Methods for Treating and Detecting Sepsis in Humans

Abstract

Biomarkers for identifying sepsis in humans are presented herein, as are related methods, uses, agents, and kits comprising same. Methods for treating, detecting, and diagnosing sepsis in humans are presented herein.

Description

RELATED APPLICATIONS

[0001] This application is a continuation application of U.S. application Ser. No. 16/526,923, filed Jul. 30, 2019, which claims priority of and benefit to U.S. Provisional Application No. 62/711,970, filed Jul. 30, 2018, the entireties of each of which is incorporated herein by reference for all purposes.

SEQUENCE LISTING

[0002] 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 Jul. 25, 2019, is named 176395-010601_SL.txt and is 185,649 bytes in size.

FIELD OF INVENTION

[0003] Polynucleotides relating to circulating nucleic acids (CNAs) indicative of sepsis are described herein, as are probes, methods, and kits for detecting and diagnosing sepsis in humans. Also encompassed herein are probes, methods, and kits for detecting CNAs described herein in samples. In a particular embodiment, the CNAs are detected in samples isolated from humans.

BACKGROUND

[0004] Sepsis, also known as systemic inflammatory response syndrome, is a leading cause of mortality in mammals and is characterized by symptoms that include fever, elevated heart and breathing rate, and organ failure. Even under the best of care, sepsis can lead to septic shock which is frequently fatal. Sepsis is linked to immune responses to infection and more particularly to an excessive inflammatory immune response. Bacterial infections are the most common infection associated with sepsis, but fungal, viral, and parasitic infections can also lead to sepsis. Sepsis arises from a complicated interactive network of the causative agent (e.g., a bacterial infection) and the host immune system response to the agent. The genetic background of the host and the status of the host immune system contribute to the potential for developing sepsis and severity of the condition once developed. Early phases of sepsis are typically associated with high levels of inflammation due to the release of inflammatory cytokines, such as high mobility group box 1 protein (HMGB1) and tumor necrosis factor-alpha (TNF-.alpha.), interleukin-1 (IL-1), and interleukin-6 (IL-6). The dramatic release of such inflammatory cytokines, wherein a positive feedback loop is established between the cytokines and white blood cells, is referred to as a cytokine storm (also known hypercytokinemia or cytokine cascade). Later phases of sepsis are somewhat paradoxically associated with prolonged periods of reduced immune system activity.

SUMMARY

[0005] Methods, reagents, and kits described herein relate to predicting and/or diagnosing sepsis in a human in advance of the appearance of sepsis symptoms in the human. In accordance with the present experimental findings, methods, reagents, and kits described herein can be used to predict/diagnose sepsis in a human subject in advance of symptomatic presentation as well as in later stages of the disease progress. Indeed, results presented herein demonstrate that methods, reagents, and kits described herein can diagnose sepsis in a human subject at least three days in advance of clinical presentation. In light of results presented in the examples, over-representation or under-representation of at least one polynucleotide relative to an internal reference region in a biological sample or body fluid sample (e.g., serum), wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148, is a positive indicator that a human subject from which the sample was isolated is developing sepsis. Accordingly, the methods, reagents, and kits described herein provide for diagnosis of sepsis at early, pre-symptomatic stages of the disease, as well as later stages of the disease. Methods for determining over-representation or under-representation of at least one polynucleotide (CNA) relative to an internal reference region, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148, in body fluids are also disclosed, as are kits for such purposes, methods for screening the diagnostic target, and detection tests for screening. In a particular embodiment, the internal reference regions is at least one of SEQ ID NOs: 59, 61, or 68.

[0006] In a particular aspect, a method is presented comprising administering to a human identified as having sepsis a therapeutically effective amount of at least one agent used to treat sepsis, wherein the human is identified as having sepsis by analyzing a biological sample isolated from the human for over-representation or under-representation of at least one polynucleotide relative to an internal reference region, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148 and wherein the over-representation or under-representation of the at least one polynucleotide in the biological sample is indicative of sepsis, thereby identifying the human as having sepsis.

[0007] In another particular aspect, a method is presented comprising administering to a human identified as having over-representation or under-representation of at least one polynucleotide relative to an internal reference region a therapeutically effective amount of at least one agent used to treat sepsis, wherein the human is identified as having over-representation or under-representation of at least one polynucleotide relative to an internal reference region by analyzing a biological sample isolated from the human for over-representation or under-representation of at least one polynucleotide relative to an internal reference region, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148 and wherein the over-representation or under-representation of the at least one polynucleotide in the biological sample is a positive indicator that the human is in need of the administering.

[0008] In another particular aspect, a method is presented for detecting sepsis in a human, comprising

(a) analyzing a biological sample isolated from the human for over-representation or under-representation of at least one polynucleotide relative to an internal reference region, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148, by contacting the biological sample with at least one synthetic probe specific for a polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148, wherein the contacting generates complexes of synthetic probes bound to specific polynucleotides when at least one polynucleotide comprising any one of 1-57 or 94-148 is present in the biological sample, (b) detecting the complexes of synthetic probes bound to specific polynucleotides, and detecting the internal reference region in the biological sample, and (c) comparing the at least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 detected in the biological sample to the internal reference region detected in the biological sample to determine relative over-representation and under-representation of the at least one polynucleotide in the biological sample, wherein detection of the over-representation and under-representation of the at least one polynucleotide serves as a positive indicator of sepsis in the human.

[0009] In another particular aspect, a method is presented for detecting sepsis in a human, comprising analyzing a biological sample isolated from the human for over-representation or under-representation of at least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 relative to an internal reference region, wherein detection of over-representation or under-representation of the at least one said polynucleotide relative to the internal reference region in the biological sample is a positive indicator of sepsis in the human.

[0010] In another particular aspect, a method is presented for detecting sepsis in a human, comprising detecting at least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 in a biological sample obtained from the human wherein over-representation and under-representation of the at least one polynucleotide relative to an internal reference region in the biological sample is a positive indicator of sepsis in the human. The method may further comprising use of a therapeutically effective amount of at least one agent used to treat sepsis for treatment of the human.

[0011] In another particular aspect, a method is presented for evaluating representation of at least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 in a biological sample of a human, the method comprising: analyzing the biological sample of the human for over-representation or under-representation of at least one polynucleotide relative to an internal reference region, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148, and wherein the over-representation or the under-representation of the at least one polynucleotide in the biological sample is determined by detecting the at least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 in the biological sample, wherein the detecting is achieved by contacting the biological sample with at least one reagent that specifically binds to any one of SEQ ID NOs: 1-57 or 94-148, detecting the internal reference region in the biological sample, and comparing the at least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 detected in the biological sample to the internal reference region detected in the biological sample to determine relative over-representation and under-representation of the at least one polynucleotide in the biological sample.

[0012] In a particular embodiment of any of the above methods, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148 over-represented or under-represented relative to the internal reference region, the at least one is at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, or at least twenty-four of the polynucleotides comprising any one of SEQ ID NOs: 1-57 or 94-148.

[0013] In a particular embodiment of any of the above methods, the biological sample is blood, a product derived from blood, or a fraction derived from blood. In a more particular embodiment, the product derived from blood is plasma or serum.

[0014] In a particular embodiment of any of the above methods, the internal reference region comprises at least one polynucleotide comprising any one of SEQ ID NOs: 59, 61, or 68.

[0015] In a particular embodiment of any of the above methods, detecting the over-representation or under-representation of the at least one polynucleotide relative to an internal reference region comprises at least one of a polymerase chain reaction (PCR)-based detection method, a hybridization-based method, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), solid-phase enzyme immunoassay (EIA), mass spectrometry, or microarray analysis. In a more particular embodiment, the PCR-based detection method is performed using at least one primer pair, wherein each primer pair of the at least one primer pair is specific for any one of SEQ ID NOs: 1-57 or 94-148, and a primer pair specific for at least one of SEQ ID NOs: 59, 61, or 68. In a more particular embodiment, the primer pair specific for any one of SEQ ID NOs: 1-57 or 94-148 and the primer pair specific for at least one of SEQ ID NOs: 59, 61, or 68 is any one of the primer pairs presented in Tables 1-3. In another particular embodiment, the method further comprises sequencing amplification products corresponding to any one of SEQ ID NOs: 1-57 or 94-148 or at least one of SEQ ID NOs: 59, 61, or 68 generated by the PCR-based detection method. In another particular embodiment, the SEQ ID NOs: (nucleic acid sequences) comprise circulating nucleic acid. In another particular embodiment, the at least one agent used to treat sepsis comprises at least one of an antibiotic, anti-fungal agent, anti-viral agent, anti-parasitic agent, or fluids suitable for intravenous administration. In another particular embodiment, the human is monitored for sepsis. In another particular embodiment, the over-representation or under-representation of the at least one polynucleotide relative to an internal reference region is determined using reagents comprising an antibody or a nucleic acid probe specific for any one of SEQ ID NOs: 1-57 or 94-148 and at least one of SEQ ID NOs: 59, 61, or 68. In another particular embodiment, the nucleic acid probe specific for any one of SEQ ID NOs: 1-57 or 94-148 and at least one of SEQ ID NOs: 59, 61, or 68 is labeled with a detectable label.

[0016] In another aspect, a probe comprising a manmade nucleotide sequence that binds specifically to a polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 and at least one manmade tag conjugated thereto and at least one of SEQ ID NOs: 59, 61, or 68 and at least one manmade tag conjugated thereto, wherein the manmade nucleotide sequence is complementary to the polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 and at least one of SEQ ID NOs: 59, 61, or 68. In a particular embodiment, the manmade nucleotide sequence that binds specifically to a polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 and at least one of SEQ ID NOs: 59, 61, or 68 exhibits at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementarity to any one of SEQ ID NOs: 1-57 or 94-148 and at least one of SEQ ID NOs: 59, 61, or 68. In a particular embodiment, the manmade tag is a detectable marker. In a more particular embodiment, the detectable marker comprises a radioactive marker or fluorescent marker. In another embodiment, an array comprising at least one probe that binds specifically to any one of SEQ ID NOs: 1-57 or 94-148 and at least one of SEQ ID NOs: 59, 61, or 68 is presented, wherein the at least one probe is bound to a solid surface. In a particular embodiment, the array is used for diagnosing sepsis. In another embodiment, a kit comprising at least one probe that binds specifically to any one of SEQ ID NOs: 1-57 or 94-148 and at least one of SEQ ID NOs: 59, 61, or 68 is presented, wherein the kit further optionally comprises instructions for use thereof. In a particular embodiment, the kit is used for diagnosing sepsis. In another particular embodiment, the kit is used for detecting the indicated SEQ ID NOs:. In embodiments thereof, the array or kit comprises comprising at least four, five, or ten different probes comprising a manmade nucleotide sequence that binds specifically to a polynucleotide comprising any one of the SEQ ID NOs: listed in Table 4 and at least one manmade tag conjugated thereto, wherein the manmade nucleotide sequence is complementary to the polynucleotide comprising any one of the SEQ ID NOs: listed in Table 4. In a particular embodiment, the array comprises a microarray, gene chip, DNA chip, or a FILMARRAY.RTM..

[0017] In another aspect, a primer comprising, consisting essentially of, or consisting of a manmade nucleotide sequence that binds specifically to a polynucleotide comprising any one of the SEQ ID NOs: listed in Table 4 and at least one manmade tag conjugated thereto is presented, wherein the manmade nucleotide sequence is any one of the polynucleotide primer sequences or a primer pair listed in Tables 1-3 or a variant thereof. In a more particular embodiment, the variant of any one of the polynucleotide primer sequences listed in Tables 1-3 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polynucleotide sequences listed in Table 4. In another particular embodiment, the manmade tag is a detectable marker (e.g., a radioactive marker or fluorescent marker.

[0018] In another aspect, a kit for detecting sepsis in a human is presented comprising at least one primer pair for amplifying a polynucleotide comprising any one of the SEQ ID NOs: listed in Table 4, wherein the at least one primer pair is listed in Tables 1-3 and optionally, instructions for use thereof. In a particular embodiment, the at least one primer pair is four primer pairs and wherein each primer pair of the four primer pairs specifically amplifies a different polynucleotide comprising any one of the SEQ ID NOs: listed in Table 4. In another particular embodiment, the at least one primer pair is five primer pairs and wherein each primer pair of the five primer pairs specifically amplifies a different polynucleotide comprising any one of the SEQ ID NOs: listed in Table 4. In another particular embodiment, the at least one primer pair is ten primer pairs and wherein each primer pair of the ten primer pairs specifically amplifies a different polynucleotide comprising any one of the SEQ ID NOs: listed in Table 4.

[0019] In another aspect, use of a polynucleotide sequence comprising any one of the SEQ ID NOs: listed in Table 4 as a biomarker for the detection of sepsis in a human is encompassed herein. In a particular embodiment, the biomarker is a biomarker in a sample obtained from the human, wherein the sample is a sample of blood, a product derived from blood or a fraction derived from blood (e.g. plasma or serum)

[0020] In an aspect, a method for treating a human suspected of having sepsis is presented, the method comprising treating the human identified as having sepsis with a therapeutically effective amount of at least one agent used to treat sepsis, wherein the human is identifiable as having sepsis by analyzing a biological sample isolated from the human for over-representation or under-representation of at least one polynucleotide, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148 and wherein the over-representation or under-representation of the at least one polynucleotide in the biological sample is a positive indicator of sepsis. In a particular embodiment thereof, the over-representation or the under-representation of the at least one polynucleotide is determined relative to an internal reference region. In a particular embodiment, the internal reference regions is at least one of SEQ ID NOs: 59, 61, or 68.

[0021] Also encompassed herein is a therapeutically effective amount of at least one agent used to treat sepsis or a composition thereof for use in a method of treating sepsis in a human identified as having sepsis, wherein the human is identified as having sepsis by analyzing a biological sample isolated from the human for over-representation or under-representation of at least one polynucleotide, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148 and wherein the over-representation or under-representation of the at least one polynucleotide in the biological sample is a positive indicator of sepsis. The human may exhibit symptoms of sepsis or may be asymptomatic. In a particular embodiment thereof, the over-representation or the under-representation of the at least one polynucleotide is determined relative to an internal reference region. In a particular embodiment, the internal reference regions is at least one of SEQ ID NOs: 59, 61, or 68.

[0022] In another aspect, a method for treating a human identified as exhibiting over-representation or under-representation of at least one polynucleotide is presented, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148 and wherein the over-representation or the under-representation of the at least one polynucleotide in the biological sample is a positive indicator of sepsis, the method comprising treating the human identified as exhibiting the over-representation or the under-representation of the at least one polynucleotide with a therapeutically effective amount of at least one agent used to treat sepsis. In a particular embodiment thereof, the over-representation or the under-representation of the at least one polynucleotide is determined relative to an internal reference region. In another particular embodiment, the human is identifiable as having sepsis by analysis of a biological sample isolated from the human for the over-representation or the under-representation of the at least one polynucleotide, wherein the over-representation or the under-representation of the at least one polynucleotide may optionally be determined relative to an internal reference region. In a particular embodiment, the internal reference regions is at least one of SEQ ID NOs: 59, 61, or 68.

[0023] In yet another aspect, a method for treating a human with early stage sepsis (e.g., pre-symptomatic sepsis) is presented, the method comprising treating the human with early stage sepsis with a therapeutically effective amount of at least one agent used to treat sepsis, wherein the human is identifiable as having early stage sepsis by analyzing a biological sample isolated from the human for over-representation or under-representation of at least one polynucleotide, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148 and wherein the over-representation or under-representation of the at least one polynucleotide in the biological sample is a positive indicator of sepsis. In a particular embodiment thereof, the over-representation or the under-representation of the at least one polynucleotide is determined relative to an internal reference region. In a particular embodiment, the internal reference regions is at least one of SEQ ID NOs: 59, 61, or 68.

