttcacggttt ctctttagtt
ttatctgaaa tacatttgta agcttagggt gcaatttgga 60ttaaaacagt tttctttagt
gtcaataatg gcctttacta 100495100DNAHomo sapiens
495gagtgaatgg atatttttcc attctggatt atcgtttaat cgaaactttg tttcctgtgg
60aaatttttct ggtttaagtt atttgatttg ggagataaat
100496100DNAHomo sapiens 496catgtaactt aataaacttt ggcatcctgg ttaactgaaa
ttgcttcatt caatatttga 60agactgaaat ctgtattgtt gcctgtacct aaattatggg
100497100DNAHomo sapiens 497ggacagacag ggagagatga
ctgagttaga tgagacgagg gggcgggctg ggggtgcgag 60aaggaagctt ggcaaggaga
ctaggtctag ggggaccaca 100498100DNAHomo sapiens
498gtggggcagg ctgcatggaa aatatccgca gggtccccca ggcagaacag ccacgctcca
60ggccaggctg tccctactgc ctggtggagg gggaacttga
100499100DNAHomo sapiens 499cctctgggag ggcgccgctc ttgcatagct gagcgagccc
gggtgcgctg gtctgtgtgg 60aaggaggaag gcagggagag gtagaagggg tggaggagtc
100500100DNAHomo sapiens 500ggggcaggcg gagcttgagg
aaaccgcaga taagtttttt tctctttgaa agatagagat 60taatacaact acttaaaaaa
tatagtcaat aggttactaa 100501100DNAHomo sapiens
501gatattgctt agcgttaagt ttttaacgta attttaatag cttaagattt taagagaaaa
60tatgaagact tagaagagta gcatgaggaa ggaaaagata
100502100DNAHomo sapiens 502aaaggtttct aaaacatgac ggaggttgag atgaagcttc
ttcatggagt aaaaaatgta 60tttaaaagaa aattgagaga aaggactaca gagccccgaa
100503100DNAHomo sapiens 503ttaataccaa tagaagggca
atgcttttag attaaaatga aggtgactta aacagcttaa 60agtttagttt aaaagttgta
ggtgattaaa ataatttgaa 100504100DNAHomo sapiens
504ttggagaagt atagaagata gaaaaatata aagccaaaaa ttggataaaa tagcactgaa
60aaaatgagga aattattggt aaccaattta ttttaaaagc
100505100DNAHomo sapiens 505ccatcaattt aatttctggt ggtgcagaag ttagaaggta
aagcttgaga agatgagggt 60gtttacgtag accagaacca atttagaaga atacttgaag
100506100DNAHomo sapiens 506ctagaagggg aagttggtta
aaaatcacat caaaaagcta ctaaaaggac tggtgtaatt 60taaaaaaaac taaggcagaa
ggcttttgga agagttagaa 100507100DNAHomo sapiens
507tggtgtaaga gatgtgccag cggctggccg aggggcgctt agggctagag cccggggcgc
60tgcagaggtt gagagtcagt gggtggggcg cagttatcaa
100508100DNAHomo sapiens 508acaccagggc ccaaaagcag gctctagata ggttccaggt
gctcaatttc tatttcacgt 60ttggagtgag ccagtggaat tgtgaagttg tggcattttg
100509100DNAHomo sapiens 509attcggttgc caagagttat
cactgggcct ttgcaggtgc caaataaatt tcaggacaga 60gcctaaggca gagctctggc
acaggaagga agtaaaacgt 100510100DNAHomo sapiens
510ttaatgagca aatggacgca tgtttccaag cggtggtagg aagacagcag tttttggttg
60tcttcctggt gatcagcatg gaaacctagt agtgctctta
100511100DNAHomo sapiens 511ctctgatcaa tacattgtcg aaggcatgta cctgatgcta
acgtaacaat aatattaaat 60attgacttta tttgctatta tttattgcta acattaagta
100512100DNAHomo sapiens 512ctgctacctg ctatgtgcta
ggtttgtctc tgaagacttt acatgtattt ttcacgttta 60attatcataa tcttaagaag
caggtaccat aattatctcc 100513100DNAHomo sapiens
513gggaaaaaga atgacgaaag gcaagacagt ggagcaagtg aggacacgct tcaccgagcc
60agatctccac tcctcccagg gtatccacag ggacaagtca
100514100DNAHomo sapiens 514cacctggcag aaagctaagt cactcagcta gaaacaggcc
cagggaattc aacagaaggc 60tgaagagcca ctgcttatgg aaataaagcc cctcctgtaa
100515100DNAHomo sapiens 515agaactgcat ggcttttccc
tcccaacccc aaacccatcc cacatctggc ttttgttgtg 60tgaatcataa actgcccttt
cttcaccaca gtgattcatg 100516100DNAHomo sapiens
516aatcctctcc cactgtggat ctgtaaaatc tagacaggtc agtcagctcc cgccctttaa
60gagtttattt tccattctgt ggaagaagca gataaggaga
100517100DNAHomo sapiens 517gctgctgtcc ttaggagaca tcctttagag gaagctggaa
gacacgggtt caggccctgc 60atcctcctct gagttgctat gtgactggga acaggatact
100518100DNAHomo sapiens 518tcacctctcc attctttctc
tccttttctc ttagggtcgg aatatggaac tagacaggaa 60agtactttgg aggttttctt
accgtaagga ggctggcatt 100519100DNAHomo sapiens
519gggccctcca cccagcctca gttctatggg ggacgtggag tcaggcgatg atgtcctctg
60aggcagcgtc catctcccct taacattaag gaataaggcc
100520100DNAHomo sapiens 520agagggttct cgctcatttg ggaaaataaa aaaagcagga
atggggcgct ggaaattcta 60taagcttttc cccaccactc acaaaaacac agctgtgaaa
100521100DNAHomo sapiens 521ataaatacca ccccccaaac
caagggtcta gggccaccaa cagtcctcct cctcctcctc 60ctcctccttc tcctcctcgt
cctccagatc cagctgccaa 100522100DNAHomo sapiens
522ccttctcctc ctcgtcctcc agatccagct gccaacagca tcccccgctc ctgaagaaat
60gcaccgccca gaagggaacg gcgaaagggg gaagaagtcc
100523100DNAHomo sapiens 523aggggacccc cggcctctgg ccgagagctt gggtgggggc
ctcggccgtc gccactcacc 60cggggagggg aaaagctcca gatcgacttt ttccgtcttg
100524100DNAHomo sapiens 524atgatggtga gagtcggctt
gagatcgacg gccgccttca tggtgccagg agtgggggac 60gtacgggatg gtagcaagtt
tgcagttact gttgtttttc 100525100DNAHomo sapiens
525tttttaatga ggattagtaa cagggggaag gggacggggg aaatccgact ttcttcccaa
60aaatctcaaa ttcccgctgc ctttctttcc cccgcgcccg
100526100DNAHomo sapiens 526gacggtgcgc gcccggcact ccaggggaag ttggcacttt
gcggcgaagt gagcgcgctc 60gggtcccagc ctcgcccgcg ccgcgcccgc tcctcctgcc
100527100DNAHomo sapiens 527gagtgagtag caaatattca
tttatgaccc agtttttgtc caccctcagg cggggcatag 60gactacagac atttttctag
attacagcta ggatattatt 100528100DNAHomo sapiens
528cctgagttta tgacaatgaa atggtttgag aaggcaatat tgtggggctt tcagagaggt
60ttgctgagtg gctaggtgca tgcatgggtt taaccattaa
100529100DNAHomo sapiens 529cttccctttt tgccttttta ttataagctg gttttgtctg
tggctgtttt tttcttttaa 60aattaattaa aacttctcaa aatttctaaa agtaaacaag
100530100DNAHomo sapiens 530gcattctcta catacatcta
catacatatt ttgcatttta aaaattggaa tatttgtcat 60ttttctgtat tacccaaaag
tatataaaca gttaccagag 100531100DNAHomo sapiens
531atttatgtga gaagacagtt gtcacattac agatgtcaga ttagctataa aattgtttca
60ttctagaaac ctaatatggt aaaaataaac cttacttatt
100532100DNAHomo sapiens 532tagccattta tcagacaatt gcttttgttc agccagtttc
ttgttctagc agtataaata 60ttctttttat agaaagttac ttggtttgag aaataaacat
100533100DNAHomo sapiens 533ataagcttaa ggtaggctag
agatgaaaaa tttcagactt gtgtttgttt tggatttatt 60gtaccctttc tactattatc
tgagaaagct atttaggagt 100534100DNAHomo sapiens
534ttaagaaata gtctagtttt aaaatagcaa tggtttgccg gacacagtgg ctcacccctg
60taatcccagc attttgggag gccgaggtgg gcagattgct
100535100DNAHomo sapiens 535gaatttgcca gttttcaata ttctgattca ctctgttaag
ctagtaaggc agtctttaaa 60ttacacagtc tgtgtgttat tttactactg ctcagagggc
100536100DNAHomo sapiens 536attggagaag gttcccttgt
gattagaact gttcatgttg agacatgaat cataaggcat 60tccaaagttg gtttaaggtg
tgtctgcttt agacactgtg 100537100DNAHomo sapiens
537cccaggacta ttcttttgct ccagttttgc cttttgatta aatcaatatt atacctgagt
60tttataaact actaagaatt tgttcccctt cctcactgtg
100538100DNAHomo sapiens 538attttcttgc agtattttct tagaagagtc aactttaata
acttacccca aagtgcacgt 60tcttgatatt atgaacttgc tattgttgtc ttcccagttt
100539100DNAHomo sapiens 539tattgtagtt tttggaaggg
ctcgttctgc ccaagagaag ttcctcctta cagctgattc 60ggctgtctac catttgcacg
ttggtgctgt tttgagtgct 100540100DNAHomo sapiens
540acctcctgct ggtgaggctt catacagcac acagatggag ccatcctctc caattctgta
60ggacacttca taggggtcaa cccagagtgt gagttcactt
100541100DNAHomo sapiens 541gggagaagcc tgaacagctc ctgactgctc agtccaatcc
gctgtgctgc ctgtccaatc 60agaggatcca ttttatggtt gatgcgaata caacggtaac
100542100DNAHomo sapiens 542ccgatccctt gcatggcttt
tctgggaacc agtgatgttt ataatgttct atagaagaaa 60agaagaacag agaaacaacg
cttaggatcg ttagctccca 100543100DNAHomo sapiens
543ctgcggattc ctcctacccc aggctccttt gaggagcgaa aatgaaaact atcaactttt
60taaaatgtcc aggattgcat ccgttgttgt gcatgtgcgg
100544100DNAHomo sapiens 544ggatggaaaa agcgggcagg gttttagaaa taacacagta
gtaccggaca aaacaatctc 60caggaaccaa ccggttgagc cgccaaaaca ggaatcaggc
100545100DNAHomo sapiens 545gcgcagcctc ggccagtcgg
gaagccactg gcacctatgg ccaggcgaga aactgtttac 60tttctccacc ccaccccaga
tgcacacaat ggagttgatg 100546100DNAHomo sapiens
546gctttggaga tgagaagcgc caccggactg ttaaccccga agggaagaaa aacaagcaac
60cctaaaccac gctctgggca gggctgttaa ttgtgccggt
100547100DNAHomo sapiens 547acgcaacggt tggagggggc tgaggaaagg ggacgtcgaa
cccaccccag ccccacggct 60cctttgtccc caaatccgcc gacggtcctc ggaccgcagc
100548100DNAHomo sapiens 548tcccgcctcg gtgggcttaa
gtttctttgt tgtgcgtgtt gtcttctcct ctccgttttg 60ccagctgggg ggaagggggc
gccctccgtc cagcccctaa 100549100DNAHomo sapiens
549agcctcgcgg ggaaccgctg ttagcggcca cccagcgcaa ccacaccggt cccgcggcgg
60ggcccaagcg cgaccggccc cggggcgctg ccgaggttcc
100550100DNAHomo sapiens 550cgcagccccg acggccggac tctgacccag ggatgtgggg
cccgcgtccc tccgacgccc 60tcgccctgct cacctgccag cagctcctgc aggctctggc
100551100DNAHomo sapiens 551tgaaggtctg cagctgtcgc
tcgctcgtga gccccttggt gcggagaaac ttggagatga 60aggacacggc ggcggcgatc
tcgcctatca tggtggcggc 100552100DNAHomo sapiens
552ccgggtgtag aagggatgca tgggggcggc gtgcgggggc ggcccggggc ggctggggct
60cggcggcgcg gccccgacgg cggagcagcc accccgggct
100553100DNAHomo sapiens 553acgccgcacc cctcccccgt gcgttctgcg gccacccagg
ccttccagga caccgtggag 60agggaacaag ggggcaggga cgcccccttc ggcaggagcc
100554100DNAHomo sapiens 554gtcggagaag ggggcccaga
ccggagggag gcgagaagcc ccactgaagc cgggcgcagg 60gtctgggacg cagttgggag
tgcaaagggc tggctgagag 100555100DNAHomo sapiens
555ccgcaggagc agcaggctgt ggcccaggcc tcctgggtga caggccctgt ctggcgggga
60agagggacca agagacaaca cggaagaggc tggacctcga
100556100DNAHomo sapiens 556acaggggcgg ctgcctcact ccctacctga gccagccgag
ggggccaagg actttagagc 60tgtttcctcc ggcataagag agacacttgc tttccagggc
100557100DNAHomo sapiens 557agcacccttt atcggagaag
gctctacagg gaaggggtct ttgcagcctg gatggccatc 60ccacattcct ttaacggagg
tctctaggcc tcagagagaa 100558100DNAHomo sapiens
558cccagagtta gaaaggaggc cagacggtcc ttgctgtccc cctggggaga gaggaagttg
60ccgcctgctg ccaggcccag gaggagctgg gcctgcaata
100559100DNAHomo sapiens 559gtgggggacc tggcccctga ggcagtggcg gccatgtcac
ggccaggcca cggtgggctg 60atgcctgtga atggtctggg cttcccaccg cagaacgtgg
100560100DNAHomo sapiens 560cccgggtggt ggtgtgggag
tggctgaatg agcacagccg ctggcggccc tacacggcca 60ccgtgtgcca ccacattgag
aacgtgctga aggaggacgc 100561100DNAHomo sapiens
561tcgcggttcc gtggtcctgg ggcaggtgga cgcccagctt gtgccctaca tcatcgacct
60gcagtccatg caccagtttc gccaggacac aggtgagcag
100562100DNAHomo sapiens 562acacccaccc catgccaccc gccccgccga gccatcacta
ccttgcagcg taggatgctg 60aaaatcccag taaatctgct gatgccaaat cccttcccca
100563100DNAHomo sapiens 563tctccctgcc tcacctccag
aaaaacaggg cagtctaacc ttgtccagtt taagacttgg 60attccaatgc agcctctgag
caagctgtag ggccttgagc 100564100DNAHomo sapiens
564gggtagatca atatctctca cagctgagtg aggattaaat aaaattgtgc tcactgagca
60cagaacctag aacagcagta gcatgggatt gtagaataag
100565100DNAHomo sapiens 565ggctttacat gcacttcctc atttgatttt tcccaagaat
cacaggcagt aagtctgtgt 60attgttgtat tattatgagt cccattttat agatgaagaa
100566100DNAHomo sapiens 566tttatagatg aagaaaccga
gtctcccaga agctgagtga tttaaactca gagctgggat 60ttaaacccag gcggttgagt
tccagaacca aagttcttaa 100567100DNAHomo sapiens
567ctggtatcct atactggctc caagtgttgg tttgtggggt ggagtcgtgc tggtggtaat
60taattgggga tggggggcgt tggtggtgtt gatggtgggg
100568100DNAHomo sapiens 568tgaggtggca atgatggagg agacagtgtt agcggttgtg
ttggtggtga ctcagtgata 60gtattgatgg tggtggggtc ttggtgacaa tggagggatg
100569100DNAHomo sapiens 569tgttggtgac attgatagtt
gtgttggtgg tggtgctgga agtggtgtga tggggtggtg 60atgatggaga aaatgagaga
atgatgttgg tggcagtctt 100570100DNAHomo sapiens
570cgtggccatg tggtgtggct ggtagccctg tgtgtggctg ttacttagtg gtattggtga
60tcctgttgtg gttgtaatga tggtgatgtt gatggttgcg
100571100DNAHomo sapiens 571ttggtggtaa tgtgatggct gatgatggag ataaaatcga
tgaggtccca ctctcaggcc 60tactctcttt tgttctggag atttgtcatc gttggggaga
100572100DNAHomo sapiens 572tgaaatggct gctgtcgggc
tgtcatctcc aggcccgggg cgctgacatt tgggccactc 60tcggtctccc tcttcattct
gggcgcgcat tagctctggt 100573100DNAHomo sapiens
573ccggccggtt ccgctgcagc tgaacagcaa gatgcggcac ccaggttacc ctgatcatcg
60cagatttctc cccggggctc tgttctgagg cctcaaaagt
100574100DNAHomo sapiens 574gctccttgta gatgggacca ggggtcattt gggcagtagc
agcgcctggt ctcagtctgg 60tactgaagtc aggaatggct taaggtgaaa tcgtggtcct
100575100DNAHomo sapiens 575ctggtgaagc tcagcgaaga
ccccctcgcc ttgtttatga caagagaact tctgggggcg 60ggaggaagag tccctgttac
gatgctgatc atcattgagc 100576100DNAHomo sapiens
576ttttgctgag cagaaaactc tttagtactc aaggtcgaga gtctctggtg gtctgcctgg
60caccaggcac cttcctacaa ccctagtttt ccaaaaggac
100577100DNAHomo sapiens 577aaagcctggg gcaggcgacg tcctagctcg catttgaaca
gggccgcggg ccagcagaga 60tgcgcgatgc ccaactcttt ccaagagcac ctcgcgtccc
100578100DNAHomo sapiens 578gaaccggtgc cttcaactcg
gagaagtcaa gagacccgca agaaacttgc acgactgcac 60ccgccgccgc gctctggggg
ctgggcaggg gcagctgggc 100579100DNAHomo sapiens
579tggctcccgg ggaacgcgac ccccccgcgc cccgcagacc ggctgtctcc catggacccc
60tcggcacctg cagcctccga ggaagggtca gcgcgcgtgt
100580100DNAHomo sapiens 580ggggggctcg ggccagccga tgtttttggc cagaagccgt
tcgtcctggg ccgcggctgc 60ctctccacac cgggagctcg tgtttgtttt gcggagggag
100581100DNAHomo sapiens 581ctgttgtttt tgttctctgc
accggggaga gggggacttg gtggcggccg cgcgtggttt 60tcgggatcac attagcgtcc
gcccggcgtg gcccggtcga 100582100DNAHomo sapiens
582cattaagggg atcgaacctt tccgcggcct cgtcggggtc tgctcggaat cggcccctgg
60gccaggcccg aggcgcaagc agatcgccag gttgggtcag
100583100DNAHomo sapiens 583agttgttgaa aactccccgc tgcctgattt caactttatt
atttttttcc cacgccttca 60ctggggtccc ggagggagag gagccgccgc aacgctggct
100584100DNAHomo sapiens 584agtagcgcct cggtctctaa
aagccactgg gggcgagcct ccggtgtggc ggtgtcacaa 60gttagctgtc ctttctgagt
caaacccaac aaaaaaggca 100585100DNAHomo sapiens
585agaggaaaat caataaagtc cacgtgctcc ccggcctcct atggaaaggg ctggctgcga
60tggccggatg cccggccgtg ggctgggttt ggctccagtg
100586100DNAHomo sapiens 586ggacaaagaa ttttcagaac cgtgagaagg ggaggctttc
caaagttgag atccaagtcg 60tcggtgtctc gggagctccc ctggtacaca gggtgcccgg
100587100DNAHomo sapiens 587tgcccgactg gagccattta
aaaatggcag aaacagctgc aggccaacac acacacgctg 60gaaaacaacc cgcagccccc
tctactgtgg gattccccgc 100588100DNAHomo sapiens
588gggaagcccg gagttgctcc cctccttgcc tcagcccctg tgcaaagaaa gaactggtgt
60ctgtgcctgg gtcccttctg tcgccggcct ggaggttggg
100589100DNAHomo sapiens 589aaacagccgg caagccgcct ttctctgctc gaggaggcgt
ggtggggcct cctactccag 60gttcccggct ggacagaggc tcctgcaccc tgacagctgc
100590100DNAHomo sapiens 590ggaggccttc cagcccgctg
accccgcggg gaccaggcct gtagttggag cttgaggggc 60tgtacctctg cgcctccctg
ggtttgggga aacaacacat 100591100DNAHomo sapiens
591cgtgtcctct gaagacctca ggctttggga tctcatggtc cagcttccag ttcacttcgt
60tgccgcgacc ttgggcatat cattgtcact tctctaacca
100592100DNAHomo sapiens 592tggtgacccg gggttttgtg cttggcttcc aggtcccctc
gggttattga ggacgattga 60ggtcatgcct ccgagagcac cgcgccctgg gcgcaggagg
100593100DNAHomo sapiens 593aatgcaaatt taacagggca
ccctgtattt tacccagagg gaagccgaag tgtttggcag 60atcatttggc cccatgagcc
ttgggtgggt ttctcctcag 100594100DNAHomo sapiens
594ccctagtgac ccctaaaatt acccccccga cccacccact gtcccctgat gcttccccca
60cccccggaaa aagctgtggc ctccctctca tttggggcag
100595100DNAHomo sapiens 595gctgcctcct gttctctttt tctggtgttt cagcaaggca
ggccagtgga ggtgaggtga 60ccagaagatg gctaaaggga aaacaaaatg gtgggcctct
100596100DNAHomo sapiens 596ccagggtttg ggggccctgt
gctggtggag gagagaagac cccagggcga tggtaggaga 60cgaaagcttg ggctgcagcg
taagcttgga ggcccgctgc 100597100DNAHomo sapiens
597ggtggctcac gcctgtaatc ccagagcttt gggaggctga gacaggagga ttgcttgagc
60ccaggagttt gagaccagcc tgggtctcaa accaaaaaaa
100598100DNAHomo sapiens 598taaatataat tttaacgcca atctgagaaa aatgacttat
tagctgtgtg attttgagca 60atgctcttaa cctcccccat gaaggatggt gtgagaacga
100599100DNAHomo sapiens 599acagaattgt agcacgtgta
tcagtctggt acacaatgtc ctatgaaggt tagctttatt 60atcaccatca ttattattgc
agaaagactt tcagttcaga 100600100DNAHomo sapiens
600ataagacagc acagttacag agacctggtt ttattttcca gcttcttaac tgagtcatct
60ttcagctcct tttaattaaa aagaaaaaac aatcagagat
100601100DNAHomo sapiens 601tcaaagacct ggcagaaatg acttcccaac cccagatgcc
cccagcagca gtatttagca 60gtcatacaat tgcctgaaat gaagaatgag taatctggat
100602100DNAHomo sapiens 602gagtcggccc tgaaatcgac
ctgcaactta cccggaacgt gagctgtctc tctctgacct 60ctgctggctg cttcacctgg
agtctgagtc cgactcatgt 100603100DNAHomo sapiens
603agcacttcac tgtccgcgtt agtttagcct tcactgtcag caactcgtca ccttgtcctc
60ttgcagcgaa ggtttggaat cccatcacgg gtgtgcagtg
100604100DNAHomo sapiens 604gttagtcctg agatcatggt ggtgctagga gaacctgcca