[0024] Also encompassed herein is a therapeutically effective amount of at least one agent used to treat sepsis or a composition thereof for use in a method of treating sepsis in a human identified with early stage sepsis, wherein the human is identified as having early stage sepsis by analyzing a biological sample isolated from the human for over-representation or under-representation of at least one polynucleotide, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148 and wherein the over-representation or under-representation of the at least one polynucleotide in the biological sample is a positive indicator of sepsis. The human may exhibit symptoms of sepsis or may be asymptomatic. In a particular embodiment thereof, the over-representation or the under-representation of the at least one polynucleotide is determined relative to an internal reference region.

[0025] Methods described herein comprise embodiments wherein the at least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 over-represented or under-represented is at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or at least twenty of the polynucleotides comprising SEQ ID NOs: 1-57 or 94-148. In a particular embodiment thereof, the over-representation or the under-representation of the at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or at least twenty of the polynucleotides comprising SEQ ID NOs: 1-57 or 94-148 is determined relative to an internal reference region. In a particular embodiment, the internal reference regions is at least one of SEQ ID NOs: 59, 61, or 68.

[0026] In a further aspect, a method for evaluating representation of at least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 in a biological sample of a human is presented, the method comprising:

[0027] analyzing the biological sample of the human for over-representation or under-representation of at least one polynucleotide, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148, and

[0028] wherein the over-representation or the under-representation of the at least one polynucleotide in the biological sample is determined by detecting the at least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 in the biological sample, wherein the detecting is achieved by contacting the biological sample with at least one reagent that specifically binds to any one of SEQ ID NOs: 1-57 or 94-148, to determine relative over-representation and under-representation of the at least one polynucleotide in the biological sample. In a particular embodiment thereof, the over-representation or the under-representation of the at least one polynucleotide in the biological sample, when compared to an internal reference is determined; for example by quantitative real time polymerase chain reaction (RT-PCR). In accordance with methods described herein, amplification of regions of the CNAs represented by SEQ ID NOs: 1-57 or 94-148 involves a determination of how many amplification cycles are called for to reach a desired detection limit. In another particular embodiment, the over-representation or the under-representation of the at least one polynucleotide in the biological sample is determined by detecting the at least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 in the biological sample, wherein the detecting is achieved by contacting the biological sample with at least one reagent that specifically binds to any one of SEQ ID NOs: 1-57 or 94-148, and detecting the internal reference region in the biological sample, and comparing the at least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 detected in the biological sample to the internal reference region detected in the biological sample to determine relative over-representation and under-representation of the at least one polynucleotide in the biological sample. The over-representation or under-representation of the at least one polynucleotide may also be referred to herein as a deviation of the level of the at least one polynucleotide in the biological sample of an infected subject relative to the level of the at least one polynucleotide in a biological sample of a healthy subject.

[0029] Detection of the at least one polynucleotide may be achieved using RT-PCR as detailed herein as well as amplification free detection methods.

[0030] Methods described herein comprise embodiments wherein the biological sample is a bodily fluid, such as, without limitation, whole blood, a blood fraction, saliva, urine, sputum, cerebrospinal fluid, tears, sweat, milk, or interstitial fluid. In a particular embodiment, the biological sample is blood, a product derived from blood, or a fraction derived from blood. In a particular embodiment, the product derived from blood is plasma or serum.

[0031] Methods described herein comprise embodiments wherein detecting the over-representation or under-representation of the at least one polynucleotide comprises at least one of a PCR-based detection method (e.g., RT-PCR), a hybridization-based method, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (MA), solid-phase enzyme immunoassay (EIA), mass spectrometry, and microarray analysis. In a particular embodiment thereof, detecting the over-representation or under-representation of the at least one polynucleotide is determined relative to an internal reference region. In a particular embodiment, the internal reference regions is at least one of SEQ ID NOs: 59, 61, or 68.

[0032] In embodiments comprising a PCR-based detection method, the PCR-based detection method comprises amplifying nucleic acid sequences in the biological sample using primers that are specific for and capable of amplifying any one of SEQ ID NOs: 1-57 or 94-148, wherein the amplifying generates amplification products corresponding to any one of SEQ ID NOs: 1-57 or 94-148 when the biological sample comprises any one of SEQ ID NOs: 1-57 or 94-148. In embodiments comprising a PCR-based detection method, the PCR-based detection method is performed using at least one primer pair, wherein each primer pair of the at least one primer pair is specific for any one of SEQ ID NOs: 1-57 or 94-148. In a more particular embodiment thereof, the primer pair specific for any one of SEQ ID NOs: 1-57 or 94-148 is any one of the primer pairs presented in Table 1 or Table 3. In an even more particular embodiment thereof, the PCR-based detection method further comprises sequencing the amplification products corresponding to any one of SEQ ID NOs: 1-57 or 94-148. In a particular embodiment, the internal reference regions is at least one of SEQ ID NOs: 59, 61, or 68 and primer pairs therefor are presented in Table 2.

[0033] Methods described herein comprise embodiments wherein nucleic acid sequences comprise circulating nucleic acids.

[0034] Methods described herein comprise embodiments wherein the at least one agent used to treat sepsis comprises at least one of an antibiotic, anti-fungal agent, anti-viral agent, anti-parasitic agent, or fluids suitable for intravenous administration.

[0035] Methods described herein comprise embodiments wherein the human is monitored for sepsis.

[0036] Methods described herein comprise embodiments wherein the over-representation or under-representation of the at least one polynucleotide is determined using reagents comprising an antibody or a nucleic acid probe specific for any one of SEQ ID NOs: 1-57 or 94-148. In a particular embodiment thereof, the over-representation or under-representation of the at least one polynucleotide is determined relative to an internal reference region. In a more particular embodiment, the antibody is a monoclonal or a polyclonal antibody. In a still more particular embodiment, the antibody is obtained from mice, rats, rabbits, goats, chicken, donkey, horses or guinea pigs.

[0037] Methods described herein comprise embodiments wherein a nucleic acid probe specific for any one of SEQ ID NOs: 1-57 or 94-148 is labeled with a detectable label.

[0038] In another aspect, a probe comprising a manmade nucleotide sequence capable of binding specifically to a polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 and at least one manmade tag conjugated thereto is presented, wherein the manmade nucleotide sequence is complementary to the polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148. In a particular embodiment thereof, the manmade nucleotide sequence capable of binding specifically to a polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 exhibits at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementarity to any one of SEQ ID NOs: 1-57 or 94-148. In another particular embodiment, the manmade tag is a detectable marker. In a still more particular embodiment, the detectable marker comprises a radioactive marker or fluorescent marker.

[0039] Also encompassed herein is an array comprising at least one probe described herein, wherein the at least one probe is bound to a solid surface. Such arrays may comprise a microarray, gene chip, DNA chip, a FILMARRAY.RTM., or a similar array.

[0040] Kits comprising at least one probe described herein and instructions for use thereof are also encompassed.

[0041] In a particular embodiment, the array or kit described herein comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or at least twenty different probes comprising a manmade nucleotide sequence capable of binding specifically to corresponding polynucleotides comprising any one of SEQ ID NOs: 1-57 or 94-148 and at least one manmade tag conjugated thereto, wherein the manmade nucleotide sequence is complementary to the polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148. In a particular embodiment, the array or kit described herein comprises at least four different probes comprising a manmade nucleotide sequence capable of binding specifically to a polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 and at least one manmade tag conjugated thereto, wherein the manmade nucleotide sequence is complementary to the polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148. In a more particular embodiment, the array or kit described herein comprises at least eight different probes comprising a manmade nucleotide sequence capable of binding specifically to a polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 and at least one manmade tag conjugated thereto, wherein the manmade nucleotide sequence is complementary to the polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148.

[0042] Also encompassed herein is a primer comprising a manmade nucleotide sequence capable of binding specifically to a polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 and at least one manmade tag conjugated thereto, wherein the manmade nucleotide sequence is any one of the polynucleotide sequences listed in Table 1 or Table 3 as a forward primer or a variant thereof or as a reverse primer or a variant thereof. In an embodiment thereof, the variant of any one of the primer sequences listed in Table 1 or Table 3 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the primer sequences listed in Table 1 or Table 3. In a particular embodiment, a variant of a primer comprises different nucleotides at the 5' end of the primer, which positions are more tolerant of variations thereto. In a particular embodiment, the manmade tag is a detectable marker (e.g., a radioactive marker, fluorescent dye), a tag that is specifically recognized (e.g., bound) by a labeled reagent (e.g., a labeled antibody), a tag that is specifically bound by a magnetic bead, or any other marker comprising detectable label.

[0043] Also encompassed herein are primers and probes for use in detecting the at least one polynucleotide described herein, wherein when using a TaqMan system, the detection of a particular polynucleotide sequence requires two specific primers (a forward and reverse primer pair specific for the polynucleotide sequence) and a probe (labeled oligonucleotide specific for the polynucleotide sequence). A labeled probe may, for example, be a TaqMan probe comprising a fluorescent label at a first terminus and a quencher at a second terminus whereby upon probe displacement during quantitative PCR (qPCR), the probe is cleaved, thus releasing the fluorescent label from the vicinity and effects of the quencher. Upon release from the vicinity/effects of the quencher, a higher fluorescence is emitted from the fluorescent label, which can be detected and quantitated to reflect the level/amount of the particular polynucleotide sequence present in a sample.

[0044] Also encompassed herein are methods for amplification-free detection of the at least one polynucleotide described herein, wherein such methods comprise a pair of oligonucleotide probes which are specific for the particular polynucleotide and are differentially labeled such that when each is bound to the polynucleotide and therefore in close proximity, the signal emitted from the probes changes. With respect to oligonucleotide probes that are labeled with fluorescent tags, close proximity of the fluorescent tags when bound to the polynucleotide is detected by an increase in fluorescence emissions.

[0045] Also encompassed herein is a primer consisting essentially of or consisting of a manmade nucleotide sequence capable of binding specifically to a polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 and at least one manmade tag conjugated thereto, wherein the manmade nucleotide sequence is any one of the polynucleotide sequences listed in Table 1 or Table 3 or a variant thereof. In an embodiment thereof, the variant of any one of the polynucleotide sequences listed in Table 1 or Table 3 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polynucleotide sequences listed in Table 1 or Table 3. In a particular embodiment, the manmade tag is a detectable marker (e.g., a radioactive marker or fluorescent marker).

[0046] Primer pairs complementary to SEQ ID NOs: 1-57 or 94-148 and suitable for PCR amplification are readily determined based on the sequences of SEQ ID NOs: 1-57 or 94-148 (5' to 3' strands) and reverse strands thereof (3' to 5' strands). Such primers are typically 8-20 nucleotides in length and are complementary (e.g., exhibit perfect complementarity or may be variants thereof that maintain a degree of complementarity sufficient to bind and act as primers in a PCR amplification) to any one of SEQ ID NOs: 1-57 or 94-148 or a reverse strand thereof. Choices regarding primer pairs suitable for PCR amplification are also determined based on the guanine/cytosine content of a potential primer sequence and the distance between the primers in a pair when bound to the target polynucleotide, with the understanding that PCR amplification products must be of a detectable size.

[0047] In another embodiment, therapeutic efficacy of a treatment regimen may be evaluated based on a change in the over-representation or under-representation of at least one of SEQ ID NOs: 1-57 or 94-148 following onset of the treatment regimen. In a particular embodiment, a decrease in representation of a CNA that is over-represented in sepsis (e.g., SEQ ID NOs: 94-148) is indicative that the treatment regimen is therapeutically effective. In another particular embodiment, an increase in representation of a CNA that is under-represented in sepsis (e.g., SEQ ID NOs: 1-57) is indicative that the treatment regimen is therapeutically effective.

[0048] Also encompassed herein is an array comprising at least one primer listed in Table 1 or Table 3, wherein the at least one primer is bound to a solid surface.

[0049] A kit for detecting sepsis in a human is also described, wherein the kit comprises at least one primer pair for amplifying a polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148, wherein the at least one primer pair is listed in Table 1 or Table 3 or a functional variant thereof or a primer that may be determined based on any one of the sequences designated herein as SEQ ID NOs: 1-57 or 94-148 and instructions for use thereof. In a particular embodiment, the at least one primer pair is four primer pairs and each primer pair of the four primer pairs specifically amplifies a different polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148. In another particular embodiment, the at least one primer pair is eight primer pairs and each primer pair of the eight primer pairs specifically amplifies a different polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148.

[0050] Additional aspects of the present invention will be apparent in view of the description which follows.

[0051] Additional aspects of the present invention will be apparent in view of the description which follows.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

[0052] The invention will now be described in relation to the drawings and tables.

[0053] FIGS. 1A-H presents Table 1, wherein each of the sequences corresponds to a CNA and is designated SEQ ID NO: 1-57 in consecutive order. SEQ ID NOs: 1-57 are positive indicators of sepsis that are more highly represented in healthy controls and thus, a decrease in copy number is observed during infection. Accordingly, a decrease in copy number of any one of SEQ ID NOs: 1-57 is a positive indicator of sepsis. Forward and reverse primers suitable for PCR amplification of each of SEQ ID NOs: 1-57 are presented in the same row as the CNA for which they are specific.

[0054] FIGS. 2A-F presents Table 2, wherein each of the Sequences corresponds to a CNA and is designated SEQ ID NO: 58-93 in consecutive order. SEQ ID NOs: 58-93 are equally represented in copy number in healthy controls and subjects afflicted with sepsis. Forward and reverse primers suitable for PCR amplification of each of SEQ ID NOs: 58-93 are presented in the same row as the CNA for which they are specific.

[0055] FIGS. 3A-I presents Table 3, wherein each of the Sequences corresponds to a CNA and is designated SEQ ID NO: 94-148 in consecutive order. SEQ ID NOs: 94-148 are positive indicators of sepsis that increase in copy number during infection. Forward and reverse primers suitable for PCR amplification of each of SEQ ID NOs: 94-148 are presented in the same row as the CNA for which they are specific.

DETAILED DESCRIPTION OF THE INVENTION

[0056] Various embodiments will be described in detail with reference to the drawings and tables. Reference to various embodiments does not limit the scope of the claims attached hereto. Additionally, any examples set forth in this specification are not intended to be limiting and merely set forth some of the many possible embodiments.

[0057] Methods, reagents, and kits described herein relate to detection and analysis of cell-free DNA available in the blood following active secretion, cell death or apoptosis. The present inventors have demonstrated that cell-free DNA indicative of sepsis can be detected in blood up to 2-3 days prior to clinical diagnosis of sepsis according to Sepsis-3-definition (defined below in accordance with current medical standards). Traditional biomarkers such as lactate, procalcitonin and others are measured only after clinical sepsis is evident from the patient's vital signs or labs. In high-risk patients, this may be too late to prevent the body's cascade of cytokine release, the harbinger of development for sepsis sequelae.

[0058] Accordingly, the lead-time provided by the present methods, reagents, and kits identifies subjects requiring heightened attention and monitoring. In particular embodiments, the present methods, reagents, and kits provide information on which basis a medical practitioner may initiate appropriate therapy and identify the site of infection, which may require source control, before the onset of irreversible organ dysfunction and widespread cellular hypoxia that occurs during the later stages of sepsis in septic patients.

[0059] Further to the above, methods described herein are based on the analysis of circulating nucleic acids, which are present in, for example, serum and plasma of humans. The composition and relative abundance of circulating nucleic acids varies depending on disease state. The present inventors identified particular genomic regions that do not change in relative abundance of specific regions of circulating nucleic acids between sepsis patients and controls (non-sepsis), which genomic regions are designated internal reference regions. Genomic regions that significantly vary in abundance between sepsis patients and controls were also identified and designated disease motifs. Relative abundance for disease motifs can be examined using Real-Time Polymerase Chain Reaction (RT-PCR) to determine if an "unknown" sample can be identified as exhibiting features characteristic of sepsis or features characteristic of non-sepsis. In a particular embodiment, identifying a sample as exhibiting features characteristic of sepsis may be used to predict onset of sepsis or diagnose sepsis.

[0060] It is to be understood that combinations of primers binding to genomic regions that significantly vary in coverage and abundance between sepsis patients can be optimized. The RT-PCR assay can be designed based at least in part on primers used and such an assay can be used to identify early features of sepsis.

[0061] Probes, methods, and kits described herein can analyze cell free DNA (cfDNA) collected at a preclinical stage of sepsis and accurately determine which patients will go on to develop sepsis. This preclinical phase includes patients that already have molecular evidence of an infection but have not yet manifested traditional criteria including vital sign abnormalities, fever and laboratory changes (according to the Sepsis-3-definition).

[0062] Early identification of sepsis, formerly known as septicaemia, is clinically valuable because sepsis has a high morbidity and mortality rate in mammals in general and, even under the best of care, in humans. Sepsis is characterized as a systemic, excessive inflammatory response, which is frequently triggered by infection and proceeds unchecked by normal regulatory controls. Sepsis is most often associated with bacterial infections in humans. Severe trauma can also lead to systemic responses that adversely affect adaptive and innate immune responses, leading to excessive inflammation having pathological consequences. Severe trauma patients, such as burn and accident victims, may subsequently be afflicted with infections, some of which lead to sepsis. Clinical symptoms of sepsis in humans include lethargy, fever/hyperthermia in early stages of sepsis, confusion, and/or hypothermia in end-stage sepsis (e.g., septic shock).

[0063] Sepsis is a leading cause of hospitalization in the United States and is one of the most expensive conditions to treat because it typically requires a stay in the Intensive Care Unit (ICU) to achieve patient recovery. It has been estimated that 19 million patients will develop sepsis worldwide each year and this estimate likely substantially underestimates the problem. Indeed, sepsis is a leading cause of death, morbidity, and medical expense. Sepsis contributes to one-third of deaths of hospitalized patients. The mortality rate is estimated to be between 20% and 80%, depending on the cause of the sepsis and progression of sepsis. Onset of sepsis in patients following a hospital stay is frequent and particularly challenging, at least in part, because such patients are likely still in a state of recovery and therefore, physiologically weakened. Such patients may be in early, pre-clinical stages of sepsis upon release, but absent the present discoveries, sepsis cannot be detected at pre-clinical stages of the disease. Re-admittance to hospitals is a significant problem as it is costly and typically not covered in full by medical insurance policies. Indeed, people with sepsis are two to three times more likely to be readmitted to the hospital as people released from the hospital who were diagnosed with a variety of conditions, including heart failure, pneumonia, and chronic obstructive pulmonary disease. It is also noteworthy that a bout of sepsis can have lifelong ramifications for patients who are successfully treated and survive. As the number of sepsis survivors grows, it has become increasingly apparent that sepsis frequently leads to long-lasting physical and cognitive impairment.

[0064] Indeed, the growing number of patients who survive the acute phase of sepsis has revealed that the danger associated with the disease extends long after discharge from the hospital. Long-term mortality following a bout of sepsis is approximately 50% in the first year and rises to >81% over five years post-discharge. Sepsis survivors are also more prone to exhibit diminished physical and/or cognitive function following the bout of sepsis than age-matched controls hospitalized for unrelated indications. As a consequence of these diminished functions, many sepsis survivors are unable to return to the workforce.

[0065] The standard of care for treatment of bacterial sepsis in humans calls for aggressive use of antimicrobial therapy (e.g., antibiotics, anti-fungal agent, anti-viral agent), anti-parasitic agent therapy, and/or intravenous fluid therapy, which may be administered in conjunction with anti-inflammatory therapy. The choice of antibiotic used for treating a septic patient depends on the type of bacteria that infected the subject, which infection subsequently led to sepsis in the subject. Methods for culturing and identifying bacteria in samples isolated from a subject are known in the art. Broad-spectrum antibiotics are recommended within one hour after sepsis recognition/diagnosis. Beta-lactam antibiotics having broad coverage administered in combination with fluoroquinolones, macrolides, or aminoglycosides are recommended for severe cases of sepsis. An exemplary inflammatory therapy involves administration of non-steroidal anti-inflammatory agents to a human afflicted with bacterial sepsis.

[0066] Fungal infections are treated with anti-fungal medications specific to the particular fungus that caused the infection. These may be used in a cream or ointment, suppository, or pill form. Candida species are the predominant agent of fungal sepsis and account for about 5% of all cases of severe sepsis and septic shock in the United States. The antifungal armamentarium has expanded considerably with the advent of lipid formulations of amphotericin B, the newest triazoles and the echinocandins. Clinical trials have shown that the triazoles and echinocandins are efficacious and well tolerated antifungal therapies.

[0067] Prospective treatments for specific viruses implicated in sepsis are being developed. Pleconaril is an antiviral against enteroviral infection which inhibits viral attachment to the hosts cell receptors and prevents uncoating of the viral nucleic acids. Examples of ativiral agents that may be beneficial in presentations of sepsis in certain situations include acyclovir, which has been proven effective in HSV infections, amantadine, rimantadine, oseltamivir, and zanamivir for influenza, and more broad-spectrum antiviral drugs like ribavirin and favipiravir.

[0068] Parasite infections can lead to sepsis. For Giardia infections, a medication like metronidazole (Flagyl), tinidazole (Tindamax) or nitazoxanide (Alinia) may be used. For Chagas disease, antiparasitic medications such as benznidazole and nifurtimox may be used. For tapeworm, the most commonly used medications are praziquantel (Biltricide), aibendazole (Albenza), and nitazoxanide (Alinia). However, if the infection has progressed and become more invasive, you may need treatment with anti-inflammatory medications, anti-seizure medications, a shunt to drain fluid from your brain, or surgery to remove cysts caused by the tapeworm. For roundworm, the most commonly used medications include medendazole (Vermox), albdendazole (Albenza) and ivermectin (Stromectol). Surgery could be required to remove the worm if there is a bowel obstruction.

[0069] The following guidelines provide the standard of care for sepsis patients as established by the American Medical Association:

[0070] Managing Infection:

[0071] Antibiotics: Administer broad-spectrum intravenous antimicrobials for all likely pathogens within 1 hour after sepsis recognition (strong recommendation; moderate quality of evidence [QOE]).

[0072] Source control: Obtain anatomic source control as rapidly as is practical (best practice statement [BPS]).

[0073] Antibiotic stewardship: Assess patients daily for de-escalation of antimicrobials; narrow therapy based on cultures and/or clinical improvement (BPS).

[0074] Managing Resuscitation:

[0075] Fluids: For patients with sepsis-induced hypoperfusion, provide 30 mL/kg of intravenous crystalloid within 3 hours (strong recommendation; low QOE) with additional fluid based on frequent reassessment (BPS), preferentially using dynamic variables to assess fluid responsiveness (weak recommendation; low QOE).

[0076] Resuscitation targets: For patients with septic shock requiring vasopressors, target a mean arterial pressure (MAP) of 65 mm Hg (strong recommendation; moderate QOE).

[0077] Vasopressors: Use norepinephrine as a first-choice vasopressor (strong recommendation; moderate QOE).

[0078] Mechanical Ventilation in Patients with Sepsis-Related Acute Respiratory Distress Syndrome (ARDS):

[0079] Target a tidal volume of 6 mL/kg of predicted body weight (strong recommendation; high QOE) and a plateau pressure of .ltoreq.30 cm H.sub.2O (strong recommendation; moderate QOE). See, for example, Howell and Davis (2017, JAMA 317:847).

[0080] As indicated hereinabove, sepsis is associated with multiple organ failure and high mortality. Commonly used markers for diagnosis of sepsis include: elevated leukocyte counts and elevated cytokine levels such as IL-6, IL-8 and IL-18, C-reactive protein, and procalcitonin. The latter two proteins are expressed at higher levels after trauma. With respect to procalcitonin, determination of procalcitonin levels is viewed as a helpful diagnostic marker, but is not viewed as a definitive marker of sepsis. Soluble urokinase-type plasminogen activator receptor (SuPAR) is considered to be a nonspecific marker of inflammation, but has prognostic value in the context of sepsis because higher SuPAR levels are associated with higher mortality in sepsis patients.

[0081] None of these markers provides a predictive indicator of sepsis, but rather collectively serve as indicators only upon onset of sepsis. Indeed, in advance of the present findings, a reliable method to predict risk for or diagnose sepsis in advance of the onset of clinical symptoms did not exist. As detailed herein, the methods, reagents, and kits described herein make it possible to diagnose patients with sepsis at pre-clinical stages and thus, make it possible to treat such patients in advance of the onset of clinical symptoms and at the least reduce the severity of acute sepsis in the patient should the patient progress toward disease despite early intervention.

[0082] Exemplary Target Patient Populations

[0083] Adult patients admitted to, for example, a hospital who are at high-risk for developing sepsis include the following:

[0084] Victims of trauma with either an Injury Severity Score of .gtoreq.15 or a Glasgow Coma Score of .ltoreq.8 on hospital presentation;

[0085] Any patient undergoing high-risk surgical procedures including any emergency surgery, high risk elective surgery procedures involving the thorax, esophagus, stomach, small bowel, large bowel; or

[0086] Any patient being admitted to any intensive care unit (ICU) setting for any reason with no current evidence or suspicion of active infection as determined by primary team of attending medical practitioners.

[0087] Methods, reagents, and kits described herein relate to predicting and/or diagnosing sepsis in a human in advance of the appearance of symptoms of sepsis in the human. In accordance with the experimental findings presented herein, methods, reagents, and kits described are useful for diagnosing sepsis in a human subject in advance of symptomatic presentation. Indeed, results presented herein demonstrate that methods, reagents, and kits described herein can diagnose sepsis in a human subject at least three days in advance of clinical presentation. In light of results presented in the Examples, detection of over-representation or under-representation of at least one polynucleotide relative to an internal reference region in a body fluid sample (e.g., serum) isolated from a human subject, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148, is a positive indicator that the human subject will develop sepsis symptoms. Accordingly, the methods, reagents, and kits described herein provide for diagnosis of sepsis at pre-clinical stages of the disease. Also encompassed herein are methods to assess over-representation or under-representation of at least one polynucleotide relative to an internal reference region, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148, in body fluids and kits for such purposes.

[0088] In addition to plasma or serum, over-representation or under-representation of at least one polynucleotide (at least one of SEQ ID NOs: 1-57 or 94-148) relative to an internal reference region may be determined in other body fluids isolated from a human subject including: whole blood, a product derived from blood, or any fraction derived from blood (in addition to plasma or serum.

[0089] Any known method may be used for the determination of over-representation or under-representation of at least one polynucleotide (at least one of SEQ ID NOs: 1-57 or 94-148) relative to an internal reference region in body fluids. Methods encompassed for such determinations include: polymerase chain reaction (PCR) amplification with sequence specific primer pairs, a hybridization-based method, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), solid-phase enzyme immunoassay (EIA), mass spectrometry, microarray analysis, and any combination thereof. Such methods when used for determining risk for developing sepsis or predicting onset of sepsis are encompassed herein.

[0090] In a particular method for determining over-representation or under-representation of any one of SEQ ID NOs: 1-57 or 94-148 relative to an internal reference region in human body fluids, e.g. serum, the method calls for PCR amplification with sequence specific primer pairs. In an embodiment thereof, the PCR amplification is performed with at least one primer pair specific for any one of SEQ ID NOs: 1-57 or 94-148. Exemplary primer pairs for amplifying any one of SEQ ID NOs: 1-57 or 94-148 are presented in Table 1 or Table 3. Conditions for performing PCR amplifications are known in the art and presented in the Examples herein below. Such conditions may be determined based in part on the composition of a primer and/or primer pair and considerations relating to same are known in the art.

[0091] In another particular method for determining over-representation or under-representation of any one of SEQ ID NOs: 1-57 or 94-148 relative to an internal reference region in human body fluids, e.g. serum, the method calls for an ELISA. In one embodiment, the ELISA for at least one of SEQ ID NOs: 1-57 or 94-148 involves a sandwich array. In such an embodiment, PCR amplification of at least one of SEQ ID NOs: 1-57 or 94-148 may be performed as an initial step. Conventional microtiter plates may be coated with a first antibody, e.g. a guinea pig polyclonal antibody, directed against any one of SEQ ID NOs: 1-57 or 94-148. The plates are then blocked and the sample or reference is loaded. After incubation with, e.g., at least one of SEQ ID NOs: 1-57 or 94-148, a second antibody against any one of SEQ ID NOs: 1-57 or 94-148 is applied, e.g. a polyclonal rabbit antibody. A third antibody that detects the second antibody, e.g. an anti-rabbit antibody, conjugated to a suitable label, e.g. an enzyme for chromogenic detection, is then added. The plate is then developed with a substrate for the label in order to detect and quantify the label, which in turn serves as a measure of any one of SEQ ID NOs: 1-57 or 94-148 in the body fluid. This determination may then be compared to that of an internal reference region measured by similar methodology. If the label is an enzyme for chromogenic detection, the substrate is a color-generating substrate of the conjugated enzyme and the color reaction is subsequently detected in a microplate reader and compared to standards.

[0092] Suitable pairs of antibodies that may be used as first and second antibodies are any combination of, e.g., guinea pig, rat, mouse, rabbit, goat, chicken, donkey or horse antibodies. In a particular embodiment, the antibodies are polyclonal antibodies. In another particular embodiment, the antibodies are monoclonal antibodies or antibody fragments. Suitable labels include: chromogenic labels (enzymes that can be used to convert a substrate to a detectable colored or fluorescent compound), spectroscopic labels (e.g., fluorescent labels), and affinity labels which may be developed by an additional compound specific for the label, thereby facilitating detection and quantification, or any other label used in standard ELISA.

[0093] Other preferred methods for detection of any one of SEQ ID NOs: 1-57 or 94-148 include radioimmunoassay or competitive immunoassay using a single antibody and chemiluminescence detection on automated commercial analytical robots. Microparticle enhanced fluorescence, fluorescence polarized methodologies, or mass spectrometry may also be used. Detection devices, e.g. microarrays, are also useful components as readout systems for any one of SEQ ID NOs: 1-57 or 94-148.

[0094] Also encompassed herein are kits for assessing over-representation or under-representation of any one of SEQ ID NOs: 1-57 or 94-148 relative to an internal reference region for determining risk for developing sepsis, which kits may comprise apparatus and reagents for detecting at least one of SEQ ID NOs: 1-57 or 94-148. Apparatus and reagents considered for PCR amplification include: suitable PCR primer pairs specific for each of SEQ ID NOs: 1-57 or 94-148, amplification reagents, and thermocycling devices. With respect to ELISA, microtiter plates for ELISA, pre-coated ELISA plates, and plate covers are encompassed. Reagents useful for ELISA include those antibodies and solutions developed and designed for detecting each of SEQ ID NOs: 1-57 or 94-148. Standard solutions comprising each of SEQ ID NOs: 1-57 or 94-148 as positive controls may be included in such kits. Kits may further comprise hardware, such as pipettes, solutions such as buffers, blocking solutions and the like, filters, and directions for use thereof.

[0095] The following definitions are presented as an aid to understand the invention.

[0096] The term "DNA" means a polymer composed of deoxyribonucleotides.

[0097] The terms "sample", "biological sample", "diagnostic sample", and the like refer to a material known or suspected of containing one or more polynucleotide or polypeptide markers. The diagnostic sample may be any tissue ((e.g., blood, and fractions thereof, including serum, etc.).

[0098] The terms "polynucleotide" and "nucleic acid", used interchangeably herein, describe a polymer of any length, e.g., greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, usually up to about 10,000 or more bases composed of nucleotides, such as deoxyribonucleotides or ribonucleotides, or compounds produced synthetically which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids in Watson-Crick base pairing interactions. Polynucleotide and nucleic acid include polynucleotides that encode a native-sequence polypeptide, a polypeptide variant, a portion of a polypeptide, a chimeric polypeptide, or an isoform, precursor, complex, modified form, or derivative of any of the foregoing, and any precursors thereof. Polynucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may be modified after synthesis (e.g., by conjugation with a label, such as a radioactive, chemiluminescent, chemiflourescent, or fluorescent label, and the like). Other types of modifications to polynucleotides known to a person skilled in the art include substitution of one or more naturally-occurring nucleotides with an analog, internucleotide modifications (e.g., uncharged linkages, charged linkages), and the like.