accaatacag aaagttgtca 60cgaatagaaa cctaagctct ggccgggtgc ggtggttcaa
100605100DNAHomo sapiens 605agatatactg ttctagacat
gtgtctgaaa ggaatcctgc aaattctgtc ttattgaaca 60ggcataaggt gtcacgtcag
gcgtaaggtg tcacagcagg 100606100DNAHomo sapiens
606cgtaaggcgt cacgtcaggc gtaaggtgtc acagcaggcg taaggcatca cgtcaggcgt
60aaggcgtcac gtcaggcgta aggtgtcaca agctcggtga
100607100DNAHomo sapiens 607acgtcagggg tgtgccttgt gttctctgtt cgttgctttc
agaagcagca gcatgtggca 60gcatctctgt gcctatgacg atattgcagt gaatatgaga
100608100DNAHomo sapiens 608aattgtacat ttcaacaaca
taaataagct gttcaagact gtctcccatg cctccaaaac 60aaataaaaac cccccacaac
tcaaatgcat ataagctgtt 100609100DNAHomo sapiens
609actatagtat aatggtgagt tatagccagt gtatgatggg attgttgata gaataatgca
60tattagagct tttagttcaa aaatttgaga tagtgattca
100610100DNAHomo sapiens 610gaaagaaaaa aaggaatgat tatcatgaat tctgtttatt
agaattctgt ttattaaaga 60gttaaagata tgttttattt ttttatcttt attatcatta
100611100DNAHomo sapiens 611aattctaatg ttggtccctt
aggatcagca gggggggacc gggaatctgt aactgcaacc 60accccaccga gaggattaca
ggaacccagt cgagagctgg 100612100DNAHomo sapiens
612ttcccaacaa tgaggttcat ttaaaaagtc gtgagggggg aggggggcca aagaaagaaa
60tagatcaaag agcgggagag tcgagaaaag aaggaagaaa
100613100DNAHomo sapiens 613tgttggggag cgctggcagc cgggctggca agtggagttt
gggaatgtgc agggagggaa 60ggaagctgaa aaattcaaac tttttaaatg ctactcttca
100614100DNAHomo sapiens 614gctcctcggc gtccctgcac
cccaaccctg cagccctggg gcgttggcag ctgcaccaac 60aggagcagca agctgggaaa
acagagcaac atgacccgac 100615100DNAHomo sapiens
615gtgttaagag aaggcaaaac acttcagcaa ttaaaaagta gcccagcagc ttcacccttt
60caaattggga gggggaggtt ggaaagaaat ttaacaacat
100616100DNAHomo sapiens 616ccatagactt ttgctatgta catttaaacc gcagtcctgg
aacattccga gtttaaaact 60tgctttttca acactggctg acaagcaaca tgttttaagg
100617100DNAHomo sapiens 617agccccccat taaatcctta
ctcgcgggac tctcgagttc aagccagcat tttgtcgcca 60cctccccccc caaccccgcc
cgcaatcgat gagccgcaat 100618100DNAHomo sapiens
618gcctcggcaa cacaggtaag cgggtcaacc tgaatgcctc tttcacccca aagtttgctg
60cacgatcggc tatcgcggga agaagcccaa cggagctagg
100619100DNAHomo sapiens 619gcggactcaa gccccactgc aaacttgttc tgcaacatct
ttttgaatca caacttggcc 60tttcttcctc gcatatcccc agctcccccc aaagagtgga
100620100DNAHomo sapiens 620ggaaaacatt gtcccgagac
tcacttcccc gagggacctc ccactcccaa ccccacgggt 60gggtaatgcc gctggacaga
cctagggcgc agactgggaa 100621100DNAHomo sapiens
621cccgatcaga ccagcaaacc tgggatccag cagcacgtta cgtaaaacag gatcgcccaa
60aacttgtccc aatcccagcc ctccccccga agcccccggg
100622100DNAHomo sapiens 622ctgccctgcc aggcaaactt cgcccctcaa aaccctggcc
tccagattca catgtaatcc 60ccgccagcaa ctgttgaaac tcaaagggtg ggaaggacgg
100623100DNAHomo sapiens 623ggccaaattc cttcaaactt
gggagaaatg ccggaggaga aaagaatcat ctcgctgcac 60cactttcccc attgccttcc
aagacccaaa cttttggggg 100624100DNAHomo sapiens
624ttctttctta aggcaaaaga aaaagacttt ttgaaaagca aatgctccgc ccccctttac
60cttgcataaa acttcgctca agtcgaagat ggtggcagac
100625100DNAHomo sapiens 625acgagggtgg tggtcatcct gtgcgttcgc gcgagccagg
ggcgaggatc tggtgtgtcg 60cgaaggtccc ggtgcgggga aggcgcagcc tctcctgtct
100626100DNAHomo sapiens 626ttattttttt atattaagat
ttattctaaa ttttgattct tctaaatata gtatatattt 60agtatatata taatgcacct
ctcttaccta atgatcattt 100627100DNAHomo sapiens
627ctaaataatc ataacaacat cgagtaaaac tatgtaataa cacatattat tattaagata
60agtataagaa atataataat aaattgtccc tgttctaaaa
100628100DNAHomo sapiens 628ggtaattata taatgctgaa tgtgtcagag gcattcgaac
cagagtgact ccattttgag 60tgagggctag gaaaatgagg ctgagacttg ctgggatgca
100629100DNAHomo sapiens 629tttaattttt atgctttctt
cagtgtatgt ttggagagag tttgaacatt ttttgactct 60ttttcattga gtaaatccaa
atacttgtaa aagacttatc 100630100DNAHomo sapiens
630tatttcttta acaaaaactt aacatggatt aaggacccat cttaaggcat cacacattaa
60aaaagtcaat attgattcaa taccggcgct tatactacga
100631100DNAHomo sapiens 631catcacttgt taaatttgtt ttctaaataa agcccagagg
tagtggaaaa tacttcacac 60tctaggccag tgtttgctat gcctggttga ccctaaactg
100632100DNAHomo sapiens 632ttgagggttc tttttaaaaa
tacagatttc tgggacccac ctgagatgat tccgataatc 60ggccatatgg atgagtcact
tagagatacc catttttaag 100633100DNAHomo sapiens
633gattaggacc ccgaagccca gaaaatgcct gctgtagtca acattatagt cacactccac
60aggcactggg tccacccctt tgaccgacat tcctttgcgg
100634100DNAHomo sapiens 634ttttcccacc cttcttccct gcctggagaa ctcctattca
tcctccagag cccggctcaa 60agtggcttca tctgtgggga tcctccctgc cccatagtga
100635100DNAHomo sapiens 635gtgctccttg agtcctcgcc
cttcctaggg catcccaagc tcccaggggc tgcccctgct 60gcctcgccat ccgctccaaa
gctggctgta cctcgatggt 100636100DNAHomo sapiens
636taagggcagc caggcgtgct gcttctcgtc caaatacacg aacttctccc aggcccacag
60gcggtccggg tggtcggtga ctgcctcccc gagtgtcggg
100637100DNAHomo sapiens 637aggaatcaga tttcaaaatg aatatgtata agaaaagaac
cggggatcag tgatcaggaa 60cagggatcca tgatctggtc cagggctcag cggtcaggaa
100638100DNAHomo sapiens 638ccctggcctg gagtcccaag
tccccagccc atcctgcccc tggagcccag tttagcttgg 60tcttgaagtc tgctctaggt
acccccaaaa tcacagtatc 100639100DNAHomo sapiens
639cagccccgct ctgcccaccg ggacagccaa gttcagctga gactggccta ccgggggagt
60cgccctctga agttcactct aagccagcct ggttcagcct
100640100DNAHomo sapiens 640ggcccaggtc agcccaggac ctccccttgc aggcagcaaa
ctcttatttc agtccagcca 60gctcaaccag cttgcttctg actcagctcc tcttagccag
100641100DNAHomo sapiens 641ttagctcagc aaagctggac
ctaaagtagc cacctcaccc cagcttcatc cagatgaata 60cagtccagat cagcttagtc
agttaagcct agcctagcta 100642100DNAHomo sapiens
642gttaaatcca gttacgacca gctcaactaa tcctgctcag gcctgctcag cccagcccag
60ctgaacccag tttagccgag gccaggccag cccagctgaa
100643100DNAHomo sapiens 643tacagttgcc cagtctagct cagcccagtc cagcactgcc
cagtttagct gagctcagcc 60tggcccagcc cagctcatat cagcccatct cagctgaacc
100644100DNAHomo sapiens 644agtttgaccc agtctaaccc
aaccccgctc agctgaaccc agcccagccc agcccagccc 60agccaaaccc agtttagcct
agctcagctc agcccatttc 100645100DNAHomo sapiens
645cctgtcctag gggtggcagg cagtctgcac ccagcctagc cctgcccagc gtggggtctc
60tgaccttctt ggtcttgggc ccagccaaga ttcccagccc
100646100DNAHomo sapiens 646ttctagcttt cctgtgtccc catgcaggga agggatgcct
agagtccacg cagtgaccaa 60gaagcttggt tgatgctgtg agggtggccc aggagtcccc
100647100DNAHomo sapiens 647cacctgctgt ccttggtcct
ggctgagagg agggccctac ggccagctct gctgaccctg 60ccctgggctc tggtgatgct
gccggcctgg acaagcccct 100648100DNAHomo sapiens
648gagctcaggt cggtcgtgcc catcctggca tcaccccaca gccggttctg ccgcatcccg
60tcatgttcct cgtgctccca gcccggtcgt cctggaggcc
100649100DNAHomo sapiens 649tgagcatgag tggggcgggc agaggcctcc gggtgaggag
acagatgggg cctgccttgc 60tgccctgggc tggggctgca cagccggggt gcgtccaggc
100650100DNAHomo sapiens 650aggagggctg agcctggctt
ccagcagaca ccctccctcc ctgagctggc ctctcaccaa 60ctgtcttgtc caccttggtg
ttgctgggct tgtgatctac 100651100DNAHomo sapiens
651accaactgtc ttgtccacct tggtgttgct gggcttgtga tctacgttgc aggtgtaggt
60ctgggtgccg aagttgctgg agggcacggt caccacgctg
100652100DNAHomo sapiens 652ggactgtagg acagccggga aggtgtgcac gccgctggtc
agagcgcctg agttccacga 60caccgtcacc ggttcgggga agtagtcctt gaccaggcag
100653100DNAHomo sapiens 653tgctacactg ccctgcacca
cctccactca gcttcattgt gctggtggcc ctggctcctg 60gcagcccatc ttgctccttc
tggggcgcca gcctcagagg 100654100DNAHomo sapiens
654ccttcctgcc tagggtccgc tggggccagc cctgggaccc tcctggtctc aagcacacat
60tccccctgca gccacacctg cccctgcctg agagctcagc
100655100DNAHomo sapiens 655cccgagccct ggaatgcctt cccttctcca tcccagctca
cccttgccaa ctgctcagtg 60ggatgggctc acactccctt cctggcacca ggaggctgca
100656100DNAHomo sapiens 656ctgcactttc accagccctc
agctgtctgc tgccagcaac tacccagctc ctgccaaaat 60ctaggagctg agtgatgcct
cccaccggcc ctgctcacct 100657100DNAHomo sapiens
657gtggttgcct tgccctgagc tctagtgcct gtcccctgct cgtcctgcct cccaccggcc
60ctgctcacct gtggctgctc tgctctgatt ccctgaggct
100658100DNAHomo sapiens 658aagcctcagt cctgctcacc ttctgatgct ctcctctgtc
ccctgagctc caggggctgt 60cccctgctcg tcctgcctcc tacctgcccc tgcttacctg
100659100DNAHomo sapiens 659agggtgctct gccctggtgc
tctgagctcc aggggctgtc ccctgctcct cctgcttcct 60accagcccct gctcacctgt
ggctgctctg ccctggtccc 100660100DNAHomo sapiens
660ctctgccctg gtcccctgag ctccaggggc ttccccctgc tcttcctgcc cccaccagcc
60cctgttcacc ttcagatgcc ctcccctggt cccctgaagt
100661100DNAHomo sapiens 661cccagagctg ccccctgttc ctcctgcctc ccaccagccc
gtgctcacct gccgctgctc 60tgccctggtc ccgagttcca ggggctgcac cctgttcgcc
100662100DNAHomo sapiens 662cacctcccac tagccatgct
cagctcttga tgctctgtcc tggtcccctg agctccagga 60gctgtcccct actcgtcctg
ccacccacca gcccctgctc 100663100DNAHomo sapiens
663acctgaggca cctgaggctg ctctgccctg gtcccctgag ctccagggtc ttccccctgc
60tcatcctgcc tcccacctgc ccttgttcac cttcagttgc
100664100DNAHomo sapiens 664tctgccctgg tctgctgagc tccaggaggt gccccctgct
ccttctgccc ccacctgccc 60tgctcacctg tggctgctcg gtcctggtac cctgaactcc
100665100DNAHomo sapiens 665gccccctgct ccttctgccc
ccacctgccc tgctcacctg tggctgctcg gtcctggtac 60cctgaactcc aatgcctgcc
ccctgctcac tctgccctcc 100666100DNAHomo sapiens
666ctcaacccgg gcagcaatgt cactcaggtc actgttgccc ccctgcctgt cctggcaccc
60tctgtccagg tttgggctgt ttttctggcc tcatttttgt
100667100DNAHomo sapiens 667tgtccagtca ggtctcccca acagagcccc ttgcccttgc
ccatgtgccc ctcctgggtg 60agctcccaga tcctcccgtc cctgcactgc tcctgctctg
100668100DNAHomo sapiens 668gaagcctctc cagaacctca
gctcctcagt ggcctctgct ctgctgggtc agctccctga 60acgcacggag cctcacccct
cccctcgccc caggcctgct 100669100DNAHomo sapiens
669gcactctggg cctttctggg cctccctgga ctcttccctc ctcccatctg tgcactcagc
60acagctctcc cctccactcc gctgctgacc acagccctgc
100670100DNAHomo sapiens 670cctttctggg cctccctgga ctcttccctc ctcccatctg
tgcactcagc acagctctcc 60cctccactcc gctgctgacc acagccctgc tccccgccag
100671100DNAHomo sapiens 671cccacggcca gcactgctga
ccctgccctg ggctccagtg atgctgctgg cctggacaag 60cccctccgtt cacctggggc
ctctcctcct ccctcgttct 100672100DNAHomo sapiens
672actgcctcct cagctcaggt gggtcctgcc catgctggca tcaccccacg gccggctctg
60ccgcatcccg tcaggttcct cgtgctccca gcctggtcgt
100673100DNAHomo sapiens 673catggaggcc tcagtcagcc tctggtgtgt cctgccctgt
tggcttggaa gcccctgccc 60acggtccccg tcatcttgca ctgggtgggc gttggtgcct
100674100DNAHomo sapiens 674agctcagccc agcctagtcc
agcccagccc agcacaggtc agcccagctt agcttagccc 60aggtcagtcc agctcagctc
agtccactta agctcaccca 100675100DNAHomo sapiens
675ggtcagctcc gtccagctca gcccagccta gcccagctta gcccagccca gcccaacaca
60ggtcagccca gctcagccta gcccagccca gctcagcaca
100676100DNAHomo sapiens 676ggtcagacca gctcagtaca gctcaggtca gcccagacca
gtccaaccca gcccagcgca 60gtccaaccca gcccagctca gctcatccaa gcctagctca
100677100DNAHomo sapiens 677gctcagccca gcccaggtca
gcctagccca gccgaaccca gctcagccca ggtcaaccca 60attcagctca gctcagccca
ggtcaaccca accaagctca 100678100DNAHomo sapiens
678gctcagccta gcccagtcca gctcagccca gctcagctca gcccagtcca gctcaatcca
60cctaagctca cccagctcag cccagtctgg ctcagcttag
100679100DNAHomo sapiens 679gtcagcccag cccagcctag cccagatcag tccagcttag
cccagcccag gtcagcccag 60cccaggtcag cccagctcag ctcagcccag cccagctcag
100680100DNAHomo sapiens 680cccagcccag ctcagcgcag
cccagcctag ctcaccccag ccaggtccag cttagcccag 60ctcagcccag cccaactcag
ctcagcccag ctcagcccaa 100681100DNAHomo sapiens
681tctgagctcc aggggctgcc cacctgctcc tcctgcttcc caccggccct gctcacctgc
60agctgctctg ccctggctcc ctgaggctga gcctcagtcc
100682100DNAHomo sapiens 682tgctcacctt ctgatgctct ccccttgtcc cctgagctcc
aggggctgac ccctgatctt 60tctgcttcct acctgcccct gctcacctgt ggctgctctg
100683100DNAHomo sapiens 683ccctgatccc ctgagctcca
ggagctgcct cctgctcttc ctgcctccca cctgcccctg 60ctcacctgca gatctgccct
ggctctctga ggtccagggg 100684100DNAHomo sapiens
684ctgccccctg ctcgcccacc tcccaccagc catgctgacg ttgtgatgct ctgccctggt
60ctcctgaggt ccaggggctg tcccctgctt attctgcctc
100685100DNAHomo sapiens 685ccacctgccc cttctcacct gaggctcttc tgccctggtg
ctctgagctc caaaagctgc 60ccacttgctc ctcctgcttc ctaccagccc ctgctctcct
100686100DNAHomo sapiens 686gtggatgatc tgccctggct
ctctgagctc caggggctgc ccacctgctc cccatgcttc 60ccacctgccc ctgctgacct
gcggctgctc tgccttggct 100687100DNAHomo sapiens
687ccctgagctc caggagcttc cccctgctca tcctgccccc cactggcccc tgttcacctt
60cagatgccct ccctggtccc ctgaagtcca ggagctgccc
100688100DNAHomo sapiens 688cctgttcctc ccgcctccca ccagcccgtg ctcacctgcg
gctgctctgc cctggtcccc 60tgagttccag gggctgcccc ctgctcgccc acctcccact
100689100DNAHomo sapiens 689agccatgctc acctcctgat
gctctgtcct ggtcccctga gctccagggg ctgccccctg 60cttgcccatc tcccactagc
catgctcacc ttctgatgct 100690100DNAHomo sapiens
690ctgccctggt cccctgagct ccagggtctt ccccctgctc atcctgccgc ccaccagccc
60ctgctcacct gaggctgctc tgccctggtc ccctgagctc
100691100DNAHomo sapiens 691cccctgagct ccagggtctt ccccctgctc atcctgccgc
ccaccagccc ctgctcacct 60gaggctgctc tgccctggtc ccctgagctc caggaggtgc
100692100DNAHomo sapiens 692ttctgccccc acctgccctg
ctcacctgtg gctgcttggt cctggtccct gagctccaat 60gcctgctccc tgctcactct
gccctccctc aacccgggca 100693100DNAHomo sapiens
693gcaatgtcac tcaggtcact gttgcccccc tgcctgtcct ggcaccctct gtccaggttt
60gggctgtttt tctgccctca tttttgattt tgcagcactt
100694100DNAHomo sapiens 694cctctgtcca ggtttgggct gtttttctgc cctcattttt
gattttgcag cacttggcgt 60gttccctatg ctgtggagca gccccagtgt ccagtcaggt
100695100DNAHomo sapiens 695agtgtccagt caggtctccc
caacagagcc ccttgccctt gcccatgtgc ccctcctgaa 60tgagctcccg gatcctcctg
tccctgcact gctcctgctc 100696100DNAHomo sapiens
696tggaagcctc tctggaacct cagctcctca gtggcctctg ctctgctggg tcagttccct
60gaacgcacgg agcctcagcc cttcccctcg ccccaggcct
100697100DNAHomo sapiens 697gctgcactct gggcctttct gggcctccct ggactcttcc
cttctcccgc ccgtgcactc 60agcacagctc tcccctcctc tccactgctg accacagccc
100698100DNAHomo sapiens 698tgctccccgc cagcaggtgc
cccaacccca tcagctggct ctgagcccag cccctgtgcc 60tcccctgtcc ctgcctctgc
ctctgggctc cttggcttcc 100699100DNAHomo sapiens
699acctgctgtc cttggtcctg gctgagagga gggccccacg gccagcactg ctgaccctgc
60cctgggctcc ggtgatgctg ccggcctgga caagcccctc
100700100DNAHomo sapiens 700cgttcacctg gggcctctcc tcctccctcg ctctgctgcc
tcctgagctc aggtcggtcg 60tgcccatcct ggcatcaccc cacggccggc tctgccgcat
100701100DNAHomo sapiens 701ccagtcatgt tcctcgtgct
cccagcccgg tcgtcctgga ggcctcagtc agcctctggt 60gtgtcctgcc ctgttggctt
ggaagcccct gcccacggtc 100702100DNAHomo sapiens
702cccgtcgtct cgcactgggt gggcatcggt gcctgaaggc tgcccacctc ccccgtgctg
60gctccgcttg ggcctccatg tggggccggc ctcgacccca
100703100DNAHomo sapiens 703cactgcactt tcaccagccc tcagctgtct gctgccggca
actacccagc tcctgccaaa 60gtctaggagc tgcgtgctgc ctcccaccgt ccctgctcac
100704100DNAHomo sapiens 704ctgtggctgc tctgccctgg
tgctctgagc tccaggagat gccccctgct cctcctgccc 60cccacctgcc cctgctcacc
tgcagcggct ctgccctggt 100705100DNAHomo sapiens
705gagctccaag agctgccccc tgctcctcct gtcccctgac cctgctcctg tttgcctatg
60gctgctctgc ccttgtcccc tgagctccag gagctgcccc
100706100DNAHomo sapiens 706tgctcattct gccgcccacc tgcccctgtt cacctgtggc
tgctcttccc tggtcctctg 60agctccatga gctgcccctt gctcctcctg ctttccacca
100707100DNAHomo sapiens 707gcccctgctc acctaccgat
gatcttcccc ggctctctga gctccagggg ctgcccacct 60gctacccctg cttcccacca
gccctgctta cctgcagctg 100708100DNAHomo sapiens
708ctctgccctg gctggcagag ctgcagaagc tgccccctgc tctgcaacct cccaccggcc
60cttctcatct tctgatgttc tcccctgttc cctgagctcc
100709100DNAHomo sapiens 709aggagctgcc ccctactcgt tctacctccc accaacccgt
gctcacctgc gactgctctg 60ccctggtccc ctgagctcca ggggctgccc cctgctcgcc
100710100DNAHomo sapiens 710tgccctgatc ccctgagctc
caggactgcc ccctgctcgt cctgcccctc acctgcccct 60gctcacctga ggctgctctg
ccctggtccc ctgagctaaa 100711100DNAHomo sapiens
711ggggctgccc cttactcatc ctgcctccca ccagcccctg ctcaccttct gatgccctcc
60cctggtcccc tgagctccag gggctgcccc ctgctcgtcc
100712100DNAHomo sapiens 712gggctgcccc ctgctcgtcc tgcctcccac cagcccctgc
tcacctgcag ctacactgcc 60ctggttccct gagctccagg agctgccacc tgcttgtcct
100713100DNAHomo sapiens 713gccttccacc agcccctgct
cacctgcagc tacactgccc tggttccctg agctccggga 60gctgccgcct gcttgtcctg
cctcccacca gcccctgctc 100714100DNAHomo sapiens
714acctgtggct acactgccct ggtgccctga gctccaggag ctgccccctg cttgcccatc