[0099] Polynucleotides can also include circulating nucleic acids ("CNA"). The term "circulating nucleic acid" or "CNA" refers to free nucleic acid, including RNA and DNA, circulating in any form in the blood. CNA can include gene transcripts, transcription factors or other polynucleotide sequences. CNA can be obtained from any applicable biological sample, including blood, plasma, serum, and the like.

[0100] "Variants" of the sequences described herein are sequences wherein at least one nucleotide differs from that of the native or wild-type sequence (or the complement thereof), by virtue of an insertion, deletion, modification and/or substitution of one or more nucleotides within the native sequence. Such variants generally have less than 100% sequence identity relative to a native sequence or its complement. Accordingly, a sequence variant may have a nucleotide sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity relative to the native or wild-type sequence or complement thereof. Variants, furthermore, may include fragments of any length that retain a biological activity of the corresponding native sequence. Variants also include sequences wherein one or more nucleotides are added to the 5' or 3' end of, or within, a native sequence or its complement.

[0101] "Percent sequence identity" is defined herein as the percentage of nucleotides or amino acid residues in the candidate sequence that are identical to the nucleotides or residues in the sequence of interest after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Methods and computer programs for the alignment of sequences are well known in the art, including, for example, "BLAST" algorithms.

[0102] "Oligonucleotides" include short, single-stranded polynucleotides that are at least seven nucleotides in length and less than about 250 nucleotides in length. The term "polynucleotides" includes oligonucleotides.

[0103] "Label" refers to a detectable compound or composition and "labelling" refers to the conjugation, fusion, or attachment of a detectable compound or composition to another. In some aspects described herein, the label is conjugated or fused directly or indirectly to a reagent, such as a polynucleotide probe or an antibody, and assists with the detection of the reagent to which it is conjugated or fused. The label itself can also be detectable (such as radioisotope labels or fluorescent labels and the like). In some aspects described herein, the label is an enzymatic label which catalyzes chemical alteration of a substrate compound or composition and results in a detectable product.

[0104] The term "diagnosis", as used herein, refers to the identification or classification of a molecular or pathological state, disease, or condition (e.g., sepsis). In a particular embodiment, sepsis is diagnosed in a subject (e.g., a human subject) in advance of onset of sepsis symptoms.

[0105] In a particular embodiment, a human subject is characterized as being "at risk for developing sepsis" because they have recently experienced an accident (e.g., vehicular, bicycle), physical trauma (e.g., due to burns resulting from exposure to, e.g., fire or chemicals; knife or gunshot wounds; blunt force trauma; explosion), surgery, and/or infection (e.g., a bacterial, fungal, or viral infection). Human subjects at risk for developing sepsis also include: subjects with weakened immune systems (e.g., immunocompromised subjects); subjects with pre-existing infections or medical conditions (e.g., type 2 diabetes, kidney disease, and asthma), obese subjects, and very young and very old subjects, and subjects injured in contaminated environments. Assays to evaluate risk for developing sepsis may also be implemented on all patients admitted to the hospital and/or released from the hospital as a matter of routine.

[0106] In a particular embodiment thereof, each patient released after a hospital stay comprising at least one overnight stay is assayed using methods described herein to evaluate whether the patient has early stage sepsis or is at risk for developing sepsis post-release. In that the expense involved in re-admittance to the hospital is substantial and insurance coverage is not commensurate with such expenses, screening all patients released after at least one overnight stay, would be a cost-effective way in which to provide better healthcare to the patient and minimize expenditures involved with same.

[0107] Further to the above, exemplary indications for use include:

[0108] At hospital admission where the best option today is to observe symptoms and run a series of blood cultures which normally takes over 24 hours for results and have high error rates;

[0109] On high risk patient groups in hospitals where early identification and treatment of sepsis could have an impact to a patient's survival;

[0110] On discharge where ruling out sepsis could lead to early discharge;

[0111] In long term care facilities where the frequency of sepsis is high and early diagnosis and appropriate monitoring and/or treatment thereafter could potentially reduce costly re-admissions and/or admissions to hospitals; and

[0112] Monitoring neonates for neonatal sepsis

Sepsis-3 Definitions

[0113] Sepsis is life-threatening organ dysfunction due to a dysregulated host response to infection

Sepsis clinical criteria: organ dysfunction is defined as an increase of 2 points or more in the Sequential Organ Failure Assessment (SOFA) score

[0114] for patients with infections, an increase of 2 SOFA points gives an overall mortality rate of 10%

[0115] Patients with suspected infection who are likely to have a prolonged ICU stay or to die in the hospital can be promptly identified at the bedside with qSOFA ("HAT"); i.e. 2 or more of:

[0116] Hypotension: SBP less than or equal to 100 mmHg

[0117] Altered mental status (any GCS less than 15)

[0118] Tachypnoea: RR greater than or equal to 22.

[0119] Septic shock is a subset of sepsis in which underlying circulatory and cellular/metabolic abnormalities are profound enough to substantially increase mortality

[0120] Septic shock clinical criteria: Sepsis and (despite adequate volume resuscitation) both of:

[0121] Persistent hypotension requiring vasopressors to maintain MAP greater than or equal to 65 mm Hg, and

[0122] Lactate greater than or equal to 2 mmol/l,

[0123] With these criteria, hospital mortality is in excess of 40%.

[0124] "Primer" refers to a polynucleotide capable of acting as a point of initiation of synthesis along a complementary strand when conditions are suitable for synthesis of a primer extension product. The synthesizing conditions include the presence of four different nucleotide bases (adenosine, cytidine, guanosine, thymidine/uridine) and at least one polymerization-inducing agent such as a reverse transcriptase or a DNA polymerase. The primers are present in a suitable buffer, which may include constituents which are co-factors or affect conditions such as pH and the like at various suitable temperatures. Primer includes single-stranded polynucleotide that is capable of hybridizing to nucleic acid and allowing the polymerization of a complementary nucleic acid, generally by providing a free 3'-OH group. Double stranded sequences can also be utilized. Primers are typically at least about 15 nucleotides. In some embodiments, primers can have a length of from about 15 to about 30, about 15 to about 50, about 15 to about 75, about 15 to about 100, or about 15 to about 500 nucleotides.

[0125] Exemplary primer pairs specific for each of SEQ ID NOs: 1-57 or 94-148 are presented herein in Table 1 or Table 3. Exemplary primer pairs specific for each of SEQ ID NOs: 58-93 are presented herein in Table 2. Such primer pairs are selected based on their specificity for a particular polynucleotide and may be optimized for use in connection with, e.g., PCR amplification. Polynucleotide-specific primer pairs may comprise primers that include variations within their sequence such that the primer is no longer 100% complementary to the polynucleotide for which it is specific. Primers comprising such variations are encompassed herein as long as the variations do not alter the ability of the primer to amplify the polynucleotide with specificity. Such variations may also include nucleotides and/or tags at the 5' and 3' ends of the primer that are not complementary the polynucleotide for which the primer is specific. It is also understood that a primer or primer pair may be complementary to sequences that flank any one of SEQ ID NOs: 1-148 in the human genome and thus, may be used to amplify one of SEQ ID NOs: 1-148 in keeping with methods described herein. Design of such primers and primer pairs is well within the capabilities of one of ordinary skill in the art having read the present specification.

[0126] In a particular embodiment, multiplex PCR based on the TaqMan approach is utilized to detect CNAs of interest. Such embodiments call for a primer pair, wherein each of the primers is specific for the particular CNA (polynucleotide) of interest and one labeled probe which is specific for the particular CNA (polynucleotide) of interest and labeled at each terminus with a different moiety. In a particular embodiment, the different moiety can be a fluorophore at the first terminus and a quencher at the second terminus. Such fluorophore-quencher pairs comprise, for example, FAM-BHQ1, HEX-BHQ1, LC610-BHQ2, CY5-BHQ-2, and CY5.5-BBQ650. Other pairs useful for this embodiment are known in the art.

[0127] A "motif" or "sequence motif" refers to a nucleotide sequence pattern that is generally conserved across multiple species. Polynucleotides can be derived from the motif. The polynucleotides can correspond to the entire sequence of the motif or a portion or portions of the motif.

[0128] "Marker" or "biomarker" refers to an indicator which can be detected in a sample, and includes predictive, diagnostic, and prognostic indicators and the like. The marker can be an indicator of a particular disease or disorder (e.g., sepsis) having certain molecular, pathological, histological, and/or clinical features. Exemplary biomarkers include, without limitation, polynucleotides, polypeptides, polypeptide and polynucleotide modifications (such as post-translational modifications and the like), carbohydrates, and/or glycolipid-based molecular markers. The "presence", "amount", or "level" of a marker associated with an increased clinical benefit to an individual is a detectable level of the marker in a sample. The presence, amount, or level of a marker can be measured by methods known to a person skilled in the art. The presence, amount, or level of a marker may be measured prior to treatment, during treatment, after treatment, or a combination of any of the foregoing.

[0129] "Internal reference region" refers to a nucleic acid fragment circulating in a bodily fluid (e.g., blood or a fraction thereof such as serum) that is present in the same amount in both control subjects (those subjects who do not have sepsis, those subjects who are not at risk for developing sepsis, and/or those subjects who do not develop sepsis in three days) and subjects who have sepsis, as determined, for example, by RT-PCR experiments. Internal reference regions provide a nucleic acid fragment which is represented in the bodily fluid at a particular level, against which representation of other nucleic acid sequences (e.g., any one of SEQ ID NOs: 1-57 or 94-148), which differ in control subjects and sepsis patients and are therefore discriminatory, can be evaluated in a relative manner. An internal reference system can be at least one region but can also be a composition of a few regions.

[0130] "Over-representation" refers to a fold increase (relative quantity RQ) relative to the internal reference region calculated as at least 2.sup.-delta-delta Cq of 2.

[0131] "Under-representation" refers to a fold decrease (relative quantity RQ) relative to the internal reference region calculated as at most 2.sup.-delta-delta Cq of 0.5.

[0132] The .DELTA.Cq method normalizes disease-specific motifs within a sample by subtracting the Cq value of the internal reference region (reference motif) from the Cq value of the disease specific motifs.

[0133] The 2-.DELTA..DELTA.Cq method calculates relative quantity (RQ) of normalized disease specific motifs between two sample types (i.e., healthy and diseased). Information from multiple reference motifs can be combined to improve accuracy.

[0134] In a PCR reaction, the Cq (Cycle Quantification) value is the PCR cycle number at which the sample's reaction curve intersects the threshold line. This value tells how many cycles it took for the PCR machine to detect a real signal from the sample. Therefore, lower Cq values indicate higher amounts of target nucleic acid fragments, whereas higher Cq values indicate lower amounts of target nucleic acid fragments.

[0135] Exemplary CNAs identified herein, which are markers of sepsis, include SEQ ID NOs: 1-57 and 94-148. See Tables 1 and 3 (FIGS. 1 and 3). Exemplary primer pairs for amplifying each of SEQ ID NOs: 1-57 and 94-148 are also presented in Tables 1 and 3 (FIGS. 1 and 3). Reference is made to the chromosomal locations to which each of these CNAs can be found in the human genome. Accordingly, longer polynucleotides comprising each of SEQ ID NOs: 1-57 and 94-148, respectively, can readily be determined via access to publicly available databases. Additional primers for amplifying each of SEQ ID NOs: 1-57 and 94-148, respectively, can be designed based on the availability of such additional sequence information.

[0136] Exemplary subsets of SEQ ID NOs: 1-57 and 94-148 comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, or at least twenty-four of the polynucleotides. Exemplary subsets and primers for amplification of each of the markers offer reagents for detecting CNAs comprising the indicated SEQ ID NOs:. See Tables 1-3. In a particular embodiment, detecting CNAs comprising the indicated SEQ ID NOs: is indicative of sepsis.

[0137] An exemplary subset may be provided in a kit for detecting at least one of SEQ ID NOs: 1-148 and subsets thereof (see, e.g., Table 4).

[0138] Table 4 presents an exemplary subset comprising, consisting essentially of, and/or consisting of the indicated SEQ ID NOs: 1-2, 4-6, 30-31, 38, 44, 46, 50, 53, 55, 59, 61, 68, 100, 108-110, 112, 114, 116, and 129. Primers for amplification of each of the markers listed in Table 4 can be found in the corresponding Tables 1, 2, or 3. Also encompassed herein are kits comprising the 24 exemplary markers presented in Table 4 and subsets thereof.

TABLE-US-00001 TABLE 4 List of 24 Exemplary Markers Listed in Final motif ID SEQ ID NO: Patent ID Table 1 Sepsis JC1 1 hu-sep-CNAD-0001 Table 1 Sepsis JC2 2 hu-sep-CNAD-0002 Table 1 Sepsis JC4 4 hu-sep-CNAD-0004 Table 1 Sepsis JC5 5 hu-sep-CNAD-0005 Table 1 Sepsis JC6 6 hu-sep-CNAD-0006 Table 1 Sepsis JC34 30 hu-sep-CNAD-0030 Table 1 Sepsis JC35 31 hu-sep-CNAD-0031 Table 1 Sepsis JC42 38 hu-sep-CNAD-0038 Table 1 Sepsis JC48 44 hu-sep-CNAD-0044 Table 1 Sepsis JC50 46 hu-sep-CNAD-0046 Table 1 Sepsis SC2 50 hu-sep-CNAD-0050 Table 1 Sepsis SC5 53 hu-sep-CNAD-0053 Table 1 Sepsis SC7 55 hu-sep-CNAD-0055 Table 2 Sepsis JU2 59 hu-sep-CNAD-0059 Table 2 Sepsis JU4 61 hu-sep-CNAD-0061 Table 2 Sepsis JU11 68 hu-sep-CNAD-0068 Table 3 Sepsis J7 100 hu-sep-CNAD-0100 Table 3 Sepsis J15 108 hu-sep-CNAD-0108 Table 3 Sepsis J16 109 hu-sep-CNAD-0109 Table 3 Sepsis J17 110 hu-sep-CNAD-0110 Table 3 Sepsis J19 112 hu-sep-CNAD-0112 Table 3 Sepsis J21 114 hu-sep-CNAD-0114 Table 3 Sepsis J23 116 hu-sep-CNAD-0116 Table 3 Sepsis J36 129 hu-sep-CNAD-0129

[0139] Exemplary CNAs identified herein, which are internal reference regions, include SEQ ID NOs: 58-93. See Table 2 (FIG. 2). Exemplary primer pairs for amplifying each of SEQ ID NOs: 58-93 are also presented in Tables 1-3 (FIG. 1-3). Reference is made to the chromosomal locations to which each of these CNAs can be found in the human genome. Accordingly, larger polynucleotides comprising each of SEQ ID NOs: 58-93, respectively, can readily be determined via access to publicly available databases. Additional primers for amplifying each of SEQ ID NOs: 58-93, respectively, can be designed based on the availability of such additional sequence information.

[0140] Candidate internal reference regions (Table 2: SEQ ID NOs: 58-93) were evaluated to identify the ones that were most consistently represented across a set of sepsis and control samples. Given a candidate reference region, the differences in median Cq values of the regions between sepsis samples and control samples were calculated. The lesser the difference in the median Cq values, the higher the consistency of the region across sepsis and non-sepsis conditions, and therefore the more suitable the region as an internal reference region.

[0141] The three most consistent regions were selected as exemplary internal reference regions (SEQ ID NOs: 59, 61 and 68). Their sequences and primers used for amplification are listed in Table 2 in the corresponding rows of the regions.