60ttccactgag ccctgctcac ctgcaactgc tctgccctgg
100715100DNAHomo sapiens 715ctctatgagc tccaggggct gccccctgct ggtcctgcct
cccacctgcc ctgcgcacct 60gtggctgcct cctcacctgt ggctgctctg ccctggtccc
100716100DNAHomo sapiens 716ctgagctcca gggtcttcct
cctgctcatc ctgcccctcc accggctcct gttcaccttc 60agatgctctc ccgtggtccc
ctgagctcca ggagctgccc 100717100DNAHomo sapiens
717cctgttcttc ctgcctccca cctgccctgt gcacctgtgg ctgcttggtc ctggtcccct
60gaactccaat gcctgccccc tgctcactct gccctccctc
100718100DNAHomo sapiens 718aacctggggc agcaacgtca ctcggtccac tgttgccccc
ctgcctgtcc tggcaccctc 60tgtccaggtt taggctgttt ttcttgcctc atttttgttt
100719100DNAHomo sapiens 719tggcaccctc tgtccaggtt
taggctgttt ttcttgcctc atttttgttt ttgcagcact 60tggcgtgttc cctatgctgt
ggagcagccc cagtgtccag 100720100DNAHomo sapiens
720tccagtcagg tctccccaac agagcccctt gcccttgccc atgtgcccct cctggatgag
60ctcccggatc ctcccgtccc tgcactgctc ctgctctgga
100721100DNAHomo sapiens 721agcctctcca gaacctcagc tcctcagtgg cctctgctct
gctgggtcag ttccctgaac 60gcacggagcc tcagcccctc ccctcgcccc aggcctgctg
100722100DNAHomo sapiens 722cactctgggc ctttctgggc
ctccctggac tcttccctcc tcccgcccgt gcactcagca 60cagctctccc ctcctctccg
ctgctgacca cagccctgct 100723100DNAHomo sapiens
723gaccacagcc ctgctcccgg ccagcaggtg ccccaacccc atcagctggc tctgagccca
60gcccctgtgc ctcccctgtc cctgcctctg cctctgggct
100724100DNAHomo sapiens 724gctctgctcc cagctcacct gctgtccttg gtcctggctg
agaggagggc cctacggcca 60gctctgctga ccctgccctg ggctccggtg atgctgccgg
100725100DNAHomo sapiens 725cctggacaag cccctcggtt
cacctggggc ctctcctcct ccctctctct gctgcctcct 60gagctcaggt cggtcatgcc
catcctggca tcaccccatg 100726100DNAHomo sapiens
726gctggctctg ccccatcccg tcatgttcct cacactccca gcccggtcgt cctggaggcc
60tcagtcagcc tctggtgtgt cctgccctgt tggcttggaa
100727100DNAHomo sapiens 727gggtagagcc cacctcgtgg cctgcaagcc agccagcccc
tgccggtcga gaaggaagcc 60tgtgtgagag cacacaactg gaggccgggc ggggaagaga
100728100DNAHomo sapiens 728aacacgtgcc aacaggccac
gcaggccagg accccagacc cggaggcagc gcccctttga 60gttcctctct ctggtctccg
atgttcttct gttgggatca 100729100DNAHomo sapiens
729tttcacctac aggcaacaga gacagtgtga aatgctttcc ctgtggtcgg gaagggagcc
60ggggcagaga tgacccagtg gggtggtgtg ggggcctccg
100730100DNAHomo sapiens 730ctttgcacac cacgtgttcg tctgtgccct gcatgacgtc
cttggaaggc agcagcacct 60gtgaggtggc tgcgtacttg ccccctctca ggactgatgg
100731100DNAHomo sapiens 731gaagccccgg gtgctgctga
tgtcagagtt gttcttgtat ttccaggaga aagtgatgga 60gtcgggaagg aagtcctgtg
cgaggcagcc aacggccacg 100732100DNAHomo sapiens
732ctgctcgtat ccgacgggga attctcacag gagacgaggg ggaaaagggt tggggcggat
60gcactccctg aggacccgca ggacaaaaga gaaagggagg
100733100DNAHomo sapiens 733actccagcta ccctgaagtc tccccaggca gacaacccag
gcctgggagt gagtataggg 60agggtgggtg tgatggggaa cgcagtgtag actcagctga
100734100DNAHomo sapiens 734ggctatccat ctatgtccaa
caagatcatg aagattggcc cagtgccatg tcctccagtt 60catcccagcc caggccagct
caatccagtt catcccagcc 100735100DNAHomo sapiens
735caggccagct caatccagcc cagcccaccc caccccagct cagcaaagcc aagctcagct
60cagcccaact cagatgagct cagaccagct cagcccagcc
100736100DNAHomo sapiens 736cagctcagct cagcccaacc cagcccagct cgctcaacct
tgctcggctc agcttagccc 60agcccagccc agctcaatcc agcctggctc agcccagccc
100737100DNAHomo sapiens 737agcccagttt ggctcaaccc
agcttggctc agcccaggtc agcctggctc aactcagccc 60agcccagccc agctctgctc
aacccagctc tgctcaactc 100738100DNAHomo sapiens
738agcccagctc atcccagctc agcccagccc agcctagctt agctcaaccc agctcagctc
60agttcagctc agccctgctc agcacagcac agcagagccc
100739100DNAHomo sapiens 739agcccggatc ggctcaaccc agcttagctc agcccaggtc
agcccagctt aactcagccc 60aggtcagccc agcttaactc agcccagccc agcccagctc
100740100DNAHomo sapiens 740tcagcccagt tcagcccagc
tcagcccagc ccagcctagc ttggctcaac acagctcagc 60tcagccagcc cagaccagct
cagctcagcc cagtccagct 100741100DNAHomo sapiens
741caacccagcc cagcccaacc cagctcggct taacccagct cggctcagcc cagatcagcc
60tggctcaact cagcccagcc cagctcaacc cagcccagtt
100742100DNAHomo sapiens 742cagctcagct gagcccagcc cagcccagtc cggctcagct
cagccccgcc ccactcagcc 60cagctcagct cagcccagct cagcccagct cagcttagcc
100743100DNAHomo sapiens 743cagcccagat catcccagct
cagctcagct cagctcggct tagcccagct caacctggcc 60cagcctggtc caggtcagcc
cagcctggac cacccagccc 100744100DNAHomo sapiens
744agctcagctc agcccagctc atcctggttc agctcagctc aacccggctc agcccaggtc
60tgctcaaccc agcccaaatc agctcagccc agcccaggtc
100745100DNAHomo sapiens 745atcccagctc agcccagcac agcctacttc agctcagctc
agctcagcct aggtcagctc 60agttgaggtc agctcaactc agcccaatcc agcctggctc
100746100DNAHomo sapiens 746agcccagctc accctagctc
agcttagctc agcccaactc aacccagccc agccttgccc 60aacccagctc agctcagccc
agcccaggtt agcccagccc 100747100DNAHomo sapiens
747agcctcggct tagctctgct cagctcggcc ctgctcgcct cagcccgttc agcccagttc
60agctcagctc agctcagccc agctcagccc agccctggtt
100748100DNAHomo sapiens 748agctcagccc agctaagctc agctcggctt ggctctgctg
agcttggccc agcttggctt 60agcctgatac aacctgctca gcccagttca gctcggctca
100749100DNAHomo sapiens 749gcccagcgta gctcagctca
gctgagccca gcccaggtta gctcagcccc agtccaggtc 60agctcaactc agcccaaacc
agcctggctc ggcccagctc 100750100DNAHomo sapiens
750accctagttc agcttagctc agcccagccc agccctgccc aacccagctc agctcagccc
60agcccaggtt agcccagccc agcctcggct tagctctgct
100751100DNAHomo sapiens 751agcccagccc aggttagccc agcccagcct cggcttagct
ctgctcagct cggcccagcc 60caggttagcc cagcccagcc tcggcttagc tctgctcagc
100752100DNAHomo sapiens 752tcggccctgc tcgcctcagc
ccgttcagcc cagttcagct cagctcagct cagcccagct 60cagcccagcc ctggttagct
cagcccagct aagctcagct 100753100DNAHomo sapiens
753cggctcagct ctgctgagct cggcccagct tggctcagcc cgacacagcc tgctcagccc
60agttcagctc ggctcagccc agcccagccc agcgtagctc
100754100DNAHomo sapiens 754agctgagccc agcccaggtt agctcagccc cagcccaggt
tagctcagcc cagctcagct 60ctgcccaggt tagctcagcc ccagtccagg ttagctcagc
100755100DNAHomo sapiens 755ccagctcagc tctgcccagg
ttagctcagc cccagtccag gttagctcag cccagctcag 60ccttgcccag gttagctcag
cccagctaag ctcaacttgg 100756100DNAHomo sapiens
756ctcagctcag cctagcttgg ctcagcccag cacagcacgc tcaacccggt tcagcttggc
60tcagcccagc ccagcccagc ctagctcagc tcagccccgc
100757100DNAHomo sapiens 757ccagctcagc gcagcccagc tcagctcagc tcagcctagc
cttgctcggc ccagctcagc 60tcagcccagc tcagcctagc cttgctcagc ccagctcagc
100758100DNAHomo sapiens 758tcagcccagc cctgcccagc
tcagcccagc ttagtgcagc caagcccagc tcagctcagc 60tcacctggtg caacttagcc
cagctcagct cagctcagct 100759100DNAHomo sapiens
759caacccagtt caactcagcc cagttcagct cagctcagcc cagttcagcc ttgtttagtc
60taggtcagct taggtcagtt ttgcccatct gagtccattt
100760100DNAHomo sapiens 760ctgaaagctg gatggagttg tcatggccag aaatggtcag
cccaccagac ctgcttgtct 60cagctaaagc catctcattg ccaggttcct gcacagccag
100761100DNAHomo sapiens 761gctggcttcc atcttttgtc
tccctctact tgatacccca gttccctgca gtcctgcccc 60agcgccacct gggttttggt
tccaaagcat taccaatcat 100762100DNAHomo sapiens
762taccaccctc cactacctgg gtggaatatt tctttgctgc tttaaagtca ttaaaacatc
60ttgagaatga gaccaagaat ttaggagcct gtgctgtgat
100763100DNAHomo sapiens 763aaaaatgagc aggtcccctt gctctagaag tggcagcata
tcttctgcac caagaggagg 60gtattgagat gctcagagcc tccaccttcc cggagcatcc
100764100DNAHomo sapiens 764cctcccttct gagtctgcag
taaacccctg cctttaaatt ccctctagat aacagtcatc 60attggaaaca accaagaaat
gcattttatc tgaatttgcc 100765100DNAHomo sapiens
765acttaaaatt ctgccattta ccataaatcg ctttggaagg catgggctac tttcaagggt
60gcgatgatga cctacagtca atgacttaga caagggcgat
100766100DNAHomo sapiens 766gccagtgggg cttggtatgt tctcaagcat cattacccat
gccatcccca ttcagaggtt 60gtggagcagc tcgtgcgacc tctccttcaa atgggcttta
100767100DNAHomo sapiens 767gggaaagtta aatgggagtg
acccagacaa tggtcactca aaagactcac ataaatgagt 60ctcctgctct tcatcaagca
attaagacca gttccccttc 100768100DNAHomo sapiens
768tagtggaaat aagacgtcaa atacaaagtt ttaagagaag caaatgcagc agcggcggct
60gcctgtctct taccatgtcg ggcgcctggt cactgcgagc
100769100DNAHomo sapiens 769cttgcaaagc tttggcatgg aatcattcct ccaagtccat
taacaagggc tggggcctga 60gcagccagtc ggcccggcag cagaagccac gcatcccagc
100770100DNAHomo sapiens 770tctgggtagt ccggggagac
ccaaagccca ggccgggcct ggcagccacc ctcccagagc 60ctccgctagg ccagtcctgc
tgacgccgca tcggtgattc 100771100DNAHomo sapiens
771ggaacagaat ctgtccttct aaggtgtctc cacagtcctg tcttcagcac tatctgattg
60agttttctct tatgccacca actaacatgc ttaactgaaa
100772100DNAHomo sapiens 772taattcagga taatgatgca cattttacct aaaacttatc
ctaaagtgag tagttgaaaa 60gtggtcttga aaaatactaa aatgaaggcc actctatcag
100773100DNAHomo sapiens 773aatatcaaag tgtttctcct
taatcacaaa gagaaaacga gttaacctaa aaagattgtg 60aacacagtca ttatgaaaat
aatgctctga ggtatcgaaa 100774100DNAHomo sapiens
774aagtatttga gattagttat cacatgaagg gataacaagc taatttaaaa aactttttga
60atacagtcat aaactctccc taagactgtt taatttctta
100775100DNAHomo sapiens 775aacatcttac tttaaaaatg aatgcagttt agaagttgat
atgctgtttg cacaaactag 60cagttgataa gctaagattg gaaatgaaat tcagatagtt
100776100DNAHomo sapiens 776aaaaaaagcc ttttcagttt
cggtcagcct cgccttattt tagaaacgca aattgtccag 60gtgttgtttt gctcagtaga
gcactttcag atctgggcct 100777100DNAHomo sapiens
777gggcaaaacc acctcttcac aaccagaagt gataaattta ccaattgtgt ttttttgctt
60cctaaaatag actctcgcgg tgacctgctt cctgccacct
100778100DNAHomo sapiens 778gctgtgggtg ccggagaccc ccatgcagcc atcttgactc
taattcatca tctgcttcca 60gcttcgctca attaattaaa aaaataaact tgatttatga
100779100DNAHomo sapiens 779tggtcaaaac gcagtcccgc
atcggggccg acagcactgt gctagtattt cttagctgag 60cttgctttgg cctcaattcc
agacacatat cactcatggg 100780100DNAHomo sapiens
780tgttaatcaa atgataagaa tttcaaatac ttggacagtt aaaaaaatta atatacttga
60aaatctctca catttttaag tcataatttt cttaaccatt
100781100DNAHomo sapiens 781tttctcagaa gccacttcaa acatatcctg tcttttaaca
gtaagcatgc ctcctaagat 60aaacaatcct tttctcttgg aaaccagctt caaggcactg
100782100DNAHomo sapiens 782aggtcctgga gcctccctaa
gcccctgtca ggacggcagc caccgtttct gggctacccc 60tgcccccaac cctgctctca
tcaagaccgg ggctacgcgt 100783100DNAHomo sapiens
783ccctcctggc tggattcacc cactccgaca gttctctttc cagccaataa agaatttaag
60atgcaggttg acacacagcg cacctcataa ttctaaagaa
100784100DNAHomo sapiens 784aatatttcac gattcgctgc tgtgcagcga tcttgcagtc
ctacagacac cgctcctgag 60acacattcct cagccatcac taagacccct ggtttgttca
100785100DNAHomo sapiens 785ggcatctcgt ccaaatgtgg
ctccccaagc ccccaggctc agttactcca tcagacgcac 60ccaacctgag tcccattttc
caaaggcatc ggaaaatcca 100786100DNAHomo sapiens
786cagaggctcc cagatcctca aggcacccca gtgcccgtcc cctcctggcc agtccgccca
60ggtcccctcg gaacatgccc cgaggaccaa cctgcaatgc
100787100DNAHomo sapiens 787tcaggaaacc ccacaggcag tagcagaaaa caaaggccct
agagtggcca ttcttacctg 60aggagacggt gaccgtggtc cctttgcccc agacgtccat
100788100DNAHomo sapiens 788gtagtagtag tagtagtaat
cacaatggca gaatgtccat cctcacccca caaaaaccca 60gccacccaga gaccttctgt
ctccgggcgt cacatggaag 100789100DNAHomo sapiens
789ctgactgtcc gtggccctgt cctgcccttc tcatggaacc ctctgctggc ctcccacgta
60ccccacattc tggcctgacc cctcagaagc cagaccactg
100790100DNAHomo sapiens 790tcggcctggg aagtccaact gcaagcagac ggctgctaag
tcacccccag gagtccaaaa 60accccggggg gcacccgtcc cagagagcgg gtgccttgga
100791100DNAHomo sapiens 791gcgggacaga gtcccaccac
gcaatcatca cgacagcccc tgagaatgct ccaggtgaag 60cggagagagg tcaccccaga
ccagccgaag gagcccccca 100792100DNAHomo sapiens
792gctgccgaca tctgtggccg gacttgggga ggacaggctg ggttcccatt cgaagggtcc
60ctctccccgg ctttctttcc tgacctccaa aatgcctcca
100793100DNAHomo sapiens 793agactctgac cctgagaccc tggcaagctg agtctcccta
agtggactca gagagggggt 60ggtgaggact cacctgagga gacggtgacc agggttccct
100794100DNAHomo sapiens 794ggccccaggg gtcgaaccag
ttgtcacatt gtgacaacaa tgccaggacc ccaggcaaga 60actggcgccc cgctacgtcc
ctgggaccct ctcagactga 100795100DNAHomo sapiens
795gcccggggag ggcccggggg ttgttgggca ttggacccca gaggcctagg gtggccctgg
60ccacagagag acccgtgctg ctgggctcag gaggaaggag
100796100DNAHomo sapiens 796catctggagc ccttgcccct cgtctgtgtg gccgctgttg
cctcagggca tcctcctgag 60ccccccagga tgctccgggg ctctcttggc aggagaccca
100797100DNAHomo sapiens 797gcacccttat ttccccccag
aaatgcagca aaacccttca gagttaaagc aggagagagg 60ttgtgaggac tcacctgagg
agacggtgac cagggttccc 100798100DNAHomo sapiens
798tggccccagt agtcaaagta gtcacattgt gggaggcccc attaaggggt gcacaaaaac
60ctgactctcc gactgtcccg ggccggccgt ggcagccagc
100799100DNAHomo sapiens 799cccgtgtccc aaggtcattt tgtccccagc acaagcatga
ctctgcccac cctttgcccc 60agcagcagag tcccagttcc caaagaaagg ccttctgctg
100800100DNAHomo sapiens 800aacgtggtcc caaacagccg
gagaaggagc cccggagggc cccacatggc ccagcgcaga 60ccaaggagcc cccggacatt
atctcccagc tccaggacag 100801100DNAHomo sapiens
801aggacgctgg gcccagagaa aggaggcaga aggaaagcca tcttacctga agagacggtg
60accattgtcc cttggcccca gatatcaaaa gcatcacaca
100802100DNAHomo sapiens 802gggacacagt ccctgttcct gcccagacac aaacctgtgc
ccgtgcagga cactcgaatg 60ggtcacatgg cccaagcaca gagcagaggc agccggcgtc
100803100DNAHomo sapiens 803cctgtcccca gccacacaga
cccccgggct gagacccagg cagggagggg tgacgttccc 60agggagacgg tggccgggct
gccctggccc cagtgctcca 100804100DNAHomo sapiens
804agcacttgta gccacactaa agcgcaggcc tggtccccgg cacatgaaca gccagcgccc
60agccccagcc caggctctgc ccacaacttc tccttcccgt
100805100DNAHomo sapiens 805ccctgccctc ggcctgcttg ctacctgtgg agggtccctg
acggggctga agcccagcgg 60ggtccctgcc tgtccttggg ggctccagct ggccccaggg
100806100DNAHomo sapiens 806ctaagtgaca gcagggctct
ggcatgcagc ccatggcgga gaccccaggg atggcagctg 60gtgtggcctc aggccagacc
caggccggct gcagacccca 100807100DNAHomo sapiens
807gatacctggc ctggtgcctg gacagagaag actgggaggg ggctgcagtg ggactcacct
60gaggagacag tgaccagggt gccacggccc cagagatcga
100808100DNAHomo sapiens 808agtaccagta gcacagcctc tgccctcctg cttctcccat
acaaaaacac accctccgcc 60ctcctgccga cctcctttgc tgagcacctg tccccaagtc
100809100DNAHomo sapiens 809tgaagccaaa gcccttgcct
ggcccagtac acctggctcc ccgctatccc cagacagcag 60actcacctga ggagacggtg
accagggtgc cctggcccca 100810100DNAHomo sapiens
810gtgctggaag tattcagcca cggtgagtca gccctgagcc aggggctaca gaaacccaca
60gcccggggtc ccgggggagc atggtttttg tagagctgcc
100811100DNAHomo sapiens 811aatcactgtg tccccagtta gcacagtggt tctcagctca
gccaaaaccc tgcggctggt 60agggggcctg tggggctggg ggctgatgtg gctgcggtct
100812100DNAHomo sapiens 812tgctgggtct gtcctctgtg
ggaggggctg ctacccaggc ccaggactgc agtggagggc 60tcactgaggg gcttttgggt
ctggcctgag ccgctgtggg 100813100DNAHomo sapiens
813gctctcaggt ctactgcggg gacactcggg tctgcccctg gcttaggtgg acagtgtccg
60tgcccacctg tgccctgagg ctccatttca ggctgatatc
100814100DNAHomo sapiens 814tgtctgtatt gtccctaccc gctgcatggc catgtccttt
tgggtttata aattgccccc 60aaatcacgca ggcatcattc aggcttttta tattccctgg
100815100DNAHomo sapiens 815tattccctgg gccaccaggt
gcctccaccc agaaagctga gatgtgggag gttctagagt 60cattctgcaa ccctggatga
gcccctgcag cctcagtgct 100816100DNAHomo sapiens
816actgaggttc cagcaagacc tggagcaggt gcagatgagg cctgaggcca ggtgaagccc
60aggccaggtg aggtccaggc cagtgaggcc caggtcagat
100817100DNAHomo sapiens 817gaggcccagg tcaggtgaag cccaggtcag gtgaaaccca
ggtcaggtga ggcccagatc 60atgtgagctc aggacaggca aggtccaagt caggtgaggc
100818100DNAHomo sapiens 818cgagctcagg tgaagcccag
aggtgaggtc taggccaggt gaggtccagg ccaggtgagg 60tccaggtcag gtgaggccca
ggtcaggcaa ggctgaggta 100819100DNAHomo sapiens
819tccaggtcag gtgaggccca ggtcaggcaa ggctgaggta gatgtatgag acttctgtaa
60ttttcagttg gtgccaaccc tgcctggtgt ccctgcccct
100820100DNAHomo sapiens 820cctcccagcc catgctctgt gcctgccaga tggcggcccc
tgcacaggtg ctgctggctg 60tggaggagct gggctctgcc tccctgtgca tgggcgtccc
100821100DNAHomo sapiens 821gcctgcagcc tgtccgggga
tgcccaggga ggtgagtgcc accacatatc aggccttttc 60tctttaaagt catttctttg
gggatacatc atcaatgtct 100822100DNAHomo sapiens
822tctaaacaca gctgtgtgca ttttcctctt cttgcaattt agaattttaa ctgctgtttt
60caaggtactg taatgtattt gttctcttct tgttaggaga
100823100DNAHomo sapiens 823cttgccaacc ctgtgtgtct cagttcatac cctcttcctt
ccccagtaga agtaacgacc 60actgtgttta tgtgatcatc cttttcttga ttttccttat
100824100DNAHomo sapiens 824tgtgatcatc cttttcttga
ttttccttat agttttccta gtggaaagtt tatcccttaa 60gaagatagtt cattttgccg