[0142] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 1, 6, 46, 50, 53, 55, 100, 109, 110, 114, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 1, 6, 46, 50, 53, 55, 59, 61, 68, 100, 109, 110, and 114 are presented in Tables 1-3.

[0143] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 1, 2, 6, 30, 53, 55, 110, 112, 114, 116, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 1, 2, 6, 30, 53, 55, 59, 61, 68, 110, 112, 114, and 116 are presented in Tables 1-3.

[0144] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 30, 31, 38, 44, 46, 50, 53, 55, 100, 129, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 30, 31, 38, 44, 46, 50, 53, 55, 59, 61, 68, 100, and 129 are presented in Tables 1-3.

[0145] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 4, 5, 30, 31, 100, 108, 109, 110, 116, 129, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 4, 5, 30, 31, 59, 61, 68, 100, 108, 109, 110, 116, and 129 are presented in Tables 1-3.

[0146] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 53, 55, 100, 108, 109, 110, 112, 114, 116, 129, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 53, 55, 59, 61, 68, 100, 108, 109, 110, 112, 114, 116, and 129 are presented in Tables 1-3.

[0147] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 30, 31, 46, 55, 108, 110, 112, 114, 116, 129, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 30, 31, 46, 55, 59, 61, 68, 108, 110, 112, 114, 116, and 129 are presented in Tables 1-3.

[0148] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 1, 2, 5, 46, 53, 100, 112, 114, 116, 129, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 1, 2, 5, 46, 53, 59, 61, 68, 100, 112, 114, 116, and 129 are presented in Tables 1-3.

[0149] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 2, 4, 6, 31, 44, 100, 109, 110, 116, 129 and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 2, 4, 6, 31, 44, 59, 61, 68, 100, 109, 110, 116, and 129 are presented in Tables 1-3.

[0150] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 5, 6, 30, 31, 44, 50, 55, 100, 110, 112, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 5, 6, 30, 31, 44, 50, 55, 59, 61, 68, 100, 110, and 112 are presented in Tables 1-3.

[0151] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 2, 31, 38, 44, 46, 55, 109, 110, 112, 129, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 2, 31, 38, 44, 46, 55, 59, 61, 68, 109, 110, 112, and 129 are presented in Tables 1-3.

[0152] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 4, 5, 6, 30, 38, 44, 53, 55, 108, 109, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 4, 5, 6, 30, 38, 44, 53, 55, 59, 61, 68, 108, and 109 are presented in Tables 1-3.

[0153] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 31, 46, 55, 108, 114, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 31, 46, 55, 59, 61, 68, 108, and 114 are presented in Tables 1-3.

[0154] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 100, 109, 112, 114, 116, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 59, 61, 68, 100, 109, 112, 114, and 116 are presented in Tables 1-3.

[0155] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 31, 38, 44, 100, 108, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 31, 38, 44, 59, 61, 68, 100, and 108 are presented in Tables 1-3.

[0156] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 1, 4, 5, 110, 112, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 1, 4, 5, 59, 61, 68, 110, and 112 are presented in Tables 1-3.

[0157] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 5, 6, 44, 55, 109, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 5, 6, 44, 55, 59, 61, 68, and 109 are presented in Tables 1-3.

[0158] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 1, 2, 5, 50, 53, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 1, 2, 5, 50, 53, 59, 61, and 68 are presented in Tables 1-3.

[0159] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 4, 5, 6, 31, 38, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 4, 5, 6, 31, 38, 59, 61, and 68 are presented in Tables 1-3.

[0160] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 1, 53, 100, 114, 116, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 1, 53, 59, 61, 68, 100, 114 and 116 are presented in Tables 1-3.

[0161] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 5, 6, 30, 31, 38, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 5, 6, 30, 31, 38, 59, 61 and 68 are presented in Tables 1-3.

[0162] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 44, 46, 50, 53, 55, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 44, 46, 50, 53, 55, 59, 61, and 68 are presented in Tables 1-3.

[0163] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 109, 110, 112, 114, 116, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 59, 61, 68, 109, 110, 112, 114, and 116 are presented in Tables 1-3.

[0164] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 4, 5, 114, 116, 119 and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 4, 5, 59, 61, 68, 114, 116, and 119 are presented in Tables 1-3.

[0165] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 4, 6, 30, 100, 108 and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 4, 6, 30, 59, 61, 68, 100, and 108 are presented in Tables 1-3.

[0166] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 38, 44, 109, 110, 112 and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 38, 44, 59, 61, 68, 109, 110, and 112 are presented in Tables 1-3.

[0167] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 1, 2, 4, 5, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 1, 2, 4, 5, 59, 61, and 68 are presented in Tables 1-3.

[0168] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 30, 53, 108, 109, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 30, 53, 59, 61, 68, 108, and 109 are presented in Tables 1-3.

[0169] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 2, 109, 110, 116, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 2, 59, 61, 68, 109, 110, and 116 are presented in Tables 1-3.

[0170] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 5, 6, 50, 53, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 5, 6, 50, 53, 59, 61, and 68 are presented in Tables 1-3.

[0171] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 1, 31, 55, 108, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 1, 31, 55, 59, 61, 68, and 108 are presented in Tables 1-3.

[0172] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 30, 38, 55, 100, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 30, 38, 55, 59, 61, 68, and 100 are presented in Tables 1-3.

[0173] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 2, 4, 112, 114, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 2, 4, 59, 61, 68, 112, and 114 are presented in Tables 1-3.

[0174] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 31, 110, 112, 114, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 31, 59, 61, 68, 110, 112, and 114 are presented in Tables 1-3.

[0175] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 5, 31, 116, 129, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 5, 31, 59, 61, 68, 116, and 129 are presented in Tables 1-3.

[0176] An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ ID NOs: 2, 4, 5, 6, and at least one of SEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 2, 4, 5, 6, 59, 61, and 68 are presented in Tables 1-3.

[0177] Machine learning classification systems, such as, e.g., a support vector or neural network are utilized herein. The system utilized herein creates a multidimensional `map` comprising basically all the markers (both sepsis and non-sepsis) and then when a sample is tested, it's position on the map it marked. After this, the system looks at the closest points and if they are sepsis, it will call the sample `sepsis`. If the closest points are not sepsis, it will call the sample `not sepsis`. In a particular embodiment, the system looks for a certain small number of neighboring samples (not the single closest one) in this virtual multidimensional space, where each sample is represented by a group of marker values on the sample. The assignment of the sample to sepsis/non-sepsis is accomplished by a majority vote of the known labels of the closest neighbors (e.g., 5 of them). If there are more sepsis samples in the neighborhood, the incoming sample is assigned to "sepsis". If there are more non-sepsis samples in the neighborhood, the new sample is assigned to "non-sepsis".

[0178] "Encode" refers to a polynucleotide "encoding" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or the polypeptide (or a fragment thereof). The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0179] "Array" or "microarray" refers to an ordered arrangement of hybridizable array elements on a substrate, such as a solid substrate (e.g., glass slide and the like) or a semi-solid substrate (e.g., nitrocellulose membrane and the like). In some embodiments, the array elements may be polynucleotide probes (e.g. oligonucleotide). Arrays may include DNA microarrays (including cDNA microarrays, oligonucleotide microarrays, SNP microarrays, etc.), protein microarrays, peptide microarrays, antibody microarrays, and the like.

[0180] "Amplification" or "amplifying" refers to the production of one or more copies of a reference nucleic acid sequence or its complement. Amplification may be linear or exponential (e.g., in a polymerase chain reaction (PCR)). A nucleic acid copy produced from amplification may not have perfect sequence complementarity or identity relative to the reference sequence. In some embodiments, the copies can include nucleotide analogs, including deoxyinosine, intentional sequence alterations (such as alterations introduced through a primer that is hybridizable, but not fully complementary, to the template), and/or sequence errors that occur during the amplification process.

[0181] The terms "expression" and "expression level", in general, are used interchangeably and generally refer to the amount of a marker in a sample. "Expression" generally refers to the process by which information (e.g., gene-encoded and/or epigenetic) is converted into the structures present and operating in the cell. Therefore, as used herein, "expression" can refer to transcription into a polynucleotide (such as mRNA and the like), translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., post-translational modification of a polypeptide and the like). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications (such as post-translational modification of a polypeptide and the like) will also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide (e.g., by proteolysis). "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (such as transfer and ribosomal RNAs and the like).

[0182] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule. The nucleic acid molecule may be present extrachromosomally or at a chromosomal location that is different from its natural location.

[0183] The term "sequencing" and its variants include obtaining sequence information from a strand of a nucleic acid molecule, typically by determining the identity of at least some nucleotides (including their nucleic acid components) within the nucleic acid molecule. The term sequencing may also refer to determining the order of nucleotides (base sequences) in a nucleic acid sample (e.g. DNA or RNA). Many techniques are available and known to a person skilled in the art, such as Sanger sequencing, high-throughput sequencing technologies (such as the GS FLX platform formerly offered by Roche Applied Science, Penzberg, Germany, based on pyro sequencing, or Illumina sequencing platforms, as offered by Illumina Inc., 5200 Illumina Way, San Diego, Calif. 92122, USA), and the like. High-throughput sequencing technologies refer to sequencing technologies having increased throughput as compared to traditional Sanger- and capillary-electrophoresis-based approaches (e.g., with the ability to generate hundreds of thousands or millions of relatively small sequence reads at a time). High throughput sequencing technologies include, but are not limited to, sequencing by synthesis, sequencing by ligation, pyrosequencing, sequencing by hybridization, and/or the like.

[0184] As used herein, "reactive" means the agent has affinity for, binds to, or is directed against a specific CNA. As further used herein, an "agent" includes a protein, polypeptide, peptide, nucleic acid (including DNA or RNA), antibody, Fab fragment, F(ab')2 fragment, molecule, compound, antibiotic, drug, and any combinations thereof. A Fab fragment is a univalent antigen-binding fragment of an antibody, which is produced by papain digestion. A F(ab')2 fragment is a divalent antigen-binding fragment of an antibody, which is produced by pepsin digestion. By way of example, the agent of the present invention can be labeled with a detectable marker. Agents that are reactive with CNAs can be identified by contacting the CNA with an agent of interest and assessing the ability of the agent to bind to the CNA.

[0185] In one embodiment of the present invention, the agent reactive with a sepsis biomarker is an antibody. Antibodies for use herein can be labeled with a detectable marker. Labeling of an antibody can be accomplished using one of a variety of labeling techniques, including peroxidase, chemiluminescent labels known in the art, and radioactive labels known in the art. The detectable marker of the present invention can be, for example, a nonradioactive or fluorescent marker, such as biotin, fluorescein (FITC), acridine, cholesterol, or carboxy-X-rhodamine (ROX), which can be detected using fluorescence and other imaging techniques readily known in the art. Alternatively, the detectable marker can be a radioactive marker, including, for example, a radioisotope. The radioisotope can be any isotope that emits detectable radiation, such as .sup.35S, .sup.32P, or .sup.3H. Radioactivity emitted by the radioisotope can be detected by techniques well known in the art. For example, gamma emission from the radioisotope can be detected using gamma imaging techniques, particularly scintigraphic imaging. By way of example, the agent of the present invention is a high-affinity antibody labeled with a detectable marker.

[0186] Where the agent of the present invention is an antibody reactive with a sepsis biomarker, a biological sample taken from a mammal (e.g., a human) can be purified by passage through an affinity column which contains the antibody having affinity to the sepsis biomarker as a ligand attached to a solid support, such as an insoluble organic polymer in the form of a bead, gel, or plate. The antibody attached to the solid support can be used in the form of a column. Examples of suitable solid supports include, without limitation, agarose, cellulose, dextran, polyacrylamide, polystyrene, sepharose, and other insoluble organic polymers. The antibody can be further attached to the solid support through a spacer molecule, if desired. Appropriate binding conditions (e.g., temperature, pH, and salt concentration) can be readily determined by the skilled artisan. By way of example, the antibody can be attached to a sepharose column, such as Sepharose 4B.

[0187] Alternatively, a biological sample from a mammal (e.g., a human) can be assayed using hybridization analysis of nucleic acid extracted from the biological sample taken from the mammal (e.g., a human) to determine the presence of a sepsis biomarker, such as a CNA. This method also can be conducted by performing a Southern blot analysis of DNA using at least one nucleic acid probe which hybridizes to CNAs (including amplified CNAs). The nucleic acid probes described herein can be prepared by a variety of techniques known to those skilled in the art, including, without limitation, the following: restriction enzyme digestion of nucleic acid; and automated synthesis of oligonucleotides having sequences which correspond to selected portions of the nucleotide sequence of the sepsis biomarker, using commercially-available oligonucleotide synthesizers.

[0188] The nucleic acid probes used herein can be DNA or RNA, and can vary in length from about 5-20 nucleotides or 10-20 nucleotides to the entire length of the nucleic acid encoding for a sepsis biomarker. In some embodiments, the nucleic acid probes are oligonucleotides. The nucleic acid used in the probes can be derived from mammalian polynucleotide sequence complementary to the sepsis biomarker. In addition, the nucleic acid probes of the present invention can be labeled with one or more detectable markers. Labeling of the nucleic acid probes can be accomplished using one of a number of methods known in the art (e.g., nick translation, end labeling, fill-in end labeling, polynucleotide kinase exchange reaction, random priming, SP6 polymerase (for riboprobe preparation)) along with one of a variety of labels (e.g., radioactive labels, such as .sup.35S, .sup.32P, or .sup.3H, or nonradioactive labels, such as biotin, fluorescein (FITC), acridine, cholesterol, or carboxy-X-rhodamine (ROX)). In some embodiments, these nucleic acid probes are used in an array or microarray.

[0189] In addition, the present invention provides a method of determining whether a human has sepsis, but is asymptomatic, or has already developed sepsis and has symptoms thereof. The method includes analyzing a biological sample of the human for the presence of at least one sepsis biomarker, and optionally, further recommending a corroborative test for sepsis if the at least one sepsis biomarker is present in the biological sample. In some embodiments, the corroborative test includes ELISA, immunohistochemistry, and Western Blot/immunoblot or a combination of more than one of any of the foregoing.

[0190] In the methods described herein, the step of analyzing a biological sample may optionally include obtaining the sample from the human; isolating nucleic acid from the sample; amplifying the isolated nucleic acid using primers that are specific for or capable of amplifying a sequence corresponding to a sepsis CNA biomarker; and sequencing the amplified nucleic acid. In some embodiments, the isolated nucleic acid includes genomic DNA, mRNA, and/or cDNA obtained from mRNA. In some embodiments, the step of determining representation of the at least one sepsis CNA marker includes use of at least one of a PCR-based detection method and a hybridization-based method. In some embodiments, the step of determining representation of the at least sepsis CNA marker includes an immunohistochemical analysis. In some embodiments, an array or a microarray is used for identifying the sepsis biomarker.

[0191] The biological sample can be assayed for expression of sepsis biomarkers in vitro or in vivo. In addition, the biological sample can be assayed for expression of sepsis biomarkers using all of the various assays and methods of detection and quantification described above.

[0192] The discovery that certain CNAs constitute sepsis biomarkers provides compositions and methods for identifying a human having early stage sepsis (e.g., pre-symptomatic sepsis), and presents the potential for commercial application in the form of a test for the diagnosis of sepsis and kits including same. The development of such a test or kit would provide general screening procedures; these procedures could assist in the early detection and diagnosis of sepsis in human subjects. Accordingly, the present invention further provides a kit for use as an assay of sepsis, comprising at least one agent reactive with a sepsis biomarker. The agent can be any of those described above, and can be used in any of the above-described assays or methods for detecting sepsis biomarkers.

[0193] Oligonucleotides complementary to a sepsis CNA biomarker can be designed based on the nucleotide sequence of the particular CNA sepsis biomarker. A nucleotide sequence complementary to the selected partial sequence of the sepsis CNA biomarker can, e.g., be chemically synthesized using one of a variety of techniques known to those skilled in the art, including, without limitation, automated synthesis of oligonucleotides having sequences which correspond to a partial sequence of the sepsis CNA biomarker nucleotide sequence, or a variation sequence thereof, using commercially-available oligonucleotide synthesizers.