gctgtaaatt ttatttagaa 100825100DNAHomo sapiens
825ctgccatcgt ttatttgcct gttttccttc agatggctgt ttgcttcatt ctcagtttgg
60ggctatgaca aacatatgtt ctgcacatct ttgcccatga
100826100DNAHomo sapiens 826ggctctcagg gagggctctg gagctggcat tgcctgcagg
gctctgcttt gttgcaggga 60gttcctgcca aggcttttca gagtgtctgt gcccagcctg
100827100DNAHomo sapiens 827aaggtacaca ctgtactttg
cccttgcatc aggcactttc cttgtgcttg cttctgtgtg 60gctccacatt ctggagaatt
tattcagatc tgtgctgcaa 100828100DNAHomo sapiens
828cttcccacac tgtcctcctg ggctcactcc cagccatcga tcttgaacac cagtttatgg
60aactatctgc acaggaaagc agaaacagca aaaggccctg
100829100DNAHomo sapiens 829ttgcgtggac cctgtttttg gtcaagggaa gtacttgctg
gtgaaggaga cctcccctcc 60tttctttctc aggagccccc tctgatgccg ttgcctggtg
100830100DNAHomo sapiens 830tttctcaggg ctggtgctgg
gggctcagca gtgtctgccc tgttccaggt gggaatgtgg 60gtctgttctg tttccacgcg
gtgttctggg gccgccagtg 100831100DNAHomo sapiens
831cagcagtgtc tgccctgttc caggtgggaa tgtgggtctg ttctgtttcc acgcggtgtt
60ctggggccgc cagtgagggg ctcgggatgt cagcggctgg
100832100DNAHomo sapiens 832tctctgtccc tatggtctgg gctccggttc actgctcccc
tgccctccag gtcggtcact 60gactcagtta ctatccagcg ggctccgtgg ctgttcagtg
100833100DNAHomo sapiens 833gggagcaaat ggagagggaa
gtggcagcgg cccgagtgcc aggcggtccc ggtttggggt 60tgatctttgt ggaacagctc
cctggcccgt gtgtaagtgg 100834100DNAHomo sapiens
834tcgggggagg cacggaggtc tggagctaca agcggtggca ggaaggcagg tcccagtctt
60gggggtctgg agcttatctt cttcctgtga actgagtgtg
100835100DNAHomo sapiens 835atggaggacc tgcctcggat gacaccccta tcttaagaag
gtcatggtgg gttccagctg 60ggaggaaggg aagtgggcca cctcctgggg gtcttccacc
100836100DNAHomo sapiens 836gtcttccacc cccaccacct
cagcctgggg cctctgtgat tcctctctgc acagacccca 60aagtctgtgc tgccgcaggg
caggaaggaa gggcctgtgg 100837100DNAHomo sapiens
837tcgaggttgg ggccacagtg gtgttcccta agcccgagtc tggtctcatg gcccgccccg
60cagcaggtcc tgagtgaggg acagagaccg gggcggggtc
100838100DNAHomo sapiens 838tttggtcctg gtggactctg gggtggattc cagtggggag
tcatcagggt cggtgtcccc 60cagggtactg gggtgtctct gctcctggag tcggctctgg
100839100DNAHomo sapiens 839cctgggtttt tgtacaggag
gtgccctggg ctgtgtcttt gtggtctgtg tgcacagtaa 60tatgtggctg tgtccacagg
gtccatgttg gtcattgtaa 100840100DNAHomo sapiens
840gtgtccttgg tgatggtgag cctgctcttc agagatgggc tgtagcgctt atcatcattc
60caataaatga gtgcaagcca ctccagggcc tttcctgggg
100841100DNAHomo sapiens 841gctgacggat ccagcccaca cccactccac tagtgctgag
tgagaaccca gagaaggtgc 60aggtcagcgt gagggtctgt gtgggtttca ccagcgtagg
100842100DNAHomo sapiens 842ctgtggagaa agcataagaa
gatgaagccc acaaacaaga aaactgatgt ttcacccgtg 60aaggagtccc tgaccacagc
actcacatga agggatggtc 100843100DNAHomo sapiens
843agcagcagga gcgtggagca aagtgtgtcc atggtggggc acaggagtca ctgagctggg
60acctgtgctc ggctttttca acccagagga gggtggagct
100844100DNAHomo sapiens 844aagtgtgtcc atggtggggc acaggagtca ctgagctggg
acctgtgctc ggctttttca 60acccagagga gggtggagct ggtggagatt tgcattcccc
100845100DNAHomo sapiens 845agatttgcat tcccctcatc
tgtgccctac tctatgggat ggagtcaggt ttcaggactc 60aggagggtgt tgcatctgtg
gtgaggacca gtgatagtaa 100846100DNAHomo sapiens
846catgatcagt gtaattcaga tggcattaat ctaaggctgg gcaagtagat tctgagtaga
60agtctttgca gaagtcatga ttatgaggtc atgttggtct
100847100DNAHomo sapiens 847gcccttcaca gagtccacat agtatttctc acttccatct
tgctttatgt tggccaccca 60ctccagcccc ttccctggag cctggcggac ccagctcatc
100848100DNAHomo sapiens 848tgagtcctct gtgctcagtg
ctgatcacca agtggaaagg ccttggagtc cagggctaag 60gctcctctct gagacctgca
gggtcagggt tgggttggtt 100849100DNAHomo sapiens
849ttcatcagta gagggagggc cctatttgca tgtctcctac tatataagaa gctctagtgg
60gatgctggag gaataggctg tacccatata agaagacggt
100850100DNAHomo sapiens 850agggccctat ttgcatgtct cctactatat aagaagctct
agtgggatgc tggaggaata 60ggctgtaccc atataagaag acggtgctct gcagaagttt
100851100DNAHomo sapiens 851gctgacaatg atggtatttg
gaaaatatgc tgtcttatga aattgtgctg tgataaacac 60tttgccctga tcaccctatt
acatttttta aaaaatgtgt 100852100DNAHomo sapiens
852caaacacaga gacaacctag tcagaaactg ccacatatat tcactgctta tctcactcac
60gtccactcaa tgtctctagt tctccataaa tcacctttta
100853100DNAHomo sapiens 853taatagcaac aaggaaaacc cagctcagcc caaactccat
ggtgagtcct ctgtgttcag 60tgctgatcac cgaatggaaa ctcctgggaa ttctggggct
100854100DNAHomo sapiens 854gtcctctgtg ttcagtgctg
atcaccgaat ggaaactcct gggaattctg gggctggggc 60tcttctccca gagctgcagg
gtctgggctc ggctggtttt 100855100DNAHomo sapiens
855tatcagcaga gggagggccc tatttgcatg tctcctacta tatagcaagc tctagtggga
60cgctggagga gagggcagtg cccagagcag atgagagggt
100856100DNAHomo sapiens 856cccggaaaac actggaggta atcctatctc tcaggaaaat
ataacttcag attatgtgat 60tgtgacttga tgatcaatta gcagtcatca tcttatttaa
100857100DNAHomo sapiens 857tgtttacata tttgcagaat
atattcagtg caagtgtcaa tgttacattt ttagagaaga 60tgaattacat acataacaga
gcagttgtgc aatgtgtcca 100858100DNAHomo sapiens
858actcacactt aatctctcta gttctccata aatcaccttt taaaatagca gcaaggaaaa
60tccagctcag cccaaactcc atggtgagtc ctctgtgttc
100859100DNAHomo sapiens 859gatgctattt aatagcccaa ttcctgaccc aggatgagaa
agagcaaata catgacacat 60ggacgacaca attgtagaag ctgagggttc aagccgtaat
100860100DNAHomo sapiens 860cctgttagag gccacgcatc
ccctacccat ccctgaactc tgtgttgaca gagcttcccc 60cactggagaa caagctcccc
caggacacgc acctcactta 100861100DNAHomo sapiens
861ggcccttcac ggagtctgcg tagtatgtgc taccaccact accactaata gctgagaccc
60actccagccc cttccctgga gcctggcgga cccagctcat
100862100DNAHomo sapiens 862ggcatagctg ctaaaggtga atccagaggc tgcacaggag
agtctcaggg accccccagg 60ctgtaccaag cctcccccag actccaacag ctgcacctca
100863100DNAHomo sapiens 863actgtttctc tcactcttat
ccattcacac tcaatttttc tatttctcca tgaattacct 60tttaaaatag ccacaagaaa
aagccagctc agcccaaact 100864100DNAHomo sapiens
864ccatggtgag ttctctctgt tcagtcctga tcaccaaatg aaaacacctg aaaatcccag
60ggctgggctc ctctctcaga gctgcagggt cagggctggg
100865100DNAHomo sapiens 865tttgcatatc tcctactata tagtaagctc tggggtgaga
ggcctttgga gatagtgggg 60ctcagagcat gtcagaatgt cctcggggag atctgtgata
100866100DNAHomo sapiens 866ttgaaagcat tgggaaattg
tgctttccta ttgtcagttt gttttgtgat aaacttaaac 60cttaaaacct aaaaatctta
taattttgta atttttattt 100867100DNAHomo sapiens
867gaggtaccat agatctacat aaactgcata tttttaaagt tagcaccaat catcttttat
60ttttacatac gcagagaaac catggtatat agtatcaata
100868100DNAHomo sapiens 868ttatttccat gttaaagatg aaaaattatc agcaaaagca
caggtgggtt ttacaatgtc 60cccagtgctc acttttggtc agagtgagcc tgggcatctg
100869100DNAHomo sapiens 869tcctacataa tgacagtgta
cacatctttc cattgctgtt ttactcaatt actcaaccca 60ttttctaaac agatttaaac
ttcataaatc ctgtcatctc 100870100DNAHomo sapiens
870ctcagcctca gcacagctgc ctcattcctc agggtttctg acgctctcag gatgtgggtt
60ttcacactgt gtctgttgca cagtaataca cggccgtgtc
100871100DNAHomo sapiens 871gctcagctcc atgtaggctg tgtctgtaga tgtgtcctcg
gtcatggtga ctctgccctg 60gaacttctgt gcgtagattg tttcaccatc ttcaggatca
100872100DNAHomo sapiens 872ttcaggatca aaacctccca
tccactcaag cccttttcca ggagcctgtc gcacccagtg 60catggataat tcagtgaggg
tgtatccgga aaccttgcag 100873100DNAHomo sapiens
873gagaccttca ctgaggcccc aggcttcttc acctcagccc cagactgtac cagctggacc
60tgggcgtggg tgcctgtgga gaggacagag gagtggatga
100874100DNAHomo sapiens 874gacaccactt aactggaccc agtcccctca tcagccctgg
aactcaggat tctcttgcct 60gtagctgctg ccaccaagaa gaggatcctc caggtgcagt
100875100DNAHomo sapiens 875gagggtggga atctgggaga
gcaaggggct tcccataagt gttctgataa aaatcctctt 60tgtttagggg gaaagtgatg
atttttttga atgatagaga 100876100DNAHomo sapiens
876atacatcacc caaacattta aaaatgtatt gtgtaaagaa gtgtaaatgg catctcagcc
60atttacacac tgcaagacac acagcttatt agtgtgcctg
100877100DNAHomo sapiens 877tggtgaatcg gcccttcacg gagtctgcat agtatttatt
acttccatca tatgatataa 60ctgccaccca ctccagcccc ttgcctggag cctggcggac
100878100DNAHomo sapiens 878acaatcactt gagttcagac
acaccaggat tcacttaatg ttatttttag ttcagaacct 60ctatcaggtt tagagggaat
cgctctgtcc cagggagtgg 100879100DNAHomo sapiens
879atcttacaat agcaaaacgg tcttagaaaa cccaacataa tctacagcga gacctcagca
60tggcaagcaa ggaatcacta aagccaccag ggagatccgg
100880100DNAHomo sapiens 880cactaaagcc accagggaga tccggatgca ctgatacgat
ccagaaacat agcgagtccg 60ggaactgatg cggactttga ggcagcctct tttttttttt
100881100DNAHomo sapiens 881gatggtgaat cggcccttca
cggagtctgc atagtattta ttacttccat cataccatat 60aactgccacc cactccagcc
ccttgcctgg agcctggcgg 100882100DNAHomo sapiens
882acccagtgca tgccatagct actgaaggtg aatccagacg ctgcacagga gagtctcagg
60gacctcccag gctggaccac gcctccccca gactccacca
100883100DNAHomo sapiens 883ctcgactctt gagggacggg ttgtagttgg tgcttccact
atgattgatt tccccaatcc 60actccagccc cttccctggg ggctggcgga tccagctcca
100884100DNAHomo sapiens 884ggctggcgga tccagctcca
gtagtaacca ctgaaggacc caccatagac agcgcaggtg 60agggacaggg tctccgaagg
cttcaacagt cctgcgcccc 100885100DNAHomo sapiens
885actgctgtag ctgcacctgg gacaggaccc ctgtgaacag agaaacccac agtgagccct
60gggatcagag gcagcatctc atatcttcat atccgcattc
100886100DNAHomo sapiens 886ctgagacact cacatctggg agctgccacc aggaggagga
agaaccacag gtgtttcatg 60ttcttgtgca ggaggtccat gactctcaga aagcacttcc
100887100DNAHomo sapiens 887gaggatttgc atgtgggtgg
tgcctttgta tggataggta aaaagggatg agggaggccc 60cagtcttttg ggctcaccct
gggaggtgta tgctggctgt 100888100DNAHomo sapiens
888agttctcttc ctgtggcctc ccctcaccaa acccagagtc ctcttcttcc aggtaggaaa
60tgtgctgaag gagctggtct gggagacaag tgtgatcatg
100889100DNAHomo sapiens 889ggtctgggag acaagtgtga tcatggatca aagacagatt
ttggaataca gttaatactg 60ttctacattt aaagattcat ataacaccaa ccatacaccc
100890100DNAHomo sapiens 890aggtcaccta aattgtcatt
taccccttca gacatattga aacagctgct gagtgtaata 60atcacagtga attgagacaa
acctggatcc atgcaatgtg 100891100DNAHomo sapiens
891tactgtagtt cagaacatcc atcatggtta gaaggatgct acctgtccca ggaagtgggt
60tatttttaaa tagtacctga gagctgccct tctgagacct
100892100DNAHomo sapiens 892tttgaaattt gagattgtgt gtgagatctc aggagaaggt
agtagaatat atctccatcc 60ttctcaatgt gtaaccctga gaatatggcc tgacctctaa
100893100DNAHomo sapiens 893acatttctgt gtgaaaagat
gtacattggg gatagcagtg acagcttcag atgaaaactc 60tatagtacat cagcactgga
ggatagtctc atcaccaaga 100894100DNAHomo sapiens
894ttagtgaaat tacctttcct gggaaccaga gaggacctct gtgagctcta ccctctgaga
60gaacaaggaa ctctggttct tccctgacag gtcacacctg
100895100DNAHomo sapiens 895aacaagtggg ctggccttct atgagacgac agagggaaag
agacagactc aatatccaga 60gcgaggtgag ctccttacct acctaccagg tggtctctgg
100896100DNAHomo sapiens 896gccatttgtt tgagcagacc
cagaagtacc ttcctcaccc tcaggagaat tatgaacatt 60gagagaaact gagatacttt
ttttatttac agggaatatt 100897100DNAHomo sapiens
897tcatcggcgt gtttacatct acctgggtgt gtacagggat gctaggatgt gctcatacac
60agaagagcaa gaattatatt tcgtggaaag aaaaccaaag
100898100DNAHomo sapiens 898agcttctgaa tttgtaggta ttgtttgctg caaatgtgtc
aggtcactag atcatgttat 60gctgctagaa gaaaaacttc ccaacattgt catggagaca
100899100DNAHomo sapiens 899aaatgcaaaa cagtaaagat
tcaactgaga ttcccttgaa aatcaccagt aatgaacagg 60ccaaaagaaa tcaaccattg
tggaaagagt ggtcattaag 100900100DNAHomo sapiens
900cccagtgtca ccttacacat cctgcaggtc acctcacaca tccaccaggt caccgcacat
60ataccccaca tcacctcaga cacaccctgg tcacctcata
100901100DNAHomo sapiens 901catacgtcag gtcacctcac gctcacccaa ggtcacctca
cacatcccgc aggtcacctc 60gtaaatcccc caggtcacca catacatgca ccagttcacc
100902100DNAHomo sapiens 902ctcttgaggg acgggttgta
gtaggtgctc ccactataat agatactccc aatccactcc 60agccccttcc ctgggggctg
gcggatccag ccccagtagt 100903100DNAHomo sapiens
903aactactact gctgatggag ccaccagaga cagtgcaggt gagggacagg gtctccgaag
60gcttcaccag tcctgggccc gactcctgca gctgcagctg
100904100DNAHomo sapiens 904gaacagaaaa acccacagtg agccctggga tcagaggcag
cctcccatat ctccatgtct 60gcatcctaga aacactcaca tctgggagcc gccaccagca
100905100DNAHomo sapiens 905ggaggaagaa ccacaggtgc
ttcattttct tgcacatgag atccatgact ctcagaaagc 60atttccctta tgagttggac
ctgaatttaa ggaaatgtgt 100906100DNAHomo sapiens
906ggtggcttcc tgtgggcgcc taagtgagga tttgcatggg ggtggtgcgt ttgtacggag
60cagtgaaaag ggatgagaga ggcgccagtc ttttgagctc
100907100DNAHomo sapiens 907accctgggag gagaatgctg gctgtgccct ttgagaactc
agttctcttc ttgggcctcc 60cctctccaag cccagagtcc tcttcttcca ggtaaagaga
100908100DNAHomo sapiens 908tgtgctgaag gagctggtct
gagagatgag tgtgatcctg gatcaaggac agattttgga 60atagggtcag tactgttcaa
cccttaaaga ttcatataaa 100909100DNAHomo sapiens
909acccaccaca cacccaggcc atctaaatag tcatttaccc tttcagacac attgaaacaa
60cagctgaatg taataatgac agtgacttca aacaatactg
100910100DNAHomo sapiens 910atgtttattg tagttcagaa catccaccat ggttacaggg
aagctcactg tccctggaag 60tgggtcattt tttaaaagca cctgagagct gtccttctgt
100911100DNAHomo sapiens 911aaggtagtgg gacatatctc
catacttctc aatgtgtgac cttgaagatg tgtcctgccc 60tctaaacact tctgattgaa
aatatgtaga ttggggatta 100912100DNAHomo sapiens
912gtggaaatgc cttggaatcc agggctaagg cacctctctg agagctgcag ggtcagggtt
60gggttggttt tcatcagtag agggagggcc ctatttgcat
100913100DNAHomo sapiens 913ggacccttga ggagtaggct gtacccagat aagacgacgg
tgccctgtag aagtttgctg 60gcaatgattg catttggaaa atatgctgtc ttattatgaa
100914100DNAHomo sapiens 914attgtgctgt gataaacact
ttgcactaat caccctattt cattttaaat attcatgtaa 60actatgttct gtaggagaca
atattttctc catttacaga 100915100DNAHomo sapiens
915acactttgca ctaatcaccc tatttcattt taaatattca tgtaaactat gttctgtagg
60agacaatatt ttctccattt acagaagtgg aagtaaaccc
100916100DNAHomo sapiens 916ctgtatgcat ctaggagctc atgtctggga tgagtgaacc
ccggtatctg gccctgtgct 60cttcatcact gtctctgaca tccccctaaa ccaactccag
100917100DNAHomo sapiens 917gacaaagctg gatgtgtcta
gtgtttttat cagaacccac tttccgtaat aagagcatgt 60gtggttttgc tgccctccag
cactcttctg aaaatatgga 100918100DNAHomo sapiens
918gagaactagg atccaggcac attaattttc aggtacttct gacattgaac ttattttttc
60tatctttcta ttactctttc cttgtctaag tttccatttg
100919100DNAHomo sapiens 919agagagaccc acagtgagcc ctgggatcag aggcacctcc
catatcccca tgtctggatc 60cctgagatac tcacatctgg gagctgccac caggagaagg
100920100DNAHomo sapiens 920aagaaccaca gatgtttcat
gttcttgcac aggaggtcca ggactctcag aaagtatttc 60ccatgtgagc tggaacctga
atttaaggaa atgtgtggtg 100921100DNAHomo sapiens
921atttgcatgt gggtggtgcc tttgtatgga gaggtgaaaa aggaggaggg aggccccagt
60cttttgggct cgccctggga gtaggatgct ggctgtgccc
100922100DNAHomo sapiens 922tttgagaact cagttgtctt cttggggtct cccctctcca
agcccagagt cctcttcttt 60caggtaaaga gacgtgctga aggacctggt ctgggagatg
100923100DNAHomo sapiens 923ctgacagtgg tgaccatggt
tgagaacttt tcatctcctc tgtgaggatc aatctgcatt 60ttctgcatag gagaataggt
tttcatatta aaacaatcat 100924100DNAHomo sapiens
924tttaaaaata tgtagaaatg accctagtaa tcacagaatt ccgaacttag gttcagtaga
60gaaactttaa gaagatgaag tcccacatcg tgacaggaaa
100925100DNAHomo sapiens 925tggagatggt gaatctgccc ttcacagagt ctgcataata
tgtgctaccc ccattactac 60taatagctga aacatattcc agtcccttcc ctggagcctg
100926100DNAHomo sapiens 926gcggacccag tgcatagcat
agctactgaa ggtgaatcca gaggctgcac aggagagtct 60cagggacccc ccaggctgga
ccaagccttc cccagactcc 100927100DNAHomo sapiens
927ttctctcact catgtccact cacactcaat atctctattt cctcatgaat cacctttaaa
60aatagcaaca aggaaaaccc agctcagccc aaactccatc
100928100DNAHomo sapiens 928atgactcttc tgtgttcagt gctgatcacc aaatgaaaac
acctgggaat cccagggcgg 60gggctcctct cccagagctg cggagtcagg gctgggctgg
100929100DNAHomo sapiens 929tagggcacat ccttcccatc
cactcaagcc cttgtgcatg ggcctggcgc acctagtgca 60tagagtaact ggtgaaggta
ggtgtatcca caagtcttgc 100930100DNAHomo sapiens
930aggagacttt cactgatgcc ccagccttct tcatctcatc cccagactgc accagctgca
60cctgggactg ggcacctgtg gagaggacac gggagtggat
100931100DNAHomo sapiens 931gaaaacttgt tcacagtagc accttcatgg aatgtttgta
tcaacgttat agagtgtggc 60cttttccact ctgtgaattt ggcttatatt acgactcttg
100932100DNAHomo sapiens 932aatggaatat ttatcttaaa
attagagtat gtacttgttt ctactgttct ttttttctca 60aatatataac ccattttgta
aacagcctta aacctaataa 100933100DNAHomo sapiens
933ctgctcagct ccatgtaggc tgtgctcgtg gatttgtccg cggtaatcgt gactctgccc
60tggaacttct gtgcgtagtt tgctgtacca aagataggga
100934100DNAHomo sapiens 934tgatccctcc catccactca agcccttgtc caggggcctg
tcgcacccag ctgatagcat 60agctgctgaa ggtgcctcca gaagccttgc aggagacctt