[0194] The present invention also provides the use of an oligonucleotide capable of identifying at least one sepsis CNA biomarker to determine the representation of same in a human. The oligonucleotide can be labelled with a detectable marker, such as a radioactive marker, fluorescent marker, the like, or a combination of any of the foregoing.

[0195] Serum from healthy individuals (controls) and diseased patients (i.e. sepsis patients) was harvested and initially stored at -80 degrees Celsius. Total DNA was extracted from the serum samples using the High Pure Viral Nucleic Acid Kit (Roche Applied Science; Cat. No. 11858874001). The DNA was amplified using the GenomePlex Single Cell Whole Genome Amplification Kit (Sigma; Cat. No. WGA4-500RXN) and purified using the GenElute.TM. PCR Clean-Up Kit (Sigma, Cat. No. NA1020-1KT). High-throughput paired-end DNA sequencing was performed by SEQ-IT Kaiserslautern Sequencing facility on NextSeq 500 sequencer and SEQ-IT machines. The resulting sequence reads were mapped to the human genome. Using an in-house Bioinformatics pipeline, which was established on the high-performance Bioinformatics infrastructure at the Institute of Computational Biotechnology at the Technical University of Graz, Austria, the present inventors have identified DNA motifs, which were present at different read count numbers in diseased humans, when compared to controls. The motifs identified are the result of the host response (i.e., the response of the human body to sepsis). The DNA motifs identified were used as targets for the development of a real-time Polymerase-Chain Reaction (RT-PCR) assay. In addition to the motifs, which are present at different levels in healthy controls and sepsis patients (markers; SEQ ID NOs: 1-57 or 94-148), motifs which were present at the same level in healthy and diseased humans were also identified to allow the normalization of the results (SEQ ID NOs: 58-93). The RT-PCR-evaluated motifs, which could be used to discriminate in a statistically significant manner between healthy controls and sepsis patients, stand-alone, or in combination with each other, are the subject basis for assays described herein.

[0196] In a particular embodiment, the Illumina sequences were analyzed using a genome assembly/mapping bioinformatics processing method. The cleaned sequence reads were mapped to the human genome using a fast mapper, genomic hotspots for sepsis were identified, the reads were comprehensively mapped to the identified hotspots, and a gene search was conducted in the hotspot region to correlate the reads with a gene. Reads that did not map to the genome were assembled to generate clusters and then analyzed using the same procedures used for the hotspots.

[0197] In an embodiment of an Illumina sequence analysis method, CNA libraries are obtained from a blood sample collection and these libraries are sequenced in an Illumina sequencing operation. After the sequencing operation, the method proceeds to an operation step, where the processing unit stores the output of the Illumina sequencing in a text-based file format, for example, a FASTQ format. It is understood that the output of the Illumina sequencing can also be stored in other file formats, such as, for example, SAM or BAM formats. The sequencing files are assembled with a reference genome to determine genome locations with significant counts of exclusively at risk or normal (not at risk) reads.

[0198] The results of the alignment are then processed. In some embodiments, the results of the alignment process are outputted in a text-based file format, such as a SAM format. A SAM formatted file is a tab-delimited text file that contains sequence alignment data. In such embodiments, the processing unit converts the SAM format to a BAM format, which is a binary version of the SAM formatted file. In other examples, the alignment process outputs a BAM formatted file, and thus, the conversion step is skipped. Preparation for and creation of an index file associated with the BAM formatted file is then created. The index file creation process may include categorizing by adding sample names as read groups, sorting, and/or merging.

[0199] The created index file and the BAM formatted file is further processed by a processing unit. In particular, processing includes reviewing the alignments and extracting the alignments, which have sufficient coverage for each contiguous sequence (=contig) of the reference genome. In an embodiment, the alignment parameters comprise a bucket size of 25 and a minimum coverage of 5, although it is understood that the bucket size and minimum coverage value may differ in alternate embodiments. Control regions and at risk regions having 50% or more proportion are extracted and compared to determine overlaps. In some embodiments, these regions are stored in a database associated with the processing unit for later retrieval and/or review. Next, the extracted control regions are subtracted from the at risk regions so that only the at risk regions remain, thereby providing sequence reads that are present in only at risk regions. In an embodiment, the control filtering parameters comprise a minimum subjects value of 3 and a minimum proportion value of 0.5, although it is understood that these values may differ in alternate embodiments.

[0200] In some embodiments, not all of the sequences are aligned. In such examples, the unaligned sequences are collected and may be stored in the database for later retrieval and/or review. In an embodiment, the unaligned sequences are aligned against other references, such as viral references, to determine if any alignments exist. If alignments exist, the sequences may be stored in the database for later retrieval and alignment with new viruses, as desired.

[0201] In an embodiment, analysis of Illumina sequencing reads can be achieved using a TimeLogic.RTM. Decypher.RTM. biocomputing platform (Active Motif, Carlsbad, Calif.) and multiple CPU servers at the sequencing center.

[0202] Sequence variability of the identified CNA motifs specific to sepsis risk was then analyzed in a sampling of different humans through PCR and Sanger sequencing of the PCR products to determine if the identified CNA motifs are capable of detecting sepsis risk generally. In healthy humans, it has been shown that approximately 97% of CNA sequences are of genomic origin [Beck et al., 2009, Clin Chem., 55(4):730-8].

[0203] Arrays comprising one of more polynucleotides of the disclosure, PCR primers and/or probes for amplifying and/or detecting polynucleotides (CNAs) described herein, and methods for detecting risk for developing sepsis comprising an array or PCR primers and/or probes are encompassed herein.

[0204] One of more polynucleotide sequences of the disclosure can be incorporated onto a sequence array, such as a biochip, DNA chip, BiofireDX filmarray and other filmarrays, microarray, macroarray, and the like, for screening, e.g., serum separated from whole blood from humans for sepsis risk. Alternatively, CNAs can be extracted from the sample for screening on the array. Arrays are generally solid supports upon which a collection of polynucleotides and/or primers and/or probes are placed at defined locations on the array, either by spotting, printing, or direct synthesis. The array can include probes corresponding to one or more of the polynucleotides described herein (e.g., at least one of SEQ ID NOs: 1-57 or 94-148 and/or primers and/or probes for amplifying and/or detecting one or more polynucleotides of SEQ ID NOs: 1-57 or 94-148 and at least one of SEQ ID NOs: 59, 61, or 68 and/or primers and/or probes for amplifying and/or detecting one or more polynucleotides of SEQ ID NOs: 59, 61, or 68).

[0205] The underlying principle of arrays is base pairing or hybridization i.e., A-T and G-C for DNA, and A-U and G-C for RNA. A sample from a mammal (e.g., a human) is allowed to hybridize with the polynucleotides and/or primers and/or probes on the array providing an expression profile/pattern of CNA. The CNA expression pattern of sepsis-specific sequences can be used to determine if a human has early stage sepsis. The array can be prepared by any method known in the art. In some embodiments, a microarray is prepared generally as disclosed in U.S. Pat. No. 7,655,397, the entirety of which is hereby incorporated by reference.

[0206] In some embodiments, the array comprises at least 2 polynucleotides selected from polynucleotides comprising or consisting of SEQ ID NOs: 1-57 or 94-148 or primers or probes specific for at least 2 of SEQ ID NOs: 1-57 or 94-148. In some embodiments, the array comprises at least 4 polynucleotides selected from polynucleotides comprising or consisting of SEQ ID NOs: 1-57 or 94-148 or primers or probes specific for at least 4 of SEQ ID NOs: 1-57 or 94-148. In another embodiment, the array comprises at least 5 polynucleotides selected from the polynucleotides comprising or consisting of SEQ ID NOs: 1-57 or 94-148 or primers or probes specific for at least 5 of SEQ ID NOs: 1-57 or 94-148. In another embodiment, the array comprises at least 10 polynucleotides selected from the polynucleotides comprising or consisting of SEQ ID NOs: 1-57 or 94-148 or primers or probes specific for at least 10 of SEQ ID NOs: 1-57 or 94-148. In some embodiments, the array comprises the 24 polynucleotides listed in Table 4 and/or primers and/or probes specific for the 24 polynucleotides listed in Table 4. The array generally includes many copies of the selected polynucleotides to facilitate detection. In some embodiments, the array comprises a million or more copies of each of the selected polynucleotides.

[0207] In a particular embodiment, the array comprises or consists of SEQ ID NOs: listed in Table 4 or primers or probes specific for the SEQ ID NOs: listed in Table 4. Also encompassed herein is an array comprising or consisting of subsets of SEQ ID NOs: listed in Table 4 or primers or probes specific for these SEQ ID NOs: as set forth herein.

[0208] Probes for detecting polynucleotides described herein can be designed and prepared using conventional methods. Software for modeling and designing probes, including determining hybridization and annealing conditions, for detecting a specific polynucleotide sequence are publically available, and include for example LightCycler.RTM. Probe Design Software (Roche Applied Science), Primer3 (Simgene), and FastPCR (PrimerDigital). See also techniques described by Illumina (Illumina Inc., 5200 Illumina Way, San Diego, Calif. 92122, USA).

[0209] The array can include positive indicator for sepsis sequences and/or probes for detecting same and negative and/or positive control sequences and/or probes.

[0210] Polynucleotides described herein can be amplified and/or detected via PCR, including but not limited to real-time PCR, multiplex PCR, nested PCR, solid phase PCR, miniprimer PCR, and the like. Primers and probes for amplifying and/or detecting polynucleotides described herein can be designed and prepared using conventional methods. Software for modeling and designing primers and probes, including determining hybridization, melting, annealing, and/or extensions conditions, for amplifying and/or detecting a specific polynucleotide sequence are publicly available, and include for example LightCycler.RTM. Probe Design Software (Roche Applied Science), Primer3 (Simgene), and FastPCR (PrimerDigital). See also techniques described by Illumina (Illumina Inc., 5200 Illumina Way, San Diego, Calif. 92122, USA). PCR conditions generally include the presence of four different nucleotide bases (adenosine, cytidine, guanosine, thymidine/uridine) and at least one polymerization-inducing agent such as a reverse transcriptase or a DNA polymerase. The primers are generally present in a suitable buffer, which may include constituents, which are co-factors or affect conditions such as pH and the like at various suitable temperatures. The primers are preferably single-strand nucleotide sequences, such that amplification efficiency of the desired polynucleotide is optimized. Double-stranded nucleotide sequences can also be utilized. The primers are typically at least about 15 nucleotides. In some embodiments, the primers can have a length of from about 15 to about 30, about 15 to about 50, about 15 to about 75, about 15 to about 100, or about 15 to about 500 nucleotides.

[0211] In some embodiments, primer sets are designed to amplify one or more of the sepsis-specific polynucleotides comprising or consisting of SEQ ID NOs: 1-57 or 94-148 and then the PCR products of the primer sets are screened for sepsis-specific sequences on an array as described herein.

[0212] Diagnostic kits comprising one or more primer pairs, and optional probes, for amplifying and detecting one or more polynucleotides described herein are also provided. The kit can optionally include nucleotide bases (adenosine, cytidine, guanosine, thymidine/uridine) and at least one polymerization-inducing agent such as a reverse transcriptase or a DNA polymerase. The kit can optionally include a suitable primer buffer, which may include constituents which are co-factors or affect conditions such as pH and the like at various suitable temperatures. The kit can optionally include an array as described herein.

[0213] The primers provided in the diagnostic kit are generally provided in pairs (forward primer and reverse primer) for amplifying/detecting a specific polynucleotide sequence. These primers can be used to amplify and detect CNAs in blood serum from humans, or any other appropriate biological sample from humans that may contain CNAs. Alternatively, CNAs can be extracted from the sample and then amplified by PCR using a diagnostic kit of the disclosure. The CNA expression pattern of sepsis-specific sequences detected by the diagnostic kit can be used to determine if a human has early stage sepsis even at a stage wherein no clinical symptoms of sepsis are apparent

[0214] In addition to polynucleotide sequence specific primer pairs, labeled probes specific for each of the CNAs may be included in kits described herein. Labeled probes and use thereof in the context of, for example, multiplex qPCR provides for enhanced specificity of detection.

[0215] In some embodiments, the kit comprises primers for amplifying at least 2 polynucleotides selected from the polynucleotides comprising or consisting of SEQ ID NOs: 1-57 or 94-148, and optionally one or more probes for detecting the amplified product. In some embodiments, the kit comprises primers for amplifying at least 4 polynucleotides selected from the polynucleotides comprising or consisting of SEQ ID NOs: 1-57 or 94-148, and optionally one or more probes for detecting the amplified product. In another embodiment, the kit comprises primers for amplifying at least 5 polynucleotides selected from the polynucleotides comprising or consisting of SEQ ID NOs: 1-57 or 94-148, and optionally one or more probes for detecting the amplified product. In another embodiment, the kit comprises primers for amplifying at least 10 polynucleotides selected from the polynucleotides comprising or consisting of SEQ ID NOs: 1-57 or 94-148, and optionally one or more probes for detecting the amplified product. In some embodiments, the kit comprises primers for amplifying the 24 polynucleotides listed in Table 4, and optionally one or more probes for detecting the amplified product.

[0216] In a particular embodiment, the kit comprises or consists of SEQ ID NOs: listed in Table 4 and/or primers and/or probes specific for the SEQ ID NOs: listed in Table 4. Also encompassed herein are kits comprising or consisting of subsets of SEQ ID NOs: listed in Table 4 and/or primers and/or probes specific for these SEQ ID NOs: as set forth herein.

[0217] The present invention is described in the following Examples, which are set forth to aid in an understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.

EXAMPLES

Example 1--Identification of Sepsis-Associated Polynucleotides in CNAs

[0218] Serum from healthy individuals (controls) and diseased patients (e.g., sepsis patients who were symptomatic or sepsis patients with early stage disease, but were asymptomatic) was harvested and total DNA was extracted from the serum samples. DNA was amplified and purified from the serum samples as described herein above. High-throughput paired-end DNA sequencing was performed and the resulting sequence reads were mapped to the human genome. Bioinformatics was used as described herein to identify DNA motifs that were present at distinctively high or low read count numbers in diseased humans, when compared to controls, corresponding to motifs that are over- and under-represented in sepsis patients. See, for example, Tables 1 and 3, which present motifs (Sequences) that are under-represented in sepsis patients (Table 1) or over-represented in sepsis patients (Table 3). The motifs identified reflect the host response to sepsis (i.e., the response of the human body to sepsis). The DNA motifs thus identified were used as targets for the development of a real-time Polymerase-Chain Reaction (RT-PCR) assay. In addition to the motifs, which are present at distinctively different levels in healthy controls and sepsis patients, motifs that were present at a highly similar level in healthy and diseased humans were also identified to allow for the normalization of the results. See, for example, Sequences presented in Table 2. The RT-PCR-evaluated motifs, which are used to discriminate between healthy controls and sepsis patients in a statistically significant manner, when evaluated alone or in combination with other motifs, are the subject basis for assays described herein.

[0219] All publications mentioned herein are hereby incorporated by reference in their entireties. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of the disclosure that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.

[0220] Specific examples of methods and kits have been described herein for purposes of illustration. These are only examples. The technology provided herein can be applied to systems other than the example systems described above. Many alterations, modifications, additions, omissions, and permutations are possible within the practice of this invention. This invention includes variations on described embodiments that would be apparent to the skilled addressee, including variations obtained by: replacing features, elements and/or acts with equivalent features, elements and/or acts; mixing and matching of features, elements and/or acts from different embodiments; combining features, elements and/or acts from embodiments as described herein with features, elements and/or acts of other technology; and/or omitting combining features, elements and/or acts from described embodiments.

[0221] The embodiments of the invention described above are intended to be exemplary only. Those skilled in this art will understand that various modifications of detail may be made to these embodiments, all of which come within the scope of the invention.