100935100DNAHomo sapiens 935caccgaggac ccaggcttct
tcacctcagc cccagactgc accagctgca cctgggactg 60gacacctgtg gagaggacac
aggggtgaat aaaatcctct 100936100DNAHomo sapiens
936cctgggactg gacacctgtg gagaggacac aggggtgaat aaaatcctct ttaactaaac
60caggatccct tcctcagcct taggactagg aagcccctta
100937100DNAHomo sapiens 937cctgtagctg ctgccaccac aaagaggaac ctccaggtcc
agtccatggt gatgagctgt 60gctcccaggg gcttcttcag aggaggaatg tggttgttat
100938100DNAHomo sapiens 938gtgatgctct cagggcacca
atatatctat atttatctca gaagacctca ggttatttgc 60atatgcatga ggcagggtat
ttcacagctc aaagcctgat 100939100DNAHomo sapiens
939tttgcatatg catgaggcag ggtatttcac agctcaaagc ctgatctagg atgagaaaga
60aaacacagat gccacatcag ctgtacaagt gtgggatgct
100940100DNAHomo sapiens 940cagaacaaac cccaacccca ggatgcactc ctcactgtga
acccacattt tattggccta 60aagattacct gggttttttg tgggaccatt gctgtctctg
100941100DNAHomo sapiens 941acattgagca ggcacctaga
cccatcctgg tcccattagg aacactcaga gctcactggt 60aacactgaaa aggtggccac
tcgttaccct acatgagtgt 100942100DNAHomo sapiens
942ccagcaggac ccatggagag ttctgagatc tgctgggcac tcccaagaca gggtccccag
60cactttcctg agggtcctga cctcccaggt ccttcagtgg
100943100DNAHomo sapiens 943ttatccattt ctatgtgttc ttttgaaaat gtctactcat
gtcctttgct cattttaacg 60gagttatttg gttcttgttg ctgttgttgt tgtagagttg
100944100DNAHomo sapiens 944ttgcaaattc ttcatattag
ttccctgtca caggcaaagt gtgcaaaagt tttctgtcat 60tctgtaaatt gcgtattcac
tctgttgttg tgaaaaaaat 100945100DNAHomo sapiens
945tatttaggtt aattaaatct catctgtcta ttttttttta ggtagcagga cctttcatgc
60tgaatctttg tcaaacagga tacagcttct gcttgcatga
100946100DNAHomo sapiens 946accactaaca ggggacatgc catttattag taaagaaaaa
ggaggaaaac aaggctctga 60gtcagatggg gatgggaaac gcacgccctg ggcaggaaat
100947100DNAHomo sapiens 947ggcatctcag ccacactatc
ctgttctgca gaagtgggga gggagcacca ctgaaaaaca 60cctgggttct tgtacaggaa
gcgccctggg ctgtgtctct 100948100DNAHomo sapiens
948gtggtatccg tgcacaataa tacgtggctg tgtccacagg gtccatgttg gtcattgtaa
60ggaccacctg gtttttggag gtgtccttgg agatggtgag
100949100DNAHomo sapiens 949acctggtttt tggaggtgtc cttggagatg gtgagcctgg
tcttcagaga tgtgctgtag 60tatttatcat catcccaatc aatgagtgca agccactcca
100950100DNAHomo sapiens 950gggccttccc tgggggctga
cggatccagc tcacacacat tccactagtg ctgagtgaga 60acccagagaa ggtgcaggtc
agtgtgaggg tctgtgtggg 100951100DNAHomo sapiens
951tttcaccagc gcaggaccag actccctcaa ggtgacctgg gataagaccc ctgtggagaa
60gacataagaa gatgaagccc acaaaggaga gaatagattt
100952100DNAHomo sapiens 952ctgtggagaa gacataagaa gatgaagccc acaaaggaga
gaatagattt tttgcttctg 60aagtactacc tgaccacagc actcacagga cgggacagtc
100953100DNAHomo sapiens 953agtagcagga gcgtggaaca
aagtatgtcc atggtggaga gcaggattca ctgagcgagg 60ccctgtcctc gtcttttgaa
cccaggggag ggtggagctg 100954100DNAHomo sapiens
954gtggagattt gcatcccctc atctgagccc tactctatgg ggtgcactca ggtctcagga
60ctcagtaggg gagtgcatct gtggtgagga gcagtgagcc
100955100DNAHomo sapiens 955tactctatgg ggtgcactca ggtctcagga ctcagtaggg
gagtgcatct gtggtgagga 60gcagtgagcc ctcaggtgtg ggggtccacg tgtgctctcc
100956100DNAHomo sapiens 956atcagggaat ctatctcatt
tcagcaccat ggctctcagt caagtcttga cgctcctgct 60tctacagaca ggatcttctt
cgatgctccc gcaccggaca 100957100DNAHomo sapiens
957tgcaaccttc tggttttagt cctagaggat tagagtagaa atcaagagag ctgccgttcc
60tcctcccttc aagaataatg atggtgggca tctggggggc
100958100DNAHomo sapiens 958aaggggctcc ccacaagcat tctgatcaaa atcctctttg
attatgggga aaagtgatga 60atttgtgtaa aaaaattgga gagaataaat aagaaaatac
100959100DNAHomo sapiens 959agttacaagt aattatgtaa
agaagtgtgt gcttagcagt gtgtgtgcac acagctgcat 60tcctagaggc atgttccatg
aaaaatcgat gttgtccttg 100960100DNAHomo sapiens
960tgccccgtca gttctgtgga gagagtagac tgcatgaatg acttcccttt tctcagccca
60tgaatgagcg gatgctttgg acaagggaat tggaagactc
100961100DNAHomo sapiens 961ctgagggagc agcaggctga ctgttgcagc cttgctctgc
acctgcactg gatgtggtct 60ctgtgctcat aaggccgtgg aaactcatca atccaggttc
100962100DNAHomo sapiens 962caaaaagggg ttaaatgatt
ttggaaaagt aagtagaaaa taaaagaagg agggagtaag 60agcggacaga agggaggaag
gcaagcaagc aatgatgaac 100963100DNAHomo sapiens
963tgtgtaaaat tttcactaat taaaagacta ttatattgaa gaggtgccta ttaggcagcc
60ttttgatgtt aaccatgtaa tatacaccat gaacaacctt
100964100DNAHomo sapiens 964gaacaacctt gtagaacaca caagagcccc ctcagagaac
tggatgggtc aggtctccca 60tccagttgcc ttaggggtta ggaacgctcc catgttgttc
100965100DNAHomo sapiens 965tctggttttt gctcctgagg
acacaaacag ccagtgtttc ctccccggat gaatagagag 60gcccctgggg agggtgtgtc
tggcagctca ctctgcacct 100966100DNAHomo sapiens
966gtttcctccc cggatgaata gagaggcccc tggggagggt gtgtctggca gctcactctg
60cacctgcacc gcggaaggtt ttagatggtc cctctcacac
100967100DNAHomo sapiens 967aataatacat ggcggcgtcc gaggccttca ggctgctcca
ctgcaggtag gcggtgctgc 60tggagctgtc ggctgagatg gtgacgtggc cttggaagga
100968100DNAHomo sapiens 968tgggctgtat ctggtatcag
agttcccagg atagatgctc cccatccact ccagttcttt 60cccgggcatc tggcgcaccc
agtggatcca gtagctggta 100969100DNAHomo sapiens
969acaggagatc ctcagagact ccccgggtct tttcacctct gctgcagact gcaacagctg
60cacctcggca aagacacctg tgtgggagac acaaaatttg
100970100DNAHomo sapiens 970gtgtctggag tatgaaccat gtatcagcac cgaaaggttc
tagaagtcag actttcgggc 60agtgtgtcac taactctcag catgctggcc tggctcggcc
100971100DNAHomo sapiens 971cacagcaagg tcttctcgcc
tccctttggg taaatactga ggggtgcctc tgcaggacgg 60gacctctgcc agactccact
ccatacccag agaagcaggg 100972100DNAHomo sapiens
972aaaccaaaat tggagtcagc cttgaggtgt agctgttgag ccctcagcag ctggggagag
60ctggcggatg ctgccctccc cccagtttcc taatggtgtt
100973100DNAHomo sapiens 973gtttaaaaag ggtcagggga cgggggaaca gatggtggga
agagcacagt gcagacacct 60ggcaccggct ctgaaggcag catggcagct acaccgttgg
100974100DNAHomo sapiens 974ctgggaaggg tgtgcccctg
aagaagtcgt ttacattctc gagtcaattt tcctggagtg 60tacaatggac ctgtgggaaa
gcctgtatga aagggtaatg 100975100DNAHomo sapiens
975atgagggacc tagcacagtg tccaatattt tataggaact ggaattgagc tcataggagc
60tcaattttat tggcattgct gttgttggat ggttaaaggg
100976100DNAHomo sapiens 976gtggtatccc ttttctcaga ctcccctgaa atgtatggtt
tgctttgaac ccagagactg 60atgacaggtc tgccggtgtg gttgggtgca gccttaagtt
100977100DNAHomo sapiens 977gctacgggaa agtgttggag
ggggagaagt cagaggtaac cttgccccct ccctcaattc 60cagatgagga aattcaggcc
tgaaaaggga aagtgaccac 100978100DNAHomo sapiens
978ctcaaagtct catgccttgg aggacccagc aggaatccaa gacctctgaa aaggaccggc
60agggctcttg ccacggctgg gggtgtggtc atggtaacac
100979100DNAHomo sapiens 979aggttttcca tccatggaag gtacctgagg gattttctct
tcctccctag ggccagcatc 60agaggagtga atagctcagt tagctcatct caggggccat
100980100DNAHomo sapiens 980gtgccctcgg aggtggtttg
ccactttcac ggttggactg agttggagag aaacagagac 60ccacccaggg gtggggacaa
gctccctgca actcaggact 100981100DNAHomo sapiens
981tgcagatcac ttgcccaagt ggctccctag ctcctggctc ctggcccggg gcctgggact
60ctccccgaag tggggctggc cactgtgagg aaccgactgg
100982100DNAHomo sapiens 982aggcagggac ctcttggatg ccccaggcag ttgggatgcc
acttctgata aagcacgtgg 60tggccacagt aggtgcttgg ttgctccaca gcctggcccg
100983100DNAHomo sapiens 983agctcagcgc tgcagaaaga
aagtgaaagg gaaaaagaac tgcggggagg cggggaggta 60ggatgaccag cggacgagct
gccacagact tgccgcggcc 100984100DNAHomo sapiens
984ccagagctgg cgggagggag aggccaccag cagcgcgcgc gggagcccgg ggaacagcgg
60taggtgacca aagtctcctc tgtaacccct aaggtcgggc
100985100DNAHomo sapiens 985tgagaatcga ggctccgaga ctgtcagcta cttgctcaag
gtcacacagc aagtctggga 60ggatgggggg atggaatatg caaaatgtag ggccgggaaa
100986100DNAHomo sapiens 986cacctcgttt ccagcatccc
cgcaacgact ctgcgcggga accaggagcc gggaacccgg 60agcttggctt gctgtgccca
gagctccggg gccgtgggcg 100987100DNAHomo sapiens
987ggtggcagga aagcctggcg gcagcttctg cagagaagcc ggagcgcaga ctgggagcgc
60ggagcagaca cactcccccg gccacccttg gccgactccg
100988100DNAHomo sapiens 988cgcgcccggg atcctgcaga ggtgcgcgcc cttcttgtac
gccagacttt ggaccagggc 60cgccgttccc tgagcttcac tttccctgtt gggtcatatt
100989100DNAHomo sapiens 989ccatctctaa ctctggaatc
ttgggtattg ggctctccag gcggggggcc ctgctcaggg 60aggcagtagg gagccaaacc
tttaaccaga ggatgggata 100990100DNAHomo sapiens
990agtcctcaac tctcgttgaa catcttggcg aaggtgtgtg ttgttgggag gggtggggga
60gggatccccc cggactgaac cgatctcttg atctctcact
100991100DNAHomo sapiens 991tctctacctc gctttggggc cctgagtcac accctctaag
gagagaggct aaagcgcccc 60ggaaagccag cgtgcgaatg ccggggtggg agtgggagat
100992100DNAHomo sapiens 992tggatctccc tggggtccag
gaaagccgga atcggagcca ccatgcttag cttagtctgg 60aactcttaaa agccgcggtc
ctcctgagtc ccacagcccc 100993100DNAHomo sapiens
993tctccaccct aggtggcaca ggagaggtgg caaaagccta gaagttcaag gcatggctcc
60ctccccagcc gcagcctgga gtgtctaact ttggcaggaa
100994100DNAHomo sapiens 994gtcttccgtt tctgctcccc actccagaga aaaaataaat
aaatacttct ccggagtgag 60attaaggaaa caggtacttc ttcctcttgg agaaagagga
100995100DNAHomo sapiens 995cttctccgga gtgagattaa
ggaaacaggt acttcttcct cttggagaaa gaggagccaa 60aggaacttga ctccaacaaa
tgatcacctt gcaaaccccc 100996100DNAHomo sapiens
996ggctccctta ggggatgacc tggtctccaa caatctcaga gcgtttggag gcagggtctt
60tggagatgac tgagtgggga atcccaggct ccccacacat
100997100DNAHomo sapiens 997gaacatcacc tgggatgatc aacctgttca ggatgtaggt
tcccgggctc acccccaggc 60ccggttggct aggcctgggg tgaggctgag atcctgcagg
100998100DNAHomo sapiens 998ttaaaccatc tatcccaggt
gactccaatg ttcgtttgtg gggcaaaagt ccctcaagtc 60agagacactg ggaggcgctg
atgtggtctc atctctttac 100999100DNAHomo sapiens
999caagaggtga gaaggggtct gcggcctcgt ctccagccga gggcgggagg cgcctcgccc
60ctacacccat ccgctccctc caacccaggc cggggagggt
1001000100DNAHomo sapiens 1000acccacatgg ttccaggcaa gtaataacaa aataacacgg
catcccagtt aatgctgcgt 60gcacggcggg cgctgccggt caaatctgga aggggaagga
1001001100DNAHomo sapiens 1001gctcaggtag
tcgcggagga cggggttgag ggggatgcga gccaggttct cgcggcccac 60ggtggccacg
atgcgctggc ggcacagctc ctgcagcggc
1001002100DNAHomo sapiens 1002cgcacgcggc gctggcgcag cggggccccc agcatgcggc
gcggcgccgc cacgtagtgc 60tccagcagct cgaagaggca gtcgaagctc tcgcggctgc
1001003100DNAHomo sapiens 1003catccaggtg
aaagcggccg gcctgaaagt gcacgcggat gctcgtgggt cccgaggcca 60tcttcacgct
aagggcgaaa aagcagttcc gctggcggct
1001004100DNAHomo sapiens 1004gtcgcgcacc aggaaggtgc ccacgggctc ggcgcgcagc
cgctcgtgcg ccccgtgcac 60gctcaggggc ccccagtaga atccgcaggc gtccaggagc
1001005100DNAHomo sapiens 1005gcgctggcgc
gcgtgatgcg ccggtaatcg gcgtgcgaac ggaatgtgcg gaagtgcgtg 60tcgccggggg
ccggggccgg gaccgcgggg cacggccgcg
1001006100DNAHomo sapiens 1006ggcgcgcggg ggccgcgggc gaggaggagg aagaggagga
aggttctggc cgccgtcggg 60gctctgctgc tgtggagact gcattgtcgg ctgccacctg
1001007100DNAHomo sapiens 1007tttaaaatca
cccaaatcaa aataatttta tcttcattaa taaataatca tcagaagttt 60aactaatttt
tactttataa tactaggttt aaaaattctt
1001008100DNAHomo sapiens 1008aatctgaatg cccaagtcgt tgattgtcgt ttgcctgttt
ccaaagattg gtagatagat 60gcctttttaa aaatctcatt tttctttaaa tctggtttac
1001009100DNAHomo sapiens 1009atggaaaacg
ttaggagagc tcatataatg aacggcaata gcaaccccct atcttgaaac 60gcgctctatc
atcccactga aattctacca cgtggaataa
1001010100DNAHomo sapiens 1010tgcttggagg gtcagagttg tggaactgcc caataaccag
tcgttactga gggttagttt 60gtgaaggagg ggacagactg cttctaaaat tctgtttaat
1001011100DNAHomo sapiens 1011gacagtcaat
taagatttct gagtctggct tgagggcctt tgcttccatc acagcccagt 60cgtccttggc
aagagagtct gtatatgggc cacagctcac
1001012100DNAHomo sapiens 1012aaaagcattg tttgaaaaaa tttattgaaa gaacattgtt
tgtaaaatga gtcccaatac 60ataggacaga ctttcctaag gtgagatgtg ttacttaccc
1001013100DNAHomo sapiens 1013agagctgtga
aaggctttac ggatggaaac tagagactga attttccaga attttaagaa 60gtctccccaa
ccaatggccc cccactttct ttttttaaac
1001014100DNAHomo sapiens 1014ggcgtgatct ccgaagccca cagtacactc atccataaag
taggaaacac tacaccctcc 60agtgctgtta gtagtgcttt ctactttatg ggtgactgca
1001015100DNAHomo sapiens 1015ctgtctgtct
gtccgtcggc gtgtactctt caggctgccc aggcctcctg actcctgctc 60caagagcccc
ccagccctcc ttgtggcttc ctaagatccc
1001016100DNAHomo sapiens 1016ccctcttccc ttccccctaa aggctccacc ccatcccccc
agtttcagag acactcaggt 60agagactagg gcctctggag gcctcacctt cagttctgtg
1001017100DNAHomo sapiens 1017aacccctggc
tggccgcttc cagccacgct agccaccctc cagcgtccaa atgaggcagc 60cacagctccc
ctgccaaggt cttggtctcc agtccacccc
1001018100DNAHomo sapiens 1018aaccgtgagg tcctgactgc ccagagcctc agtccccacc
cttcagcctc cccaccagcc 60caagatcctg accccccagg gcctaagtcc ccagcctccc
1001019100DNAHomo sapiens 1019caacagccca
gggtcctgac cccccagggc ctcaggccct ggcctcccca ccagcccaag 60gtcttgaaca
caccagggcc tcaattccca gcctccccac
1001020100DNAHomo sapiens 1020cagctcaagg tcctgactcc cccagagcct cagtcccagc
ctccatagca gcccaaggtc 60ctgacccccc agggcctcag tccccagcca ctccaccagc
1001021100DNAHomo sapiens 1021cccaaagtcc
tgactcccca gagccttgat tctcggcctc cccaccagcc caaagtcctg 60actccctcac
tgccctgctg ttcccctggc aggagcccaa
1001022100DNAHomo sapiens 1022ggctatccca acaaaaatgg tggccatgtt gggcggagga
agaggctggc gccccttgag 60acactggtcc cacttctcag cctctgcgta ccctctgcca
1001023100DNAHomo sapiens 1023tccccgcctt
actctccagc cctcctcctt ggacacctct ttccccgcct ggggtcccgg 60agccatttta
ccttccttca ctagagaggg tttcaaggcg
1001024100DNAHomo sapiens 1024ctaagatttt caagaagtta aacgtagaat taagattgtt
ctaattctgg ttgtaaactg 60ctattttaaa aaacaaaaca aacagaaaac atcaaaaaca
1001025100DNAHomo sapiens 1025aaacaaacag
aaaacatcaa aaacacaaaa agatattaaa acagcaagtc ttttgtacat 60cactgtagca
taagctgctt gaggttgtca tgcagaatag
1001026100DNAHomo sapiens 1026tatccttcac gtcacggaaa acaaggcgga tgttctccgt
gttgatagca gtggtgaagt 60ggtggtataa gggcttctgt tgctggtccc ggcgtttgtt
1001027100DNAHomo sapiens 1027ccggaaacat
tccaccagga atttttggac gtctcttaag cagtggggat ccccttcaaa 60ttctaggaaa
tagtctttga tgctcacaat ttgcaccttc
1001028100DNAHomo sapiens 1028tcctcaagca agtctgtctt gtttaagaac agaattatgg
agacattgct gaaaacccgg 60ttattgacga ttgtttcaaa aatgttcaga gactctgtaa
1001029100DNAHomo sapiens 1029ggcgattggt
cagtcgatct tccataagca cctggtcaaa ttcacttgag gaaacaagga 60aaagtattga
tgtcacactg tcgaaacatt caaaccaacg
1001030100DNAHomo sapiens 1030tttcctttct gatctctgac cacctacatc aaccattttg
aaaggaacat tttttatttc 60aaagtcgtat tcatggatgc ctttggtggg tcttctggca
1001031100DNAHomo sapiens 1031agcagaatat
cttgttgtga tggaatataa tcctggaaaa gaaaaaactt gttttatacc 60tattaatccc
gaagtaatgc gaatttttaa tggactacta
1001032100DNAHomo sapiens 1032tgtaaatatt tggccaacta agctgagtgg ctaagttctc
ctgctgcccg gagcttcttg 60gaacatgttt ccttttcgca aggggtttcc ctggcttcca
1001033100DNAHomo sapiens 1033ggagggccag
gaagaaattc gaattggcca ccgctttctc taaaatcact ccgctcaagt 60tatcacccct
ctgggctccc gaagaccggc tggctggagg
1001034100DNAHomo sapiens 1034ctggagatag tctcaatgct cgaaatgccg taaccgaagc
tccccgcggc gccggcactg 60ggatccaggg agctgctgct acagcgcagc tctggattcc
1001035100DNAHomo sapiens 1035tggatgtgtt
ggatatgtgc agggcgttcc tgggaggagc ggggagggag ggtgctgctg 60gcggggctgg
tctgcgtgtg ctttgcttct ctacaatggc
1001036100DNAHomo sapiens 1036atgctgcgtg tcggccatgc agaggcatgt cagtgagcag
gggctgaggg atctccctaa 60cggacctgct ttcagagggt cttttcatgc tgggagaacc
1001037100DNAHomo sapiens 1037ccagagacta
aatcatgcag ccaacggggt ggtccccggc ctcaaagcag ggaggggcga 60ggagctttgt
aggcaatgcc atctgctcct gaaacgccgt
1001038100DNAHomo sapiens 1038cagcctcctt agtagctacc gccttagtaa gtaccactta
gtaagtaccg ccttagtaag 60taccacttag tagctacctc cttagtaagt accacttagt
1001039100DNAHomo sapiens 1039aagtacctcc
ttagtaagta ccacttagta ctaccaccac gcctggctaa tttcgtattt 60tttttttttt
agtagagacg gggtttctcc atattggtca
1001040100DNAHomo sapiens 1040aggtcaggcg catactgcat gcgggtctcg cggtcgtgct
ccagccacag cacggacatc 60tggaagagcg ccagctccga ctccacgggg ggcggcagcg
1001041100DNAHomo sapiens 1041agtccagcag
ggcgcgcatc tcctcgaagt tgagcagcag cacatcctcc accaggtact 60tgttggccag
cttcttggtc tcctccaggc cgtgcagcgc
1001042100DNAHomo sapiens 1042ggcgatcttg cacacctgct tgtagttctg caccgagatc
tggtcgttga ggaactgcac 60gcagagcttg gtgacctggg ggatgtgcag gatcttgctg
1001043100DNAHomo sapiens 1043accgacagca
cctcctccac cgtgtccagg gacagggtca cgttggccgt gtagaggtac 60tcgagcacca
ggcgcagccc gatggacgag cagccctgca
1001044100DNAHomo sapiens 1044gcaccaggtt gttgatggcc cgggggctgg tcagcagctt