Sequence CWU 1

1

4781166DNAHomo sapiens 1aaacgtccgc ttgcagatac tacaaaaaga gcgtttcaaa cctgctctat gaaaggcaat 60gttcaactct gtgacttgaa tgcagacatc acagagcagt ttctgagaat gcttctgtct 120agattttata ggaagatatt cccgtttcca acgaaatctt cacagc 1662146DNAHomo sapiens 2ggaaatatgg caaagtattt tctgagtatg ctgctgtgta cgttttatat tgcatcccgt 60ttccaacgaa atcctcaaag cgatccaaat atccacttgc agattccaaa aaaagagtgt 120ttcaaactgc tctgtcagta caaagg 1463261DNAHomo sapiens 3tctttgaggc tttcgttgga aacgggattt cttcacataa tgctagacag aagaattctc 60agtaacttct tttgggatgt atgtattcaa ctcagagagt tgaaccttcc tttagacaga 120gcggattgga aacacgcttt ttgcggaatt ttcaggtgga gattccaaga gccttgaggc 180cagtggtaga aaaggctatc ttcgtataaa aactagaggg aatcattctc agaaactgct 240ttgtgatgtg tgcattaaac t 2614199DNAHomo sapiens 4tgtgaactca gctaacagag gtggatcttt cttttgatag agcagttctg aaaaacactt 60tttgttgaat ctgcaagtgg acatttggat agatttgaag atttcgttgg aaacgggaat 120atcttcatat caaatctaga cagcagcatt cccagaaatt tctttcggat atttccattc 180aactcataga gatgaacat 1995214DNAHomo sapiens 5cttgtggcct tcgttggaaa cgggatttct tcatattatg ctagacagaa gaattctcag 60taacttcctt gtgttgtgtg tattcaactc acagagttga acgatccttt acacagagca 120gacttgaaac actctttttg tggaatttgc aagtggagat ttcagccgct ttgagttcaa 180tggtagtata ggaaatatct tcctatagaa acta 2146171DNAHomo sapiens 6atatttggat agctgtgaag atttcgttgg aaacgggaat atcttcctat aaaatctaga 60cagaagcatt ctcagaaact gctctgtgat gtttgcattc aagtcacaga gttgaacatt 120gcctttccta gagaggtttg aaacgctctt ttggtagtat atggaagtgg a 1717160DNAHomo sapiens 7ctcagctaac agaggtggat ctttcttttg atagagcagt tctgaaaaac actttttgtt 60gaatctgcaa gtggacattt ggatagattt gaagatttcg ttggaaacgg gaatatcttc 120atatcaaatc tagacagaag cattctcaga atcttctttg 1608140DNAHomo sapiens 8agtggagaac acacatcaca atcaaggttc tgagaatgct tctgtctaaa ttttctatga 60agacattccc gtttccaacg aaatcctcac agctatccaa atatccactt gcagattcta 120caaaaagtgt ggttcaaaac 1409261DNAHomo sapiens 9aatatcttcc cctacaagct agaaagaagc attctgtgaa acttgtttgt gatgtgtgta 60ctcaactaac agagaagaac cttccttttg acagagcagt tttgatacac tctttttgta 120gaatctgcaa gtggatattg ggatagctgt gaagatttcg ttggaaacgg gaatatcttc 180ctataaaatc tagacagaag cattctcaga aactactctg tgatgtctgc attcaagtca 240cagagttgaa cattgccttt c 26110137DNAHomo sapiens 10cctttatgac gtatgcactc acctaacaga gaagaacctt ccttttgaca gagcagtttt 60gatacactct ttttgtagaa tctgcaagtg gatatttgga tagctgtgaa gatttcgttg 120gaaacgggaa tatcttc 13711254DNAHomo sapiens 11tttttgttgt atctggatgt ggacatttgg agcgctttca gccctatggt gaaaaaggaa 60atatcttctc ctgaaaacta gacagaagca ttctcagaat cttatttgtg atgtgcgccc 120tcaactaaca gtgttgaagc tttcttctga tagagcagtt ttgaaacact cttttcgtaa 180aatctgcaag aggatatttt gatagctttg aggatttcgt tggaaacggg attgtcttca 240tataaactct agac 25412288DNAHomo sapiens 12ctttaaggtc aatggcagaa aaggaaatat cttcgtttca aaactagaca gaatcattcc 60cacaaactgc gttgtgatgt gttcgttcaa ctcacagagt ttagcctttc ttttcataga 120gcagttagga aacactctgt ttgtaaactc tgcaagtgga tattcagacc tctttgaggc 180cttcgttgga aacgggattt cttcatacta tgctagacag aagaattctc agtaacttcc 240acgtgttgtg tgtattcaac tcacagagct gaacgatcct ttacacag 28813208DNAHomo sapiens 13aagaattctc agtagcttct ttgtgtgtgt attcaactca cagagttgaa ccttccttta 60gacagagcag acttgaaaca ctctttttgt ggaatttgca agtggatatt tcagccgctt 120tgaggtcaat ggtagaatag gaaatatctt ccaatagaaa ctagacagaa tgattctcag 180aaactccttt gtgatgtgtg cgttcaac 20814126DNAHomo sapiens 14gaagaattcc cgtttccaac gaaggccaca agatgtcaga atatccactt acagacttta 60caaacagagt gtttcctaac tgctctatga acagaaaggt taaactctgt gagttgaacg 120aacaca 12615254DNAHomo sapiens 15gagttcaacc tttcttatga tacagcagtt tggaaacact ctttttatag aatttgcaag 60ctgatacatg gatagcccta actatttcgt tggaaacggg aatatcttca cataaaacct 120agacagaagc actctcagaa actactttgt gatatctgca ttgatatcag agagttgaat 180attccctttc taagggcagg cttgaaagcg tcttttcgtg gaatctgcag gaggatattt 240ggatagcttg gagg 25416171DNAHomo sapiens 16attcttctgt ctagcatagt atgaagaaat cccgtttcca acgaaggcct caaagaggtc 60tgaatatcca cttgcagagt ttacaaacag agtgtttcct aactgctcta tgaagagaaa 120ggttaaactc tgtgagttga acgcacacat cacaaagaag tttctgagaa t 17117219DNAHomo sapiens 17gtaaagtctg caagtggatg tttggacctc tttgaggcct tcgttggaaa cgggatttct 60tcatataatt ctagacagca gaattttcag taacttcctt gtgttgtgtg tattcaactc 120acagagttga acgatccttt acacagagca gacttgaaac actctttttg tggaatttgc 180aagtggagat ttcagccgct ttgaggtcaa tagtagaaa 21918211DNAHomo sapiens 18ctccaccata gccctcaaag cgctccaagt gtccgctagc agattccaca gaaacagtgt 60ttcaaaactg ctctaacaaa agaaagattc aactccgtga tttgaatgca cacatcacaa 120agcattttct gtgaatcctt ctgtctagtt tttatatgag gatatttcct tttctaccat 180gggcatcaaa gggttccaat tatccaattg t 21119234DNAHomo sapiens 19atgacgcttc tgagaatgct tctgtctaga gtttatatga agacaatccc gtttccaacg 60aaatcctcaa agctatccaa atatcctctt gcagatttta caaaaagagt gtttcaaaac 120tgctctatca aaagaaagct tcaacactgt tagttgaggg cgcacatcac aaataggatt 180ctaagaattc ttctgtctag tttttatttg aagatatttc ctttctcacc atag 23420221DNAHomo sapiens 20ggaatgttca actctgtgac ttgaatgcag acatcacaga gcagtttctg agaatgcttc 60tgtctagatt ttataggaag atattcccgt ttccaacgaa atcttcacag ctatccaaat 120atccacttgc agattcaaca aaaagtgttt ttcagaactg ctctatcaaa agaaagatcc 180acctctgtta gctgagttca cacatcacaa acaagtttat g 22121195DNAHomo sapiens 21catccctgtc ttgtgccagt tttcaaaggg aatgcttcca gtttttgccc attcagtatg 60atattggctg tgggtttgtc atagatagct cttattattt tgagatatgt cccaccaaca 120ccaaacacac ccaacacaag acaagtatgt cctctctcac cactcctatt caacatagtg 180ttggaagttc tggcc 19522174DNAHomo sapiens 22aaaacagact ttaaaccaac aaagatcaaa agagacaaag aaggccatta cataatggta 60aagggatcaa ttcaacaaga agagctaact atcctaaata tatatgcacc caaaaaccaa 120acacacccaa cacatacagt aaaattggaa cacacaatca gacgtaaaac aatc 17423292DNAHomo sapiens 23aaatcctcca attaaaagac acagactggc aaattggata aagagtcaag acccatcaga 60gtgctgtatt caggaaaccc atctcacatg cagagacaca cataggctca aaatgaaggg 120atggaggaag atctaccaag caaatggaaa acaaaaaaag gcagaggttg caatcctagt 180ctctgataaa acagacttta aaccaacaaa gatcaaaaga gacaaagaag gccattacat 240aatggtaaag ggatcaattc aacaagaaga gctaactatc ctaaatatat at 29224228DNAHomo sapiens 24gcagtagagg atataactgc ccataaaaac tagacagtag cattcccagg aaacactttg 60tgacgattga gttcaactca cagagctgaa cattcctttg gatggagcag tttcaaaaca 120cactttctgt agaatctgca agtggatatt tggacctctc tgaggatttc gttggatacg 180ggagaaaact cacctatcta aagagaagca ttctcagaac cttcttcg 22825264DNAHomo sapiens 25gctctgtgta aaggatcgtt caactctgtg agttgaatac acacaacaca aggaagttac 60tgagaattct tctgtctagc cttatatgaa aaaaacccgt ttccaacgaa ggcctcaaag 120aggtctgaat atccacttgc agactttaca aacagagtgt ttcctaactt gtgttgggtg 180tgtttggggt ttctgagaat gcttctgtct agattttaac tgaagacaat cccgtttccc 240acgaaatcct caaagctatg caaa 26426224DNAHomo sapiens 26ggggtttcca cttttgcatc ttgcttattt tctcttgcca ctgccaatta agaagtgcct 60tttgggcagg ggttgcaatc ctagtctctg ataaaacaga ctttaaacca acaaagatca 120aaagagacaa agaaggtcat tacataatgg taaagggatc aattcaacaa gaagagctaa 180ctatcctaaa tatatatgca cccaatacag gagcacccag attc 22427199DNAHomo sapiens 27gaataatgcc gcaataaaca tacgtgtgca tgtgtcttta tagcagcatg atttatagtc 60ctttgggtat atacccagta atgggatggc tgggtatata cctaaaggat tataaatcat 120gctgctataa agacacatgc acacgtatgt ttattgcggc actattcaca atagcaaaga 180cttggaacca acccaaatg 19928151DNAHomo sapiens 28gtttataaag tctgcaagtg gatattcaga cccctttgag gccttcgttg gaaacgggat 60ttcttcatat tatgctagac agaagaattc tcagtaactt ccttgtgttg tgtgtattca 120actgacagag ttgaacgatc ctttacctgt c 15129251DNAHomo sapiens 29gtgttaaagt ctcccattat taacgtgtgg gagtctaagt ctctttgtag gtcactcagg 60acttgcttta tgaatctggg tgctcctgta ttgggtgcat atatatttag gatagttagc 120tcttcttgtt gaattgatcc ctttaccatt atgtaagacc ctctttctca agatctcatc 180aaataaacac gaatggtcaa ccacaagaga aaagactgga gtcatcatca tgcccagaca 240gacatttcat c 25130221DNAHomo sapiens 30gtttggaaac actctgtctg taaagtctgc aagcagatat ttggatctct ttgagccctt 60cgttggaaac ggggtttctt catattatgc tagacagaag aattctcagt aacttccttg 120tgttgtgtgt attcaactca cagagttgaa cgatccttta cacagagcag acttgaaaca 180ctctttttgt ggaatttgca agtggagatt tcacaaaaaa c 22131171DNAHomo sapiens 31gctctgcgat gtgtgcgttc aactctcaga gtttaacttt tcttttcatt cagcagtttg 60gaaacactct gtttgtaaag tctgcacgtg gataatttga ccacttagag gccttcgttg 120gaaacgggat ttcttcatat tctgctagac agaagaattc tcagaatctt c 17132284DNAHomo sapiens 32agtctaagtc tctttgtagg tcactcagga cttgctttat gaatctgggt gctcctgtat 60tgggtgcata tatatttagg atagttagct cttcttgttg aattgatccc tttaccatta 120tgtaatggcc ttgtctcttt tgatctttgt tgggtgtgtt tgggtttttg ttttccattt 180gcttggtaga tcttcctcca tccctttatt ttgagcctat gtgtgtctct gcacgtgaga 240tgggtttcct gagtacagca cactgatggg tcttgactct atcc 28433234DNAHomo sapiens 33gtgttaaagt ctcccattat tattgtttgg gcgtctaagt ctctttgtag gtttctaagg 60atctgcttta tgaatctagg tgctcctgta ttgggtgcac atatatttag gatagttagc 120tcttcttgtt gaattgatcc ctttaccatt atgtaatggc cttctttgtc tcttttgatc 180tttgttggtt taaagtctgt tttatcagag actaggattg caaccccaca ccaa 23434233DNAHomo sapiens 34ggtggtgtgt gcgttcaact cacagagttt aacctttctt ttcatagagc agttaggaaa 60cactctgttt gtaaactctg caagtggata ttcagacctc tttgaggcct tcgttggaaa 120cgggatttct tcatactgtg ctagacagaa gaattctcag taacttcctt gtgttgtgtg 180tattcaactc aaagaggtct gaatatccac ttgcagagtt tacaaacaga gtg 23335151DNAHomo sapiens 35gacttgcttt atgaatctgg gtgctcctgt attgggtgca taaatattta ggatagttag 60ctcctcttgt tgaattgatc cctttaccat tatgtaatgg ccttctttgt ctcttttgat 120ctttgttgct ggccagggca atcaggcagg g 15136225DNAHomo sapiens 36tttgtctagc tttgaggatt tcgttggaaa cgggattaca tataaaaagc agacagcggc 60attcccagaa acttctttgt gatgtttgca ttcaagtcac agagttgaac attccctttc 120atagagcagg tttgaaacac tctttctgta gtatctggaa gtgaacatta ggacagcttt 180caggtctatg gtgagaaagg aaatatcttc aaaaaaaaac caaac 22537151DNAHomo sapiens 37gtttggtggg ggaattcttc tgtctagcct tatatgaaaa aaacccgttt ccaacgaagg 60cctcaaagag gtctgaatat ccacttgcag actttacaaa cagagtgttt cctaactgct 120ctatgaaaag aaaggttaaa ctctgtgagt t 15138238DNAHomo sapiens 38agttcaacca ttgtggaaga cagtgtggtg attcctcaag gatctagaac tagaaatacc 60atttgaccca gccatcccat tactgggtat atacccaaag gattataaat gattctacta 120taaagataca tgcacatgta tgtttattgt agcactcttc acaatagcaa agactgggaa 180ccaacccaaa tgcccatcaa taatagactg gataaagaaa atgtggcaca tagatacc 23839290DNAHomo sapiens 39acctttgact tcaaagcggc tgaaatctcc acttgcaaat tccacaaaaa gagtgttaca 60agtctgctct gtgtaaagga tcgttcaact ctgtgagttg aatacacaca acacaaggaa 120gttactgaga attcttctgt ctagccttac atgaaaaaaa cccgtttcca acgaaggcct 180ctaagtggtc aaattatcca cgtgcagact ttacaaacag agtgtttcca aactgctgaa 240tgaaaagaaa agttaaactc tgagagttga acgcacacat cacaaaggag 29040244DNAHomo sapiens 40cccattatta ttgtgtggga gtctaagtct ctttataggt ctctaaggac ttgctttatg 60aatctgggtg ctcctgtgtt gggtgtgttt ggttttttgt tgaattgatc cctttaccat 120tatgtaatga caaagaaggc cattacataa tggtaaaggg atcaattcaa caagaagagc 180taactatcct aaatatatat gcacccaata caggagcacc cagattcata aagcaagtcc 240ttag 24441151DNAHomo sapiens 41ggattattcc attccattcc attagatgat tccattcggg tccattcgat gattctcttc 60gattccattc gataattccg tttttttccg tttgatgttg attccattcg attccattcg 120atgataattc cattcgattc tatgcgatga t 15142172DNAHomo sapiens 42ggatcgttca actctgtgag ttgaatacac acaacacaag gaagttactg agaattcttc 60tgtctagcag aatatgaaga aatcccgttt ccaacgaagg cctcaaaggg gtctaactaa 120tcacttgcag actttacaga cagagtcttt ccaaactgct ctatgaagag aa 17243228DNAHomo sapiens 43gacgtttctg agaatgcttc tgtctagatt tgatatgaag atattcccgt ttccaacgaa 60atcttcaaat ctatccaaat gtccacttgc agattcaaca aaaagtgttt ttcagaactg 120ctctatcaaa agaatggatc aacactgtta gttgagtacc cacatcacaa acgtgattct 180ttgcgatgtt tgcattcaac tcatagagtt gaacattccc tttgagag 22844151DNAHomo sapiens 44gaatcgaatg gaatcatcga atggactcga atggaataat cattgaacgg aatcgaatgg 60aatcatcatc ggatggaaat gaatggaatc atcatcgaat ggaatcgaat agaattatgg 120aatgaaatcc agtgtgatca tcatcgaatg g 15145274DNAHomo sapiens 45aacattccgt ttcagagagc agctttgagg cactcttttt gtagtatgtg caagtggata 60tttggagcgc tctgaggcct acggtgaaaa agcaaatatc ttcccataac caccagacag 120aaacattctc agaaactcct ttatgacgta tgcactcacc taacagaaaa gtccacttcc 180atatactaca aaaagagcgt ttcaaacctg ctctatgaaa ggcaatgttc aactctgtga 240cttgaatgca gacatcacag agcagtttct gaga 27446245DNAHomo sapiens 46agatatagaa aaggcctttg acaaaattca acaactcttc atgctataaa ctctcagtaa 60attaggtatg gatgggaaat atctcaaaat aataggagct atctatgaca aacccacagc 120caatatcata ctgaatgggc aaaaactggg agcattccct ttgaaaactg gcacaagaca 180gggatgccct ctctcaccac tcctattcaa catagtgttg gaagttctgg ccagggcaat 240taggc 24547151DNAHomo sapiens 47attcatttcc atccgatgat gattccattc gattccgttc aatgattatt ccattcgagt 60ccattcgatg attccattcg attccattcg atgatgattg cattcgagtc catggattat 120tccattccat tccattagat gattccattc g 15148151DNAHomo sapiens 48aagaattttc tgagaatgat tctgtctggt ttcttcctat agaaactaga cagaatgatt 60ctcagaaact cctttgtgat gtgtgcgttc aactcacata gtttaacctt tcttttcata 120gagcagtttg gaaacactct gtttgtaaag t 15149200DNAHomo sapiens 49tcaacaccac cttcttcgac cccgccggag gaggagaccc cattctatac caacacctat 60tctgattttt cggtcaccct gaagtttata ttcttatcct accaggcttc ggaataatct 120cccatattgt aacttactac tccggaaaaa aagaaccatt tggatacata ggtatggtct 180gagctatgat atcaattggc 20050200DNAHomo sapiens 50gtggatattc ggacctcttt gaggccttcg ttggaaacgg gatttcttca tattatgcta 60gacagaagat ttctcagtaa cttctttgcg ttgtgtgtat gcaactcaca gagttcaacc 120ttcctttaga cagagcagat ttgaaacact ctttttgtgg aatttgcaag tggagatttc 180aagcgcttcg atgccaatgg 20051200DNAHomo sapiens 51gaaccatttg gatacatagg tatggtctga gctatgatat caattggctt cctagggttt 60atcgtgtgag cacaccatat atttacagta ggaatagacg tagacacacg agcatatttc 120acctccgcta ccataatcat cgctatcccc accggcgtca aagtatttag ctgactcgcc 180acactccacg gaagcaatat 20052150DNAHomo sapiens 52acagaacagg ctcctctaga gggatatgaa gcaccgccag gtcctttgag ttttaagctg 60tggctcgtag tgttctggcg agcagttttg ttgatttaac tgttgaggtt tagggctaag 120catagtgggg tatctaatcc cagtttgggt 15053200DNAHomo sapiens 53caataacttg accaacggaa caagttaccc tagggataac agcgcaatcc tattctagag 60tccatatcaa caatagggtt tacgacctcg atgttggatc aggacatccc gatggtgcag 120ccgctattaa aggttcgttt gttcaacgat taaagtccta cgtgatctga gttcagaccg 180gagtaatcca ggtcggtttc 20054200DNAHomo sapiens 54caactaggac tcataatagt tacaatcggc atcaaccaac cacacctagc attcctgcac 60atctgtaccc acgccttctt caaagccata ctatttatgt gctccgggtc catcatccac 120aaccttaaca atgaacaaga tattcgaaaa ataggaggac tactcaaaac catacctctc 180acttcaacct ccctcaccat 20055200DNAHomo sapiens 55ctggcatttt gtagatgtgg tttgactatt tctgtatgtc tccatctatt gatgagggtc 60ttactctttt agtataaata gtaccgttaa cttccaatta actagttttg acaacattca 120aaaaagagta ataaacttcg ccttaatttt aataatcaac accctcctag ccttactact 180aataattatt acattttgac 20056200DNAHomo sapiens 56aatcattttt attgccacaa ctaacctcct cggactcctg cctcactcat ttacaccaac 60cacccaacta tctataaacc tagccatggc catcccctta tgagcgggcg cagtgattat 120aggctttcgc tctaagatta aaaatgccct agcccacttc ttaccacaag gcacacctac 180accccttatc cctatactag 20057200DNAHomo sapiens 57tattcgagcc gagctgggcc agccaggcaa ccttctaggt aacgaccaca tctacaacgt 60tatcgtcaca gcccatgcat ttgtaataat cttcttcata gtaataccca tcataatcgg 120aggctttggc aactgactag ttcccctaat aatcggtgcc cccgatatgg cgtttccccg 180cataaacaac ataagcttct 20058218DNAHomo sapiens 58atattctgtt gatttggggt ggagagttct gtagatgtct attaggtcca cttggtgcag 60agctgagttc aattcctggg tatccttgtt gactttctgt ctcattgatc tgtctaatgt 120tgacagtggg gtgttaaagt ctcccattat taatgtatgg gagtctaagt ctctttgtag 180atcactcagg acttgcttta tgaatctggg tgctcctg 21859256DNAHomo sapiens 59atatgaactt taaagtagtt ttttccaatt ctgtgaagaa agtcattggt agcttgatgg 60ggatggcatt gaatctataa attaccttgg gcagtatggc cattttcatg atattgattc 120ttcctatcca tgagcatgga atgttccatt tgtttgtgtc ctcttttatt tcattgagca 180gtggttcgta gttctccttg aagagtcctt cacatccctt gtaaggtgga ttcctaggta 240ttttattctc tgaagc 25660248DNAHomo sapiens