gtcgtcgggg gaggaagaag 60gagtcccggg ctcctcctgc ggcggcggct gctgctgctg
1001045100DNAHomo sapiens 1045tgacggctgc
tgctgcggcg gctgctgctg gtccttgggg gcccccaggc cgtcctggcc 60gccgacccct
cccccgagag gggggtggct ggagaagagc
1001046100DNAHomo sapiens 1046gagacttcag ccggagctgg ctattccaga gatggacctc
agaggattcc ttagtctaat 60taccttctgg gctggggtag aagatggtgt ctggagggaa
1001047100DNAHomo sapiens 1047gcacagaacc
aagttcccta ctgccgcact agctatgcaa atactgcagg gcacctgtgg 60gctcatgtcc
ctcctgcaag aaggtgtggt cagtccagta
1001048100DNAHomo sapiens 1048attcaaaaga cgtacttctg aaataggtgg agaaatgcat
ttatagcaaa aagtgctaaa 60aatatgttaa tagttatgct atttggttca ccaggttagt
1001049100DNAHomo sapiens 1049gtaataaacc
ataacaagag agactaaagg ccgtatctat atgaccttga aatctcatct 60tcagcgggct
tattcattca gtaaccaaac tatttttgta
1001050100DNAHomo sapiens 1050aggtgctgag tatttagctt aaagctaaat aagacacatg
ccctgcccta tagtaactgc 60ttggtaatat tcccagtggc ttccatgggc ctgataattt
1001051100DNAHomo sapiens 1051tcttagtact
gaattcaaag cactttgtgt cttgtctgca ggcccatttg cccagcagtg 60gccttgccag
gagagaacag gcccatgctc ctgtcctcat
1001052100DNAHomo sapiens 1052caaacaaaca attcaagaag aggatttaaa ttttagaaat
ttaaattggg gcattttagt 60taatcttact tttaaacacc aaacagtggc atcaatattt
1001053100DNAHomo sapiens 1053tgtcaacttt
ggtcaaataa gatcagatgt tcacatcaat catctacttt tcttggcctt 60ttctctattt
ggcctcctag tatgagcaca ctttgtaaaa
1001054100DNAHomo sapiens 1054tgtaataaaa acatgtggtg tgcttcttga catctaatcc
acttgcagta atttctaggc 60tttttgctcc tgttaggtcc tataaaataa tgacattagt
1001055100DNAHomo sapiens 1055atagatacct
agatgcaaat ttttttcagc cgaccacaaa attaggtcca ctctgagtgg 60tgaaaaacaa
aagattctaa cattctagca aactggtaaa
1001056100DNAHomo sapiens 1056ccatacacaa attatagaat acaaagaatg cagccgatgc
aaattctgtc actgacaagg 60tagcaaagcc atagcctgat actcctcagg acacctcatc
1001057100DNAHomo sapiens 1057acgcccactg
ggaacatggc acacactgga gattccagtc caaggacttt ggaatgtcaa 60cttagctctt
tacaaacaca actaagtttt tcagggaaaa
1001058100DNAHomo sapiens 1058agacttacat tggttttcct cttttggaaa attttaccga
ttgatgatgc ccttggtctt 60ctgtggagtc tattcttcta atcgggttgt tctccaattt
1001059100DNAHomo sapiens 1059tagtgtacaa
cgggcttgtt tcaggggagc ttgtttggga tgcagactgt caagacccaa 60cctggtatct
ggttcataag cagtccctga aacctccctc
1001060100DNAHomo sapiens 1060cggttccaac aagctgctca agccaggaaa cggtggtcct
ggggactcct ggaccttcag 60cttgagaaac actgaagggg taccatttac caccacatcc
1001061100DNAHomo sapiens 1061tactggatta
caaacgctag atctttggat ctccacgact agcaagcaag ttaaagactt 60ttagatggca
ggcgttatcg gtcaggttgg gagtgaacgc
1001062100DNAHomo sapiens 1062tttgtccaga ggaggaggta gggacgccgg gaagcaacaa
ctctgatttt atttcgccgg 60ctccacagcc tcccattgcc ccaggagccc acccgcactc
1001063100DNAHomo sapiens 1063caacccccgc
atctcggacc tgtggcctca gcccagactc acatcaccaa gtgcacctac 60ccagcctccg
ttatcctgga tccaggtgtg caggtgccgg
1001064100DNAHomo sapiens 1064ttcaggtact cagtcatcca cagggcgatg ttgtccacca
ggggcgacat ctcccggttg 60acgctctcca cacacatgac cccaccgaac tcaaagaagg
1001065100DNAHomo sapiens 1065ccacaatcct
cccccagttc accccgtccc tgaagagctc ctccaccacc gtggcaaagc 60gtccccgcgc
ggtgaagggc gtcaggtgca gctggctgga
1001066100DNAHomo sapiens 1066catctcggcg aagtcgcggc ggtagcggcg ggagaagtcg
tcgccggcct ggcggagggt 60caggtggacc acaggtggca ccgggctgag cgcaggcccc
1001067100DNAHomo sapiens 1067gcggcggcgc
cgggggcagc cggggtctgc agcggcgagg tcctggcgac cgggtcccgg 60gatgcggctg
gatggggcgt gtgcccgggc tgggaggaga
1001068100DNAHomo sapiens 1068agatgcccgg tgcgggggcg gcccccgggg gcgcggcgcc
cacatctccc gcatcccact 60cgtagcccct ctgcgacagc ttataatgga tgtacttcat
1001069100DNAHomo sapiens 1069cactatctcc
cggttatcgt accctgttct cccagcgtgc gccatccttc ccagaggaaa 60agcaacgggg
gccaacggca cctctcgccc cagctcccac
1001070100DNAHomo sapiens 1070cccacggccc ccagagaaag aagaggagtt ataatccagc
tattttattg gatgtgcttt 60gcattcttgg acgagggggt gtcttcaatc acgcggaaca
1001071100DNAHomo sapiens 1071cttgattctg
gtgtttcccc cttggcatga gatgcaggaa atttttattc caattccttt 60cggatcttta
tttcatgagg cacgttatta ttagtaagta
1001072100DNAHomo sapiens 1072ttgttaatat cagtctactt cctctgtgat gctgaaaggt
taaagaaaaa acaaactaat 60aagtaaaaaa tcaggtgcgt ttccctgtac acactgagtg
1001073100DNAHomo sapiens 1073aaagcagggc
atacacacta caagtaacac ggctaaaaag aatgtattaa gctgcctgga 60aattaaattt
actcgaatgc actttaagta aaaaatctca
1001074100DNAHomo sapiens 1074aaggtttcca ttgaaagtta cattaaacca atttcctgtg
cagagaactt acttgtattt 60tttaagtaca gcatgatcct ctgtcaagtt tcctttttgt
1001075100DNAHomo sapiens 1075aaaaccaaaa
caaatgcata aggcaacgat cccatcaatc ttcagcactc tccagttata 60gctgatttga
aacttcccaa tgaatcagga gtcgcgggga
1001076100DNAHomo sapiens 1076gagggagtaa aaattaggag gatttccaga tcgattccca
gacttctgct tcacagaaat 60gtcaatccgc aggaatccca accggagatc tcaagagctc
1001077100DNAHomo sapiens 1077gagaaaaaaa
aaaggcagcg gcggcggcag atgaattaca attttcagtc cggtattcgc 60agaagtcctg
tgatgttttc cccttctcgg caatttacac
1001078100DNAHomo sapiens 1078tgaaggagcc ggggacggag gcaggaatcc tcttctgatt
aaactccgaa cagcaaatgc 60attttccgaa aagctgctgg ataaatgaag gcaggacgcg
1001079100DNAHomo sapiens 1079cctggcccgc
cggtgccgag cgctagaagc ccgcgctgtg tgtggtgcgg cgaggggtgg 60ggagaaggag
gtggtggggg agggttttat tttttccctc
1001080100DNAHomo sapiens 1080ttttcctaaa aaggatgact gctacgaagt tctcccccct
ggaccccctc ttccgctgca 60ccccaccggc gcaccccgcc tccgggctgc gcaccctttc
1001081100DNAHomo sapiens 1081gtgtgtgtct
cgcctggacc ttttctagcc gtgtatgtgg gagtgtgtgt gtcgcctgga 60ccctttctag
ccgtgtatga gagtgtgtac acgcgcctac
1001082100DNAHomo sapiens 1082acacacacac gttgtgttac cggcgctcgg ccgccggggg
aagacccagg ccaatgccgc 60cccccaccgc ccccagcagt gggacctcag cgctgccctg
1001083100DNAHomo sapiens 1083ctgtgaagac
aggtgactct gcacgtttta agcaatgtct agggacgccc cgagcgtggt 60gtttactttc
aagtagcttc ctaggtgtcc gcgcactaca
1001084100DNAHomo sapiens 1084cacgcacgcg catccccgcc cgtgtccacc tgaacaccta
gtccgtggcc caggccatgc 60agaactcagc gctccaggga aggggtttat caagggcttt
1001085100DNAHomo sapiens 1085acgacagttt
aagtcaatgt tttccctctg tccctaacac cttttacact ggtttagtgc 60tacacgatga
ggacttccat atagtaactt tcaggcccac
1001086100DNAHomo sapiens 1086cgtcctaacg ctggggtggg tgggctccta aaggtctcca
cctttgcctc gtagccaatc 60ctagttggcc gcactttctc aaatgaggta catagataca
1001087100DNAHomo sapiens 1087gtgtctccat
ggagatggca gcaggacccg accccgtgct ggcccgcact ctcggcctcc 60ttatctggtt
taggaatgcg cggtatccac gctcgctcgc
1001088100DNAHomo sapiens 1088gcgggagcca cgcctcctct cccccccgcc cccgagaccg
ccacacgcgc ggggccccca 60cgtctccaag cggcactgga aggattcctc tccgtcccgc
1001089100DNAHomo sapiens 1089caggggtccc
gcctcgagat tctgggaaga ctgggggtgg gggaccagat cgcagcagca 60gctgcaccgc
gagttccgcg cctggccgtg tcgccccacg
1001090100DNAHomo sapiens 1090agggggactg tgggctcagc gcgtggggcc cggagcatct
gacaaggaca gagacagagg 60agggggtgga aatccccggg tgagtcaacc cgtgcctgag
1001091100DNAHomo sapiens 1091aagggggcga
gttccgacgc tccgcccggc tcggggccac gcgaggtccg cgccacgcgc 60gccttcaccc
acgacccatc cctgagccgg agttgaaaga
1001092100DNAHomo sapiens 1092ggaggcgtct gagccacgca gtcactttct ctttccttac
aaaacaaagc cacgcccccc 60gccgggggac cggaggaggc aaacaacttg gggaaaccga
1001093100DNAHomo sapiens 1093cccactttcc
ccttctgtcc ctaaagtttt ttcttcctct tgcctccccc agcccttttg 60aaagctcccc
gcgtcgtcct cctgctgccc cggctcctta
1001094100DNAHomo sapiens 1094gcagcttctg ggacgcacgg gagggaaaag ccgcggggac
cccccccacc ccagcctccc 60agccgggtga gatttggttg ctgtgtttcc tcctcacttg
1001095100DNAHomo sapiens 1095ccaccccagc
ctcccagccg ggtgagattt ggttgctgtg tttcctcctc acttgggcat 60ttaaaaaata
ttttaacacg aattgtccgc ggaattttca
1001096100DNAHomo sapiens 1096catggcctgg acccctctcc tcctccagct tctcaccctc
tgctcaggtg actgcctgtg 60gaatgccaaa gtgattattg gggacacatg ggatgacttt
1001097100DNAHomo sapiens 1097tctcttatat
tttaacattg tggggtgggt agtgaaccca gactcacctc tctgtgcctg 60cctcctctgt
tccagggtcc tgggcacagt ctgcgctgac
1001098100DNAHomo sapiens 1098ccaggaagcc tcggtgtcag ggaccgtggg acagaaggtc
accctctcct gtactggaaa 60cagcaacaac gttggaagtt atgctgtggg ctggtaccaa
1001099100DNAHomo sapiens 1099cagatttctc
acggtgctcc caaaactgtg atgtttggaa attctctgcc ctcagggatc 60cctgaccgct
tctctggctc aaagtctggg accacagcct
1001100100DNAHomo sapiens 1100ccctgactat ctcgggcctc tagcctgagg acgaggctga
ttattactgt tcaacatggg 60actacagcct cagtgctcac acagtgctgc aggcacatgg
1001101100DNAHomo sapiens 1101ggaaccgaga
caaaaacctg cccttggcct gtcccgaggc tgatcactcc atacttgcct 60atgacaaaca
aagagggtgc ctgtggctga tcgtacagtt
1001102100DNAHomo sapiens 1102gaaatgttgt ttgctcttgt ccttccttca ggccataatg
agcgtctctg ttttcagggt 60ctctctccca gcctgtgctg actcaatcat cctctgcctc
1001103100DNAHomo sapiens 1103tcaagctcac
ctgcactctg agcagtgggc acagtagcta catcatcgca tggcatcagc 60agcagccagg
gaaggcccct cggtacttga tgaagcttga
1001104100DNAHomo sapiens 1104aggtagtgga agctacaaca aggggagcgg agttcctgat
cgcttctcag gctccagctc 60tggggctgac cgctacctca ccatctccaa cctccagttt
1001105100DNAHomo sapiens 1105gaggatgagg
ctgattatta ctgtgagacc tgggacagta acactcacac agtgatacag 60gcagatgagg
aagtgggaca aaatcctcaa cctgctgagg
1001106100DNAHomo sapiens 1106aaggtcacca tctcctgctc tggaagcagc tccaacattg
ggaataatta tgtatcctgg 60taccagcagc tcccaggaac agcccccaaa ctcctcattt
1001107100DNAHomo sapiens 1107atgacaataa
taagcgaccc tcagggattc ctgaccgatt ctctggctcc aagtctggca 60cgtcagccac
cctgggcatc accggactcc agactgggga
1001108100DNAHomo sapiens 1108tcagccagac tcacctgcac cttgcgcagt ggcatcaatc
ttggtagcta caggatattc 60tggtaccagc agaagccaga gagccctccc cggtatctcc
1001109100DNAHomo sapiens 1109tgagctacta
ctcagactca agtaagcatc agggctctgg agtccccagc cgcttctctg 60gatccaaaga
tgcttcgagc aatgcaggga ttttagtcat
1001110100DNAHomo sapiens 1110agagatctgg gggaagctca gcttcagctg tggtagagaa
gacaggattc aggacaatct 60ccagcatggc cggcttccct ctcctcctca ccctcctcac
1001111100DNAHomo sapiens 1111tcactgtgca
ggtgacagga tggggaccaa gagaggggcc ctgggaagcc catggggccc 60tgctttctcc
tcttgtctcc tttcgtctct tgtcaatcac
1001112100DNAHomo sapiens 1112catgtctgtg tctctctcac ttccagggtc ctgggcccag
tctgtgctga ctcagccacc 60ctcagcgtct gggacccccg ggcagagggt caccatctct
1001113100DNAHomo sapiens 1113tgttctggaa
gcagctccaa catcggaagt aattatgtat actggtacca gcagctccca 60ggaacggccc
ccaaactcct catctatagt aataatcagc
1001114100DNAHomo sapiens 1114ggccctcagg ggtccctgac cgattctctg gctccaagtc
tggcacctca gcctccctgg 60ccatcagtgg gctccggtcc gaggatgagg ctgattatta
1001115100DNAHomo sapiens 1115atttgcataa
agcagcacac agcacacccc ctccgtgcgg agagctcaat aggagataaa 60gagccatcag
aatccagccc cagctctggc accaggggtc
1001116100DNAHomo sapiens 1116ccttccaata tcagcaccat ggcctggact cctctctttc
tgttcctcct cacttgctgc 60ccaggttaag agagatttca aataccagcc tttggaggga
1001117100DNAHomo sapiens 1117tccctttttc
tccctttcta attcctaata tatgtctgtt ttttttgttt cagggtccaa 60ttcccaggct
gtggtgactc aggagccctc actgactgtg
1001118100DNAHomo sapiens 1118ggacagtcac tctcacctgt ggctccagca ctggagctgt
caccagtggt cattatccct 60actggttcca gcagaagcct ggccaagccc ccaggacact
1001119100DNAHomo sapiens 1119gatttatgat
acaagcaaca aacactcctg gacacctgcc cggttctcag gctccctcct 60tgggggcaaa
gctgccctga cccttttggg tgcgcagcct
1001120100DNAHomo sapiens 1120gaggatgagg ctgagtatta ctgcttgctc tcctatagtg
gtgctcggca cagtgacaga 60cccatgagag gaaccaagac ataaacctcc ctcggccctt
1001121100DNAHomo sapiens 1121ggtcagccac
ccagcctgat tctgactctt ctggcaaaga tccctgaaaa actttaccct 60ggtttctgcc
ttagcaccca ttaatgtctg tgtttccagg
1001122100DNAHomo sapiens 1122ttccctctcg caggctgtgc tgactcagcc gtcttccctc
tctgcatctc ctggagcatc 60agccagtctc acctgcacct tgcgcagtgg catcaatgtt
1001123100DNAHomo sapiens 1123gcatcagcca
gtctcacctg caccttgcgc agtggcatca atgttggtac ctacaggata 60tactggtacc
agcagaagcc agggagtcct ccccagtatc
1001124100DNAHomo sapiens 1124tcctgaggta caaatcagac tcagataagc agcagggctc
tggagtcccc agccgcttct 60ctggatccaa agatgcttcg gccaatgcag ggattttact
1001125100DNAHomo sapiens 1125acagatgggg
aagtgggaca aaaacctcac cctgctctgg gtcttgctct gtaccaattt 60ttaaatttta
aaataactgg cctaggcaca aactatattt
1001126100DNAHomo sapiens 1126gcccagtctg tgctgactca gccaccctca gcgtctggga
cccccgggca gagggtcacc 60atctcttgtt ctggaagcag ctccaacatc ggaagtaata
1001127100DNAHomo sapiens 1127ctgtaaactg
gtaccagcag ctcccaggaa cggcccccaa actcctcatc tatagtaata 60atcagcggcc
ctcaggggtc cctgaccgat tctctggctc
1001128100DNAHomo sapiens 1128tgctgctcag gcctggcctg tggcttctgc tgctgcagct
tccttcatgg gtccaggggc 60atccagggcc ctgcctgaga gtggaggctc ctcctcccct
1001129100DNAHomo sapiens 1129tccagcactg
gagcagtcac cagtggttac tatccaaact ggttccagca gaaacctgga 60caagcaccca
gggcactgat ttatagtaca agcaacaaac
1001130100DNAHomo sapiens 1130ccctccttgg gggcaaagct gccctgacac tgtcaggtgt
gcagcctgag gacgaggctg 60agtattactg cctgctctac tatggtggtg ctcagcacag
1001131100DNAHomo sapiens 1131tgacagactc
ataagaggaa ccaagacata aacctccctc ggcccttgtg atgtggagat 60tgtgtgatca
tacacaccag ctctcaagac agcctacatg
1001132100DNAHomo sapiens 1132acataaacct ccctcggccc ttgtgatgtg gagattgtgt
gatcatacac accagctctc 60aagacagcct acatgtggac cagccataga aaggggaagg
1001133100DNAHomo sapiens 1133atagaaaggg
gaaggaaagg gtctgaattg atttctatcc ctccttgtgc cctgaagtgg 60aggaaatgtg
agagtgattt gcagtaattg aatgagacaa
1001134100DNAHomo sapiens 1134agcaaaagtt atttgtttta tatgaaaaaa aaaaacagaa
acagcaggat cagatctaaa 60ggctgagtct aaatgcattt cctccagaca gaagcttctt
1001135100DNAHomo sapiens 1135cagatctaaa
ggctgagtct aaatgcattt cctccagaca gaagcttctt caaacgatgg 60gctttctgag
ctaagagcaa agaaaataaa ctctccacgg
1001136100DNAHomo sapiens 1136gtatattatt aaagtttatt ttattgagtt actttcaaag
caatccatga ctattatata 60aagtcagaaa gtattaaaaa tcaccaagtt ctctgctaag
1001137100DNAHomo sapiens 1137ctaccttatc
ccatgcaatc aaaataagta cttttcttca tttggatgca ttttttattt 60ctgtttttaa
tatttccaca atggtgatta aacctggtgc
1001138100DNAHomo sapiens 1138acagggtcag gggaggggtc caggaagccc atgaggccct
gctttctcct tctctctcta 60gaccaagaat caccgtgtct gtgtctctcc tgcttccacg
1001139100DNAHomo sapiens 1139gtcctgggcc
cagtctgtgt tgacgcagcc gccttcagtg tctgcggccc caggacagaa 60ggtcaccatc
tcctgctctg gaagcagctc cgacatgggg
1001140100DNAHomo sapiens 1140aattatgcgg tatcctggta ccagcagctc ccaggaacag
cccccaaact cctcatctat 60gaaaataata agcgaccctc agggattcct gaccgattct
1001141100DNAHomo sapiens 1141ctggctccaa
gtctggcacc tcagccaccc tgggcatcac tggcctctgg cctgaggact 60aggccgatta
ttactgctta gcatgggata ccagcctgag
1001142100DNAHomo sapiens 1142agcttgcaca gtgctccagg ccaatgggga actgagacaa
gaaccctctt cctcctccgc 60caggagggtg agtgcctgca gctgctgctc acacctgacc
1001143100DNAHomo sapiens 1143tgtagcttct
gctgctgtag cttcccccat gggcctcggg gcatccaggg ccttgcctag 60gagtggaggc
tccaccactt ttgtcctcag agtcaggaac
1001144100DNAHomo sapiens 1144agggacccca ggagacagaa tatcctgctc ctcagcttgg
gacacagggt ctctgcactg 60aaatcgtggg ctgaggtggc aggtccaact gtgtcttcac
1001145100DNAHomo sapiens 1145ctctgcactg
aaatcgtggg ctgaggtggc aggtccaact gtgtcttcac agtccttcct 60gtgcctgccc
atggtgtggg gacggagtga ggaagtgtgg
1001146100DNAHomo sapiens 1146tcctcactct cctcgctcac tgcacaggtg actggataca
ggtccagggg aggggccctg 60ggaagcctat ggattcttgc tttctcctgt tgtctctaga
1001147100DNAHomo sapiens 1147agccgaataa
tgatgcctgt gtctctccca cttccagggt cctgggccca gtctgtgctg 60acgcagccgc
cctcagtgtc tggggcccca gggcagaggg
1001148100DNAHomo sapiens 1148tcaccatctc ctgcactggg agcagctcca acatcggggc
aggttatgat gtacactggt 60accagcagct tccaggaaca gcccccaaac tcctcatcta
1001149100DNAHomo sapiens 1149ctccaggctg
aggatgaggc tgattattac tgccagtcct atgacagcag cctgagtggt 60tccacagtgc
tccaggcccg gggggaactg agacaagaac
1001150100DNAHomo sapiens 1150gctcctcact ctcctcactc aggacacagg tgacgcctcc
agggaagggg tcttggggac 60ctctgggctg atccttggtc tcctgctcct caggctcacc
1001151100DNAHomo sapiens 1151ttccagggtc
ctgggcccag tctgccctga ctcagcctgc ctccgtgtct gggtctcctg 60gacagtcgat
caccatctcc tgcactggaa ccagcagtga
1001152100DNAHomo sapiens 1152tgttgggagt tataaccttg tctcctggta ccaacagcac
ccaggcaaag cccccaaact 60catgatttat gagggcagta agcggccctc aggggtttct
1001153100DNAHomo sapiens 1153aatcgcttct
ctggctccaa gtctggcaac acggcctccc tgacaatctc tgggctccag 60gctgaggacg
aggctgatta ttactgctgc tcatatgcag
1001154100DNAHomo sapiens 1154gctgaggacg aggctgatta ttactgctgc tcatatgcag
gtagtagcac tttccacagt 60ggtccaagtt catggggaac tgagaccaaa acctgcccag
1001155100DNAHomo sapiens 1155ggccttcaga
cttcctcctt gctctgaaga tgcttcctca cccggtgcaa gaggcttgct 60gcagcgcggc
cttgagaatt cttctctctc agctccttcc
1001156100DNAHomo sapiens 1156ctttccacca tgaattccaa caggaaacct gccctgtggt
ttcccatcca ggacagggac 60agcttcctga tgcttgtgtg ctgtggtccc tgaatgtgca
1001157100DNAHomo sapiens 1157actcttccca
gctcttcaaa tgcagggaca gtgacaagga gctgcctgat tggtgcagtc 60actgcttttt
tcagggatgt cttcacccta catgtatcat
1001158100DNAHomo sapiens 1158catcccctac actgtgggta gaattttagc aactacattc
taatggttat cgccacaact 60ttgatcttag aaataacagt gcagtgaaca tccctatgca
1001159100DNAHomo sapiens 1159ggctcctttg
agttcctgtg tgaatacgac cataggattc atttctaaaa gtgaaattgc 60gggtcagaaa
gatgtgtgtt tgtgattttc acccaatgtt
1001160100DNAHomo sapiens 1160accagcagaa gccaggccag gcccctgtgc tggtcgtcta
tgatgatagc gaccggccct 60cagggatccc tgagcgattc tctggctcca actctgggaa
1001161100DNAHomo sapiens 1161cccagcctcg
gtcaccctct tgctccagcc ccgggaagcc tgttgataaa gccatgagtg 60aatctggccc
agttcacctg gatctgagcc tttcaggttg
1001162100DNAHomo sapiens 1162cccttccctc cagccccctc caggagtctc tacagaagat
acatcaggca taaatatggc 60ctggaagggc cagaatcatc tggtgacttg gggctgttgt
1001163100DNAHomo sapiens 1163ggtcctgggc
ccagtctgcc ctgactcagc ctgcctccgt gtctgggtct cctggacagt 60cgatcaccat
ctcctgcact ggaaccagca gtgacgttgg
1001164100DNAHomo sapiens 1164aaagccccca aactcatgat ttatgaggtc agtaatcggc
cctcaggggt ttctaatcgc 60ttctctggct ccaagtctgg caacacggcc tccctgacca
1001165100DNAHomo sapiens 1165aggctcagtg
cccatagacc ccaagttggc cctgccctga accctgtgca aagcccagac 60acagtcttag
ggtaggaccc ctgggaatgg gctcttgatc
1001166100DNAHomo sapiens 1166ttcaagcccc ctctcctgtt ttccttgcag tctctgaggc
ctcctatgag ctgacacagc 60caccctcggt gtcagtgtcc ccaggacaaa cggccaggat
1001167100DNAHomo sapiens 1167agaagtcagg
ccaggcccct gtgctggtca tctatgagga cagcaaacga ccctccggga 60tccctgagag
attctctggc tccagctcag ggacaatggc
1001168100DNAHomo sapiens 1168caccttgact atcagtgggg cccaggtgga ggatgaagct
gactactact gttactcaac 60agacagcagt ggtaatcata gcacagtgac actggcagat
1001169100DNAHomo sapiens 1169ggggaagtga
gacacaaacc ccttcttcat ctattttacc ctctccctcc agccccagga 60ccgctgtgga
ccaacccata agcaggtctg gcagaattca
1001170100DNAHomo sapiens 1170aggctcacct gggcccagca ctgactcact agactgtgtt
tctccctttc cagggtcctg 60ggcccagtct gccctgactc agcctccctc cgcgtccggg
1001171100DNAHomo sapiens 1171catctcctgc
actggaacca gcagtgacgt tggtggttat aactatgtct cctggtacca 60acagcaccca
ggcaaagccc ccaaactcat gatttatgag
1001172100DNAHomo sapiens 1172gtcagtaagc ggccctcagg ggtccctgat cgcttctctg
gctccaagtc tggcaacacg 60gcctccctga ccgtctctgg gctccaggct gaggatgagg
1001173100DNAHomo sapiens 1173aggctgagga
tgaggctgat tattactgca gctcatatgc aggcagcaac aatttccaca 60gtgttttaag
tcaatgagga agtaagatca aaacctgccc
1001174100DNAHomo sapiens 1174tcaggctcag aacccatagg atcctgagct gggcctgccc
aaacatgagt tcatcccagg 60cacaacctca gggtgggacc ccctgggaac agattcatca
1001175100DNAHomo sapiens 1175tttacaagcc
tcctctcctg tcctctcttg caagctccta tgagcttaca cagccaccct 60cagtgtcagt
gtcaccagga caggcagcca tgatcacctg
1001176100DNAHomo sapiens 1176ctcttgagat aacctcaaag atgagtatgt ttactggttc
tggcagaagc cagaccaggc 60ccatactggt gatatatgaa ggcagcaagc ggccctcagg
1001177100DNAHomo sapiens 1177aatttctgat
tttctgagtc cagctcaggg aacatggcca ccctgaccat cagcagggct 60cagactgagg
acgaggctga ctattactgt cacaggtaca
1001178100DNAHomo sapiens 1178atagaaacag tgatgagccc acagtgacac aggcagatta
ggaagtgaga cacaaacccc 60ttcccaatct gtgtcaccct ctttctccag ccccaggatg
1001179100DNAHomo sapiens 1179gggatgagaa
gggaccaggg gcctgggatt gagctgtgaa gggaaccaaa aggcaggagg 60gacagggcag
gggctgtcag ctatgactca ggggaggttc
1001180100DNAHomo sapiens 1180ctgggcctca ggatcctccc tctgaggcca ccagggggcg
ggggtggcac atgcctggac 60ctgggaggtc cctgctgggc ttcaccctgg gtgggtccta
1001181100DNAHomo sapiens 1181atgcctggac
ctgggaggtc cctgctgggc ttcaccctgg gtgggtccta ggagctcctt 60cctcctaagt
ccccctaaag agacagaggc attctggggt
1001182100DNAHomo sapiens 1182cctaaatctg tcatgccccc ataaatgcat ttctacgagg
gccaataaat gaactccagg 60tttatccaag cagcagcttc aggcgtctgc agacacagag
1001183100DNAHomo sapiens 1183cggggaggaa
ttagccaacc tgaggcaccc tagaagggct gaagggggct gaaggggact 60gaagggtccc
tgtggggcct gtggtcctgg ggaggggaga
1001184100DNAHomo sapiens 1184gctggggtgt ctcccagcca ctctgggccc tgtcctgaca
cttctcccac aaagaaggga 60agggaaatcc tgggacccca cagccaggac caaccgtgaa
1001185100DNAHomo sapiens 1185ccacaggaca
ggaaggacag ggacccccaa ggctggctcc atttcccagg cactgtcatg 60ggctgagtct
caggaaatcc aagtcaagga gtttcaatcc
1001186100DNAHomo sapiens 1186ccaaggaaac agaagtctac gggcccaggc ccaggtgagg
gtggggtaag aagaggagct 60taggatgcag atttgcatgg aggccccgcc ctcctctgag
1001187100DNAHomo sapiens 1187gcatcagggt
aagacaaggc tgggggcagg cccagtgctg gggtctcagg aggcagcgct 60ctggggacgt
ctccaccatg gcctgggctc tgctcctcct
1001188100DNAHomo sapiens 1188ctcagggcac aggtgacgcc tccagggaag gggcctcggg
gacccttggg ctgatccttg 60gtctcctgct cctcaggctc acctgggccc agcactgact
1001189100DNAHomo sapiens 1189ttgggagtta
tgactatgtc tcctggtacc aacagcaccc aggcacagtc cccaaaccca 60tgatctacaa
tgtcaatact cagccctcag gggtccctga
1001190100DNAHomo sapiens 1190tcgtttctct ggctccaagt ctggcaatac ggcctccatg
accatctctg gactccaggc 60tgaggacgag gctgattatt agtgctgctc atatacaagc
1001191100DNAHomo sapiens 1191tgaggacgag
gctgattatt agtgctgctc atatacaagc agtgccactt aaccacagtg 60gtccaagttc
ttggggaact gagacgaaaa cctgccctgg
1001192100DNAHomo sapiens 1192cctgggctct caggctccct ttttgctctg aagatgtttc
ctcacccagt gcaacgggct 60tcctgaagca cagccttgag aattcttctc cctcagcaac
1001193100DNAHomo sapiens 1193tctcttttcc
caccatgaaa tccaaaggaa acctgctctg tggtttctca tccaggacag 60ggacagcttc
cttttgcttg tgtgttgtgg tccctgagtg
1001194100DNAHomo sapiens 1194ggtgcaactc ttcctagctt tttaaattat gggagggtga
caatgagctc cctgactggt 60gcagtccctg ctgttttcag gaacatcctc atcctaaatg
1001195100DNAHomo sapiens 1195catctgaatc
tcccactgtg tgcagaccaa tctggacaga tgttattagg gggagtttcc 60agaagccaca
tcttactcaa ctctgtatcc accacactct
1001196100DNAHomo sapiens 1196tgcctcagcc atggcatgga tccctctctt cctcggcgtc
cttgcttact gcacaggtgc 60tgcccctagg gtcctagcca ctggtccagt cccagggctc
1001197100DNAHomo sapiens 1197tgggtccagc
ctggccctga ctctgagctc agcagggccc ccgcctgtgg tgggcaggat 60gctcatgacc
ctgctgcagg tggatgggct cggcggggct
1001198100DNAHomo sapiens 1198tgggcaggat gctcatgacc ctgctgcagg tggatgggct
cggcggggct gaaatccccc 60cacacagtgc tcatgtgctc acactgcctt agggctcttt
1001199100DNAHomo sapiens 1199catccctgga
tctgtgtcca ggccaggcac gtgggaagat ttacttggag ttcagctcct 60cagtttcaag
ccttttctct cccgttttct ctcctgtagg
1001200100DNAHomo sapiens 1200atccgtggcc tcctatgagc tgactcagcc accctcagtg
tccgtgtccc caggacagac 60agccagcatc acctgctctg gagataaatt gggggataaa
1001201100DNAHomo sapiens 1201caggacagac
agccagcatc acctgctctg gagataaatt gggggataaa tatgcttgct 60ggtatcagca
gaagccaggc cagtcccctg tgctggtcat
1001202100DNAHomo sapiens 1202ctatcaagat agcaagcggc cctcagggat ccctgagcga
ttctctggct ccaactctgg 60gaacacagcc actctgacca tcagcgggac ccaggctatg
1001203100DNAHomo sapiens 1203gatgaggctg
actattactg tcaggcgtgg gacagcagca ctgcacacag tgacacaggc 60agatgcggaa
gtgagacaga aaccagccac ctcggcctgg
1001204100DNAHomo sapiens 1204ctcacaagac ccttccctct ctcctgccct gtcacactga
gcaggaggga gccttccatg 60tggaatggaa gtttccagtc ctatccctgc ccttatgttc
1001205100DNAHomo sapiens 1205ctgagagacg
ggagcaagtt cctgcccacc tctaggctca gcttatccca gaataaactg 60agctagtcat
tttgatgatc aaatgccagc tcccaaaaga
1001206100DNAHomo sapiens 1206ccccagaaac cctgatatct aagtagcacc gactctatta
gtatcaaggg agactagccc 60tagggtggaa tcattttagt gtctcagaag gcacagggca
1001207100DNAHomo sapiens 1207atggaaagtg
tttatgaggt ttcaggatat gcacgtgagc agttaaaggc aggtcttaca 60aggaaggaac
ctactagaat tggggcccat ctgtgacatc
1001208100DNAHomo sapiens 1208acatccctct gctttgggag agaagggcca gggcgggacc
cagagagctc tgcagaggca 60ccacagaccc tcagcagggg gtctgccaaa caggacagct
1001209100DNAHomo sapiens 1209ggacttggct
gcttctgccc aggcctggat ccagcccttg cacatctcag ggcaggggat 60aggcctgggt
ggccagagct gcagctgcac ctgctgggga
1001210100DNAHomo sapiens 1210ggcctagtcc agtcctccag ggtccccaga cagactcgga
tttccgactg cagccaccat 60ggaaggatgt ggtctgcggt gacgatgtct atccagaggc
1001211100DNAHomo sapiens 1211ccgaatatcc
aaggagccca agatcagagg caggaatagg ccaagctccc cagtggagaa 60gctgtgctgg
accaggggtt tcccagggcc ctcccttgtg
1001212100DNAHomo sapiens 1212ccctgaatga tgtctgttag ggcacctaca ccctgttact
gctcagtgcc ttgcctattt 60tgaaggacag ggatgtgtgg tgattatttg tataatccag
1001213100DNAHomo sapiens 1213cccccagcac
ctggtcctca aaagttaccc aagcaatgtg tataaagatc cagcctggag 60atctttgaaa
accgattcga tgagtcgaac cattaagtca
1001214100DNAHomo sapiens 1214tgatcaccat cctcaacttc atctctttct tcctcctcct
cctcattatc atcaccttca 60agaactgtta agagtctgag acttcatcct atttgcagac
1001215100DNAHomo sapiens 1215tcctcctcct
cctcattatc atcaccttca agaactgtta agagtctgag acttcatcct 60atttgcagac
taaaaagtaa gcctgccaca gtgccatgga
1001216100DNAHomo sapiens 1216tgctggcaga agatacaaga ctcctgggtc agagacaacg
aataatctgt ttttcacagc 60aatagcagtt gccaaggtat cagcattgtc ttgcaccagt
1001217100DNAHomo sapiens 1217tccacaaggt
gatgcaaaga gggccaggtg acatctgcat gccagagctc agggatccca 60aatatttcat
acttgacagt aagcatatat ctgtgttttg
1001218100DNAHomo sapiens 1218ctccaaagag aggcattctc tgtaccttcc gaggttgttc
actccacaaa cactcttgaa 60aagataatcc acaatcagtg cctttgcccg agagacatgc
1001219100DNAHomo sapiens 1219agaaatgcag
agatccatag tagaccactg tctcccaaca accatcaact ttatcaatga 60aatgaagtct
caggctattt gtctgttacc atagcccaca
1001220100DNAHomo sapiens 1220aaaatgtctg gcttgattgt caccaaatgt atcaaggaag
ttaaggagta tctgacacaa 60aatgtgaacc aagcaattct caaaggagcc tcccaggaaa
1001221100DNAHomo sapiens 1221ttcactttag
gaagtcctag gaggctcctc tgagagttgc taaaacaaaa cattgagagt 60cctagagggc
tgcagatctg aacttgagca gatattttta
1001222100DNAHomo sapiens 1222aagattttgt ggcagaaaaa gaaactggaa agcaagaggg
cagaccctca ttgcagttct 60gtaatgtaag ggggcagagc aggggccttt ctcaccagag
1001223100DNAHomo sapiens 1223gatattggac
cctgcattca tcttctctgg atggtaattt tctcacctgt aaaacagaga 60cactggcccc
aaggacaccc cacaagtagt tgtgaatccc
1001224100DNAHomo sapiens 1224aaagtaagag aagaacaaaa aaagaaccag aatttattca
acacccactg agtgcttagc 60aaacacatgg tttctttaac tctcataagc ttcatgctgc
1001225100DNAHomo sapiens 1225agaggaactc
tccccatttt acagataagg aaactgaggc ccagaggtaa cctaggtcta 60gatagactcc
acatttatga cttcaccact cttccttgcc
1001226100DNAHomo sapiens 1226aaactgaggc ccagaggtaa cctaggtcta gatagactcc
acatttatga cttcaccact 60cttccttgcc tgaaggatat agaatcactc cctgcagggc
1001227100DNAHomo sapiens 1227tcttgcctga
ctcaggaaag ggccacagga tagccagcca ggcttaacca acccagccaa 60gaaagggctg
gtcccaactg gctggagtgc agtgtacagg
1001228100DNAHomo sapiens 1228gttggtagat gcccctctgg gagagatccc caggggtgac
agccatggac cctggaaggg 60cctgggctag ggacagggac cagagccagt ccagggagag
1001229100DNAHomo sapiens 1229gacagagcca
atggactggg gtgtactgta acagccctgc tggcgagagg gaccagggca 60ccgtcctcca
gggagcccat gctgcaagtc gggccagagg
1001230100DNAHomo sapiens 1230tgcccctgaa cctgaaggcc aatgagaccc aagacaggcc
aagtgggttg tgagacccct 60gaggagctgg gccctggtcc caggcagcgc tggcccctgc
1001231100DNAHomo sapiens 1231tgctgctggg
tctggccatg gtcgcccatg gcctgctgcg cccaatggtt gcaccgcaaa 60gcggggaccc
agaccctgga gcctcagttg gaagcagccg
1001232100DNAHomo sapiens 1232atccagcctg cggagcctgt ggggcaggta aggggcaaga
gattccaggg gatgtggggg 60tcctgcagca gagctgggaa agggtgacca aggggagaca
1001233100DNAHomo sapiens 1233agccagagga
gtgaggagga aggttaaccc ctaagagggg cctgggctga cactggcttt 60agtaatgggt
tgatattttg tccatcacag atttgtttga
1001234100DNAHomo sapiens 1234attactgttt ttaatatcat attacgatat tatttttctt
gatttctgag ttttctggcg 60ccacttaaat tttcaccagg gtcagtgcct caatcaccta
1001235100DNAHomo sapiens 1235gtcctagtcc
tctgggtagg gaaggaacag aggcagggac aggacatcca cagggggtgg 60tggccactgt
ccccacaggg tgcccaggcc tgttcctccc
1001236100DNAHomo sapiens 1236cctcctcctc tctgcccatg tgcctcctgc ccagtgaggg
caggggccac tccctggaga 60aggcagcaag ggcttggttt ggtctccccc aaggctgtct
1001237100DNAHomo sapiens 1237gttcaccaac
ttgcacataa atgcttactg gggccaggct caaggacaca gggagggtgg 60gatgaaccga
ggggagctgt ccagtcattg gaacaggccc
1001238100DNAHomo sapiens 1238acggcccatg tttggagcaa taaagggaga ggggatctcc
ctctgggatg atgcccaggc 60tggtctcaca gatcgagggg cactggctgg tgatgggtgc
1001239100DNAHomo sapiens 1239tggtctcaca
gatcgagggg cactggctgg tgatgggtgc ccccaaaaga cagagcagcg 60tcagaggaga
ggagagcaca ggatgaggct gggagctcct
1001240100DNAHomo sapiens 1240gggtgactgg gaaggggagg caagaagacc atagggtccg
tgcaccattc ccagtccagg 60acgagtcctt ggatggattt aggtagattg attatcagag
1001241100DNAHomo sapiens 1241tcagatttgt
gtttttggaa aaatcagcac cggattggag gctgatgcga cgcccgatta 60gaggagggag
gagagggggt gatggccaag tccagggtag
1001242100DNAHomo sapiens 1242gtggggatcc tggaggaagc cgtgccttgg ggatggggag
gacactcaga ttcagagcac 60ccaggggccc agtttcctat gaaatgggag catgaagttg
1001243100DNAHomo sapiens 1243aagtgagggc
tgagcagagg ggagcagaca cgctcgggga ctgtctatgg gcattaaaaa 60tgtataacca
ttttagcaac aggcggcgag tcaaaaaaca
1001244100DNAHomo sapiens 1244aagtgtgttt atctaaactg ggcaattcca cttctaggaa
tttatcctaa gggttggttg 60ggggaataat caaagctgta accaaatctt tataacaagg
1001245100DNAHomo sapiens 1245gtggttagct
cagcattatt agtgatggga gaaaactgga aaaaatccaa atatctacca 60gaaagggtgt
gaaaaaacac aattgtattt gggggactgt
1001246100DNAHomo sapiens 1246tggctaattt tgattaggat tattattagt ttagagacag
agcctcgcta tattgctcag 60gcctgtctca aattcctaag ctcaagcaat ctttctgcct
1001247100DNAHomo sapiens 1247actgcacctg
acccaactgt gtttttaaag tatatatgca ttttcaaaaa cctgtcagaa 60aatatagaaa
aatgtcaatg gtgtgtctgg ctggctgatg
1001248100DNAHomo sapiens 1248ggatttcacc taattttaat gtggctttat aattttctgg
ttttgtgaag ttgttcacaa 60aaagagacat ttcttctaat ataattttta atacaacagt
1001249100DNAHomo sapiens 1249aatgtactca
tgtgcattac tctttttgta atgagtatat tacaaaatgt aatgactttt 60gtacattact
cttttttctt gccaaaaaaa aaaaagatta
1001250100DNAHomo sapiens 1250agcagagaag tatataaagt aaaagcaagt gcttctgctt
accatctctc acctcttccc 60agagatagcc actgtcaggt tggtcaatat acttccagaa
1001251100DNAHomo sapiens 1251cttttcctgt
gtgtgtgtgt gtccctgaaa acacacacac acacacacac acacacacac 60acagttggtg
ctgggatttt attttgcaaa agtaagagcc
1001252100DNAHomo sapiens 1252cacacacagt tggtgctggg attttatttt gcaaaagtaa
gagccatatt ctgcatatta 60ccaactttta atctattatt gacactttct gtatcagtcc
1001253100DNAHomo sapiens 1253atatggatta
accacattca ttgcttataa actttgtttt ataagcaaag tttagatgag 60ccagaattta
tttccactaa aaaatctaaa tgacaaatga
1001254100DNAHomo sapiens 1254tgctgcagtg gaaatttgtg tgtgtgtgtg tgtgtgtgtg
tgtgtgtgtg tgtgtgtgtg 60tgtatgtgta caaagtgcac ttatatatct ccccaggata
1001255100DNAHomo sapiens 1255tgacctgggt
gtttttcttt ttctctgtag gatgttaata gtatcttgtg tcatgctagg 60atgtctagga
cagagggcaa tacaatgagg ggaaggcatt
1001256100DNAHomo sapiens 1256ctgcgatgtc cccaggcctc tggcttgaag agtaacttgc
tgaagtgagg actctgtgga 60ggagcaagtt atacagaaag aagtttagtt gtgatctgtt
1001257100DNAHomo sapiens 1257gagttggagg
tgtctacagg gcatccaagc agacataggt tgaggaggca gaatatatgt 60gaatctggag
ccaagaagag aggtaagggc tggaaatagg
1001258100DNAHomo sapiens 1258gatctaagac ccctggacag ttgtgagtgt gcacaatgag
ggtcagatgc agagaaaatt 60aggagactac agagagcaga acccagggtg gggatctggg
1001259100DNAHomo sapiens 1259agtcagcagt
tgggcatggg cctggtagaa agggaagcca aggaggagga gagggggcag 60tctcagacac
caaggagggg agagtgacta gaaagaaaac
1001260100DNAHomo sapiens 1260cttcttgcag agacataggg gatggggaag aactgcagac
tgaactgggg caaaggactg 60ttggccttaa ccagagagat ttgagggaga gatgaggctg
1001261100DNAHomo sapiens 1261agagccaggg
gatcctgcca tgtcccagca taaaaacagt acctgacaca gatgggtgct 60tgggagctgt
tgtcggatga atgagtggac agatgcatgg
1001262100DNAHomo sapiens 1262atggacggat ggatggaagg atgatagatt gatggacaaa
cagatgaaca gatgaatagc 60tggatggaca actggatgga tgggtagaca gaatgatctc
1001263100DNAHomo sapiens 1263agagatcaga
aaaagcttca tgcactaagt gggactgaac cgcgtctcca tgggtagaaa 60gcagaggaat
ctccacttga gtcaggaatg acccagtgct
1001264100DNAHomo sapiens 1264ctcaatccag ggagaaagcc agcctggctt cactggggac
acttgtgtgg gggactcaga 60ggccctttaa atgaggccag acgaggttgg acaggtccaa
1001265100DNAHomo sapiens 1265gccaactcag
cactcctctg ccacactgca caggagggga tgtgtcactc agggagttgc 60tgggacctat
gggtcccagt gttgtcatca gcaccgacag
1001266100DNAHomo sapiens 1266cctcagagag gaaagacaca cactggggta actccaaggc
tgtgtgtggc acttgccttg 60gacagcagac aggcacaggg acacctctag ggggctggcc
1001267100DNAHomo sapiens 1267acccccctgc
ctcatgtcta ggtcccagcc ccgcccactg caaccctgtg cccgtcatgc 60ccagcaggct
cctgctccag cccagccccc agagagcaga
1001268100DNAHomo sapiens 1268cactgcaacc ctgtgcccgt catgcccagc aggctcctgc
tccagcccag cccccagaga 60gcagacccca ggtgctggcc ccgggggttt tggtctgagc
1001269100DNAHomo sapiens 1269ctcagtcact
gtgttatgtc ttcggaactg ggaccaaggt caccgtccta ggtaagtggc 60tctcaacctt
tcccagcctg tctcaccctc tgctgtccct
1001270100DNAHomo sapiens 1270ggaaaatctg ttttctctct ctggggcttc ctcccctctg
tcctcccagc cttaagcact 60gacccttacc tttctccatg gggcctggag gaggtgcatt
1001271100DNAHomo sapiens 1271agtctccggg
taaccggcag gaagggcctc cacagtggga gcagccggat gcagcctggt 60cccggggcct
gagctgggat tgggcagggt cagggctcct
1001272100DNAHomo sapiens 1272cctctcttcc agggcagatg tctgagtgag ggacagaggc
tggttctgat gaggggccct 60gcagtgtcct tagggacatt gcccagtgac tcctggggtc
1001273100DNAHomo sapiens 1273ggacagaggc
tggttctgat gaggggccct gcagtgtcct tagggacatt gcccagtgac 60tcctggggtc
aaggacagag gctgctgggg tgggcctggg
1001274100DNAHomo sapiens 1274agctgctgag tctcatagtc taggggagca gccccaagaa
cagctgaggg tctaggctga 60ggactggatg ccaatccagc ctgggagggc cacacggcct
1001275100DNAHomo sapiens 1275tctcatagtc
taggggagca gccccaagaa cagctgaggg tctaggctga ggactggatg 60ccaatccagc
ctgggagggc cacacggcct ggtgacacag
1001276100DNAHomo sapiens 1276aggtcacccc aaggggagac caatggaggg cacagagagg
gctctgggtc taggctgcag 60ctctgtggcc tgtgctgggt catgaggaca tggggacaca
1001277100DNAHomo sapiens 1277tgtgctgggt
catgaggaca tggggacaca gagggacggg tgagactggg tgaggtgcca 60gaatccaacc
ctcccaggac agtcaccaga aaggagacag
1001278100DNAHomo sapiens 1278tctcttaggg cagagatgtg tctgtccctg gagccccgtc
acctctgggg cccagtgtct 60ctctgttcac ggatcggcct cctgccttcc tcaaagggca
1001279100DNAHomo sapiens 1279tgttagactc
aggaaatgac cagaggggag tgaatgaggg gtgcagagaa ctccatggct 60accaggtgaa
gtttggggtc atcacaggct gctggggtgg
1001280100DNAHomo sapiens 1280catagtctgt gggagcagcc ccaggaacag ctgaggtgaa
gggttctgtg gtcgggcttg 60tggagacagg aaacatctca gagcctcaga ggagccctga
1001281100DNAHomo sapiens 1281ggcttgtcta
ggtggagccc actccttgcc aggagagcca agtgggctgg gctggggcag 60agcccggtgc
ctgtgaggga taggaagctc cagttcaaag
1001282100DNAHomo sapiens 1282caggcttggg tctccccaca cactgcctgc caggacagtc
ctacaggatg agcaggggac 60ccacagttca cggaggaggc tctaggtcct ggaagaataa
1001283100DNAHomo sapiens 1283agtgggtgat
ggaggggggt atagggatgg aaatgaggga tccaggggtc aaggccagat 60tctaaactca
gactccagag atcagagaag aaggaacaca
1001284100DNAHomo sapiens 1284gcctgccctg ggtatatgga gaaattgagg ctgtagagga
gaggggctgg gccaggacac 60ctgtgaaagg tgacttggga gggctcctag gaaggcacag
1001285100DNAHomo sapiens 1285tgaaagcccc
actgctatga ccaggtagcc gggacgtggg gtggatgcca gaaaagactc 60cacggaataa
gagagagccc aggacagcag gcaggctctc
1001286100DNAHomo sapiens 1286cgatcccccc aggcccttgc cccatacacg ggctccagaa
cacacatttg gctggaacag 60cctgagggac caaaaggccc cagtatccca cagagctgag
1001287100DNAHomo sapiens 1287gagccaggcc
agaaaagtaa ccccagagtt cgctgtgcag gggagacaca gagctctctt 60tatctgtcag
gatggcagga ggggacaggg tcagggcgct
1001288100DNAHomo sapiens 1288gagggtcaga tgtcggtgtt gggggccaag gccccgagag
atctcaggac aggtggtcag 60gtgtctaagg taaaacagct ccccgtgcag atcagggcat
1001289100DNAHomo sapiens 1289atgcaggaca
gtccggagag ggaaatcagg agaagtgaag gggtctctgg ggagcccaga 60tgtgggctag
aggcagaagt aagggtgaag agcacctatg
1001290100DNAHomo sapiens 1290agtcaatgtc atggtctcag caggaacaca gttgaaaatc
cccattccac acaagaccgt 60ttagcaggaa aggagtccat acttgtgctg ccaccaggat
1001291100DNAHomo sapiens 1291gtcctgagaa
gccttggaga atgaaacata caggtgcatt tcctagactt gacaatgcac 60gttagccaag
taaaggcaat gaaaagttct ctactaggga
1001292100DNAHomo sapiens 1292tttgtttgtt tctgtatctt gtctcaactt gtggtcagcc
tttctccctg catcccaggc 60ctgagcaagg acctctgccc tccctgttca gacccttgct
1001293100DNAHomo sapiens 1293tgcctcagca
ggtcactaca accacttcac ctctgaccgc aggggcaggg gactagatag 60aatgacctac
tgagcctcgt ctgtctgtct gtctgtctgt
1001294100DNAHomo sapiens 1294ctgtttgtct ctctgtctgt ctgacaggcg caggctgggt
ctctaagcct tgttctgttc 60tggcctcctc agtctgggtt cttgtcggaa cagctttgcc
1001295100DNAHomo sapiens 1295cttgggttac
ctgggttcca tctcctgggg aattgggaac aaggggtctg agggaggcac 60ctcctgggag
actttagaag gacccagtgc cctcggggct
1001296100DNAHomo sapiens 1296agagttcgct gtgcagggga gacacagagc tctctttatc
tgtcaggatg gcaggagggg 60acagggtcag ggcgctgagg gtcagatgtc ggtgttgggg
1001297100DNAHomo sapiens 1297gccaaggccc
cgagagatct caggacaggt ggtcaggtgt ctaaggtaaa acagctcccc 60gtgcagatca
ggacatagtg gaaaacaccc tgacccctct
1001298100DNAHomo sapiens 1298gcctggcata gaccttcaga cacagagccc ctgaacaagg
gcaccccaac acctcatcat 60atactgaggt caggggctcc ccaggtggac accaggactc
1001299100DNAHomo sapiens 1299agaatattcc
gtgagaaggt ggccccacag cgctgggtca cacgccatcc cccaagacag 60gcaggacacc
acagacaggg tggtgggtct cagaaaactc
1001300100DNAHomo sapiens 1300aggccctaaa cgtggatgct taccaattcc tccactggag
gaagacctca gagcagatgc 60ccaggacagg gacttctggt agggacggtg actgggacgg
1001301100DNAHomo sapiens 1301gtgcctgttt
gtcagggaaa acccactgga gagtcagatc ccccagataa cttctcacga 60catggagact
ctttcgaaca gacaaagctc cacgttcagc
1001302100DNAHomo sapiens 1302tcagggagta aaaaaaaaat gcctcaaatg gaggcctttg
atctactgga atccagcccc 60caggactgac accctgtctc accaggcagc ccagaggggt
1001303100DNAHomo sapiens 1303cagggtccac
cagaaggcat ctcagaacca gccagcagtg gccctgattg tcagcaggac 60cccagggagg
ggggtggcca ggacagggct ctgaagcccc
1001304100DNAHomo sapiens 1304caccccagga ccttccctgg gcagaacgag ttggtgaggg
agtgatgagc aaccacaggc 60ctcctaactt cccaagctgg cgattctgag aggcctcaag
1001305100DNAHomo sapiens 1305gctgagacac
ggttcagcct tttaggccct cctgaacgtg tcccctgtct ccacagcctg 60ggaatgcact
ctcttttgac ccagaaatcc tgctcataag
1001306100DNAHomo sapiens 1306ctgtcattgt acaacacatc atttcacttt gtttttcaaa
catagtgaat tctttcctaa 60ttaaagaaga aaagagtata aagagaaagt ttccagtgca
1001307100DNAHomo sapiens 1307gtataaagag
aaagtttcca gtgcagcctg gagatctgta ctggttgtat ctggaattcc 60agactcagcc
ttgcatttca catagcagat agatgatgat
1001308100DNAHomo sapiens 1308gatggagaag gagaagaaga aggaggagga ggaggaaaga
aggaagaaga agaagaagag 60gaggaggaag aagaagacga agggaagaag aagaaggatg
1001309100DNAHomo sapiens 1309tccaggtctg
ccaggtgtag gggaggtgtg actggttcca tcatggaccg gttcctccat 60ggaccggttc
ctccgtggac cggttccgcc atggaccggt
1001310100DNAHomo sapiens 1310tccgccatgg accactcctg ccctggacca ctcctgccct
ggaccggttc tgccgtggac 60tggttcccgc cgtggaccag ttcccgctgt atactggttc
1001311100DNAHomo sapiens 1311tgccctggac
tggttcccgc tgtggactgg ttccttgggg ctctaagtgc ggaagggccc 60agagctggtc
cctgcccagc gccctgctag ggctgtgtcc
1001312100DNAHomo sapiens 1312tcgtactcgt gcgcctcgct tcggtgagcc ccagggcccc
tgcctccttc ctcctgccgt 60cctgcctccg tccccgccct ttcatcatcc gcgtccctgt
1001313100DNAHomo sapiens 1313gaaggcattc
cctaaatccg agcccgagtg gttctccccg ggaaggctac tttggggagc 60tggggggatg
cgaaacaccc tagatactgg ataatggggt
1001314100DNAHomo sapiens 1314ggggaaatcg atgatttaag aacaaaaccg aaaaactggc
gttttgccgt gccgctcgga 60ggggacatta aaaaatttct tagtgtttgc ccgcaaaggt
1001315100DNAHomo sapiens 1315tagtgtttgc
ccgcaaaggt attgtgcgtt gccttggagg ctgagatatg ggggaataga 60caagtccttt
gttctgaggt tcatcttccg agccccgagc
1001316100DNAHomo sapiens 1316ctcctcccag cctcggacgg ctgcgcgggc tgcatctgtg
cagcctggcg gcggcggggc 60tgtgctatga catctttaca gtccttcttg cagagacatg
1001317100DNAHomo sapiens 1317tgtgccaggg
atgccgaatt gccgggagag caggcaagac cggcttcggg gcgcgcggcg 60gccgctttgt
gtgcggggct gcattgtgac gcgggcgatg
1001318100DNAHomo sapiens 1318aagccggtag ggcggtggtc ggaagctcca gccgcggccg
ccgcctttgt gagaggacta 60gaaagccgga tccggcccgc atccttgcgg agaggccgcg
1001319100DNAHomo sapiens 1319gctaggaaat
ggaaacgctt ttcctacctg ggctccattt taggaattct tgccgatttt 60tcccacttga
atttggaagt ggctttcctc ttctttcctt
1001320100DNAHomo sapiens 1320gtcctagcca gcctttaatt ttaaacgctg taattaacaa
ttcgcagtgg tcaatttcct 60ttattctgca agattcggct ttgagaggca tccgccctct
1001321100DNAHomo sapiens 1321ttggtccaca
gcgttttgaa atatggggag gaggggcgcg gggggtgtcg cctctttttc 60tgtagaaaga
ggaagctcgt gagcgcggaa cggcagcagt
1001322100DNAHomo sapiens 1322aagtgcagtt cccagcccag agacagcggg gcgggtggct
cttcctcacg ctcgctcttg 60gcttgctccc tgcagctttt cctccgcaac catgtctgac
1001323100DNAHomo sapiens 1323aaacccgata
tggctgagat cgagaaattc gataagtcga aactgaagaa gacagagacg 60caagagaaaa
atccactgcc ttccaaagaa agtgagctcc
1001324100DNAHomo sapiens 1324agacgcaaga gaaaaatcca ctgccttcca aagaaagtga
gctccgaccc acccccatct 60ttagaaaggc tgggtgggag cggccggtgg gagggcggga
1001325100DNAHomo sapiens 1325tttatagaaa
ggcatatgga acaggagtca tccaaatata tcccaggggt tgcaaattga 60ccaaaagagt
cacctttagg gaagcctgct tctgaatgct
1001326100DNAHomo sapiens 1326tgtggaattt atcattcttc tgaatggctg ttgcatttat
ctgcagcttt tactcaccag 60atgagacctc agacatttca aattctgcgg aggctggcta
1001327100DNAHomo sapiens 1327cacaccttca
taggaaagct ttttgctgat ttccctgttg gtacttttct cttacacatt 60ctatggggta
tggtaaacct ggaggtagag tcatagccaa
1001328100DNAHomo sapiens 1328gcacagataa agcaggcaca gaatctctga ccagcctcac
aaaagcagac aaacacacaa 60tctttttgca cctgtttctt ccactccggt tgccgtgaat
1001329100DNAHomo sapiens 1329tagaaatggt
tcaaccagtc caatatcaat atagctgctt attactctat tcacttactt 60caaagtggca
tttgttttga gtaagacttt atttaattct
1001330100DNAHomo sapiens 1330taccgttagc ttgaaaccat agagatcttc tctctatttg
ccctacttcc ttcaaaagtc 60aaatgacctc ctacaaataa aagacgttct tattttcatt
1001331150DNAHomo sapiens 1331cgactacgac
tcggtgcagc cgtatttcta ctgcgacgag gaggagaact tctaccagca 60gcagcagcag
agcgagctgc agcccccggc gcccagcgag gatatctgga agaaattcga 120gctgctgccc
accccgcccc tgtcccctag
1501332150DNAHomo sapiens 1332cgactacgac tcggtgcagc cgtagttcta ctgcgacgag
gaggaaaact tctaccagca 60gcagcagcag agcgagctgc agcccctggc gcccagcgag
gatatctgga agaacttcga 120gctgctgccc accccgcccc tgtcccctag
1501333150DNAHomo sapiens 1333cgactacgac
tcggtgcagc cgtagttcta ctgcgacgag gaggaatact tctaccagca 60gcagccgcag
agcgagctgc agcccctggc gcccagcgag ggtatctgga agaacttcga 120gctactgccc
accccgcccc tgtcccctag
1501334150DNAHomo sapiens 1334cgactacgac tcgttgcagc cgtagttcta ctgcgacgag
gaggaatact tctaccagca 60gcagccgcag agcgagctgc agcgcctggc gcccagcgag
ggtatctgga agaacttcga 120gctacagccc accccgcccc tgtcccctag
1501335150DNAHomo sapiens 1335cgactacgac
tcgttgcagc cgtagatcta ctgcgacgag gaggaatact tctacctgca 60gcagccgcag
agcgagctgc agcgcctggc gcccagcgag cgtatctgga agaacttcga 120gctacagccc
accccgccct tgtcccctag
1501336150DNAHomo sapiens 1336cgacaacgac tcgttgcacc cgtagatcta ctgcgacgag
gaggaatact tctacctgca 60gcagccgcag agcgagctgc agcgcctggc gcccagcgag
cgtatctgaa agaacttcga 120gctacagccc acgccgccct tgtcccctag
1501337150DNAHomo sapiens 1337cgacaacgac
tcgttgcacc cgtagatcta ctgcgacgag gaggaatact tctacctgca 60gcagccgcag
agcgagctgc agcgcctggc gcccagcgag cgtatctgaa agaacttcga 120gctacagccc
acgccgccct tgtcccctag
1501338150DNAHomo sapiens 1338gctcacctgt acaaatctgg ctccgcaggt ttcgcatttg
tagggcttct ctccagagtg 60aattcgagtg tgggttttca ggttggctgg ccggttgaac
tgggccccac agatgttgca 120acgatagggt ttctcaccta ttaccaagaa
1501339150DNAHomo sapiens 1339gctcacctgt
acaaatctgc ctccgcaggt ttcgcatttg tagggctcct ctccagagtg 60aattcgagtg
tgggttttca ggttggctgg gcggttgaac tgggccccac agatgttgca 120acgctagggt
ttctcaccta ttaccaagaa
1501340150DNAHomo sapiens 1340gctcacctgt acaaatctgc ctccgcaggt ttcgcctttg
tagggctcct ctccagagtg 60aattcgagtg taggttttca agttggctgg gcggttgaac
tgggccccac ggatgttgca 120acgctagggt ttctcaccta ttaccaagaa
1501341150DNAHomo sapiens 1341gctcacctgt
acaaatctgc ctccgccggt ttcgcctttt tagggctcct ctccagagtg 60aattcgagtg
taggttttca agttggctgg gcggttgaac tgggccccac ggatgttgca 120acgctagggt
ttctcaccta tttccaagaa
1501342150DNAHomo sapiens 1342gctcacctgt acaagtctgc ctccgccggt tacgcctttt
tagggctcct ctccagagtg 60aattcgagtg taggttttca agttggctgg gcggttgaac
tgggctccac ggatgttgca 120acgctaggga ttctcaccta tttccaagaa
1501343150DNAHomo sapiens 1343gctcacctgg
acaagtctgc ctccgccggt tacgactttt tagggctcct ctccagagtg 60aattcgagtg
taggctttca agttggctgg gcggttgaac tgggctccac ggctgttgca 120acgctaggga
ttctcaccta tttccaagaa
1501344150DNAHomo sapiens 1344gctcacctgg acaagtctgc ctccgccggt tacgactttt
tagggcacct ctccagagtg 60aattcgagtg taggctttca agttggctgg gagcttgaac
tgggctgcac ggctgttgca 120acgctaggga ttctcaccta tttccaagaa
1501345150DNAHomo sapiens 1345ctaggggaca
ggggcggggt gggcagcagc tcgaatttct tccagatatc ctcgctgggc 60gccgggggct
gcagctcgct ctgctgctgc tgctggtaga agttctcctc ctcgtcgcag 120tagaaatacg
gctgcaccga gtcgtagtcg
1501346150DNAHomo sapiens 1346ctaggggaca ggggcggggt gggcagcagc tcgaagttct
tccagatatc ctcgctgggc 60gccaggggct gcagctcgct ctgctgctgc tgctggtaga
agttttcctc ctcgtcgcag 120tagaactacg gctgcaccga gtcgtagtcg
1501347150DNAHomo sapiens 1347ctaggggaca
ggggcggggt gggcagtagc tcgaagttct tccagatacc ctcgctgggc 60gccaggggct
gcagctcgct ctgcggctgc tgctggtaga agtattcctc ctcgtcgcag 120tagaactacg
gctgcaccga gtcgtagtcg
1501348150DNAHomo sapiens 1348ctaggggaca ggggcggggt gggctgtagc tcgaagttct
tccagatacc ctcgctgggc 60gccaggcgct gcagctcgct ctgcggctgc tgctggtaga
agtattcctc ctcgtcgcag 120tagaactacg gctgcaacga gtcgtagtcg
1501349150DNAHomo sapiens 1349ctaggggaca
agggcggggt gggctgtagc tcgaagttct tccagatacg ctcgctgggc 60gccaggcgct
gcagctcgct ctgcggctgc tgcaggtaga agtattcctc ctcgtcgcag 120tagatctacg
gctgcaacga gtcgtagtcg
1501350150DNAHomo sapiens 1350ctaggggaca agggcggcgt gggctgtagc tcgaagttct
ttcagatacg ctcgctgggc 60gccaggcgct gcagctcgct ctgcggctgc tgcaggtaga
agtattcctc ctcgtcgcag 120tagatctacg ggtgcaacga gtcgttgtcg
1501351150DNAHomo sapiens 1351ctaggcgaca
agggcggcgt gggctgtagc tcgaagttct ttcagatacg ctcggtgggc 60gccaggcgct
gcagcacgct ctgcggctgc tgcaggtaga agtattcctc ctcgtcgcag 120tagatctacg
ggtgcaacga gtcgctgtcg
1501352150DNAHomo sapiens 1352ttcttggtaa taggtgagaa accctatcgt tgcaacatct
gtggggccca gttcaaccgg 60ccagccaacc tgaaaaccca cactcgaatt cactctggag
agaagcccta caaatgcgaa 120acctgcggag ccagatttgt acaggtgagc
1501353150DNAHomo sapiens 1353ttcttggtaa
taggtgagaa accctagcgt tgcaacatct gtggggccca gttcaaccgc 60ccagccaacc
tgaaaaccca cactcgaatt cactctggag aggagcccta caaatgcgaa 120acctgcggag
gcagatttgt acaggtgagc
1501354150DNAHomo sapiens 1354ttcttggtaa taggtgagaa accctagcgt tgcaacatcc
gtggggccca gttcaaccgc 60ccagccaact tgaaaaccta cactcgaatt cactctggag
aggagcccta caaaggcgaa 120acctgcggag gcagatttgt acaggtgagc
1501355150DNAHomo sapiens 1355ttcttggaaa
taggtgagaa accctagcgt tgcaacatcc gtggggccca gttcaaccgc 60ccagccaact
tgaaaaccta cactcgaatt cactctggag aggagcccta aaaaggcgaa 120accggcggag
gcagatttgt acaggtgagc
1501356150DNAHomo sapiens 1356ttcttggaaa taggtgagaa tccctagcgt tgcaacatcc
gtggagccca gttcaaccgc 60ccagccaact tgaaaaccta cactcgaatt cactctggag
aggagcccta aaaaggcgta 120accggcggag gcagacttgt acaggtgagc
1501357150DNAHomo sapiens 1357ttcttggaaa
taggtgagaa tccctagcgt tgcaacagcc gtggagccca gttcaaccgc 60ccagccaact
tgaaagccta cactcgaatt cactctggag aggagcccta aaaagtcgta 120accggcggag
gcagacttgt ccaggtgagc
1501358150DNAHomo sapiens 1358ttcttggaaa taggtgagaa tccctagcgt tgcaacagcc
gtgcagccca gttcaagctc 60ccagccaact tgaaagccta cactcgaatt cactctggag
aggtgcccta aaaagtcgta 120accggcggag gcagacttgt ccaggtgagc
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