60agagaataaa atacctagga atccaactta caagggatgt gaaggacctc ttcaaggaga 60actacaaact gctactcaag gaaatagaag aggatacaaa caaatggaag aatattccat 120gctcatgggt aggaagaatc aatatcgtga aaatggccat actgcccaag gtaatttaca 180gattcaatgc catccccatc aagctaccaa tgactttctt cacagaattg gaaaaaacta 240ctttaaag 24861277DNAHomo sapiens 61accttgggca gtatggccat tttcaggata ttgattcttc ctacccatga gcatggaatt 60ttcttccatt tgtttgtatc ctcttttatt tcactgagca gtggtttgta gttctccttg 120aagaggtcct tcacatccct tgtaagttgg attcctaggt attttattct ctttgaagca 180attgtgaatg ggagttcact catgatttgg ctctctgtct gttgttggtg tataagaatg 240cttgtgattt ttgtacattg attttgtatc ctgagac 27762232DNAHomo sapiens 62cctattcaac ataatgttgg aagttctagc caggacaatc aggcaggaga aagaaataaa 60gggtactcaa ttaggaaaag aggaagtcaa attgtccctg tatgcagata acatgattgt 120atatttagaa aaccccatca tctcagccca aaatctcctt aagctgataa gcaacttcag 180caaagtctca ggatacaaaa tcaatgtgca aaaatcacaa gcattcctat ac 23263270DNAHomo sapiens 63ctttaaagta gttttttcca attctgtgaa gaaagtcatt ggtagcttga tggggatggc 60attgaatcta taaattacct tgggcagtat ggccattttc atgatattga ttcttcctat 120ccatgagcat ggaatgttct tccatttgtt tgtgtcctct tttattttgt tgaacagtgg 180tttgtagttt tccttgaaaa ggtgcttcgc attccttgta agttggattc ctagatattt 240tattctcttt gtagcaattg tgaatgggag 27064210DNAHomo sapiens 64atgttcttcc atttgtttgt ctcctctttt atttccttga gcagtggttt gtagttctcc 60ttgaagaggt ccttcacatc ccttgtaagt tggattccta ggtattttat tctctttgaa 120gcaattgtga atgggagttc acccatgatt tggctctctg tttgtctgtt gttggtgtat 180aagaatgctt gtgatttttg tacattgatt 21065206DNAHomo sapiens 65cacaataata atgggagact ttaacacccc actgtcaaca ttagacagat caatgagaca 60gaaagttaac aaggataccc aggaattgaa ctcagctctg caccaagcag acccaataga 120catctacaga actctccacc ccaaatcaac agaatataca tctttttcat caccacacct 180attgcaaaat tgaccacata gttgga 20666243DNAHomo sapiens 66ctgtgatctg agagcctgtt tgttatgatt tctgttcttt tccatttgct gacgagtgtt 60ttgcttccaa ttatgtggtt gattttagaa taagtgctat gtggtgctga gaagaatata 120tattctgttg atttggggtg gagagttctg tagatgtcta ttaggtccac ttggtgcaga 180gctgagttca attcctggat atccttgtta actttctgtc tcgttgatct gtctaatgtt 240gac 24367213DNAHomo sapiens 67tgccatcccc atcaagctac caatgacttt cttcacagaa ttggaaaaaa ctactttaaa 60gttcatatgg aaccaaaaaa gagcctgcat tgccaagtca atcctaagcc aaaagaacaa 120agctggaggc atcatgctac ctgacttcaa actatactac aagattacag taaccaaaac 180agcatggtac tggtaccaaa acagagatat aga 21368188DNAHomo sapiens 68ttttggtatc agtaccatgc tgttttggtt actgtagcct tgtagtatag tttgaagtca 60ggtagcgtga tgcctccagc tttgttcttt tggcttagga ttgacttggc gatgcgggct 120cttttttggt tccatatgaa ctttaaagta gttttttcca gttctgtgaa gaaagtcatt 180ggtagctt 18869262DNAHomo sapiens 69ctgtagcctt gtagtatagt ttgaagtcag gtagtgtgat gcctccggct ttgttctttt 60ggcttaggat tgacttggca atgcggggtc ttttttggtt ccatatgaac tttaaagtag 120ttttttccaa ttctgtgaag aaagtcattg gtagcttgat ggggatggca ttgaatctat 180aaattacctt gggcagtatg gccattttca cgatattgat tcttcctatc catgagcatg 240gaatgttctt ccatttgttt gt 26270191DNAHomo sapiens 70aaataaaaga agacacaaat aaatggaaga acattccata ctcgtggata ggaagaatca 60atattgtgaa aatggccata ctgcccaagg taatttatag attcaatgcc atccccatca 120agctaccaat gactttcttc acagaattgg aaaaaactac tttaaagttc atatggaacc 180aaaaaacagc c 19171247DNAHomo sapiens 71ccctctctca ccactcccat tcaacatagt attggaagtt ctggccacag caatcacgca 60agagaaagaa ataaagagta ttcaagtagg aaaagaggaa gtcaaattgt ccctgtttgc 120agatgacatg attgtatatc tagaaaaccc catcatctca gcccaaaatc tccttaagct 180gataagcaac ttcagcaaag tctcaggata caaaatcaat gtgcaaaaat cacaagcatt 240cctatac 24772199DNAHomo sapiens 72ataagcaact tcagcaaagt ctcaggacac aaaatcaatg tacaaaaatc acaagcattc 60ttatacacca ataacagaca aacagagagc caaatcatga gtgaactccc attcacaatt 120gcttcaaaga gaataaaata cttaggaatc caacttacaa gggacgtgaa ggacctcttc 180aaggagaact acaaaccac 19973279DNAHomo sapiens 73ctcttttttg gtacaagtac catgctgttt tggttactgt agccttgtag tatagtttga 60agtcaggtag catgatgcct ccagctttgt tctttttact tagcattgtc ttggcaatgt 120gtgctgtttt ttggttccat atggacttta aagtagtttt ttccaattct gtgaagaaag 180tcattggtag cttgatgggg atggcattga atctataaat taccttgggc agtatggcca 240ttttcatgat attgattctt cctatccatg agcatggaa 27974282DNAHomo sapiens 74ataagaatgc ttgtgatttt tgcacattga ttttgtatcc tgagactttg ctgaagttgc 60ttatcagctt aaggagattt tgggctgaga cgatggggtt ttctagatat acaatcatgt 120catctgcaaa cagcgacaat ttgacttcct cttttcctaa ttgaataccc tttatttcct 180tctcctgttt cattgccctg gccagaactt ccaacactat gttgaatagg agtggtgaga 240gagggtgtcg ctgtgttgtg ccagctttca aagggaatgc tt 28275253DNAHomo sapiens 75tttgattgca ctgtggtctg agagacagtt ggttgtgatt tctgttcttt tacatttgct 60gaggagtgct ttacttccaa ctatgtggtc agttttggaa taaatgtggt gtggtgctga 120aaaaaatgta tattctgttg atttggggtg gagagttctg tagatgtcta ttaggtctgc 180ttggtgcaga gctgagttca attcctgggt atccttgttg actttctgtc tcgttgatct 240gtctaatgtt gac 25376283DNAHomo sapiens 76accccactgt caacattaga cagatcaacg agacagaaag ttaacaagga tatccaggaa 60ttgaactcag ctctgcacca agcggaccta atagacatct acagaactct ccaccccaaa 120tcaacagaat atacattctt ctcagcactg catcgcactt attccgaaat tgaccacata 180gttggaagta aagccctcct cagcaaatgt aaaagaacag aaattataac aaactgtctc 240tcagaccaca gtgcaatcaa actagaactc aggattaaaa aac 28377211DNAHomo sapiens 77aaaacagcat ggtactggta ccaaaacaga gatatagacc aatggaacag aacagagccc 60tcagaaataa taccacacat ctacaaccat ctgatctttg acaaacctga caaaaacaag 120aaatggggaa aggattccct atttaacaaa tggtgctggg aaaactggct agccatatgt 180agaaagctga aactggatcc cttccttaca c 21178172DNAHomo sapiens 78caaatggaag aacattccat gctcatgggt aggaagaatc aatatcgtga aaatggccat 60actgcccaag gtaatttaca gattcaatgc catccccatc aagctaccaa tgactttctt 120cacagaattg gaaaaaacta ctttaaagtt catatggaac caaaaaagag cc 17279192DNAHomo sapiens 79atttcgttga gcagtggttt gtagttctcc ttgaagaggt ccttcacatc ccttgtaagt 60tggattccta ggtattttat tctctttgaa gcaattgtga atgggagttc actcatgatt 120tggctctctg tctgttattg gtgtatagga atgcttgtga tttttgcaca ttgattttgt 180tatcctgaga ct 19280254DNAHomo sapiens 80ggtaatttat agattcaatg ccatccccat caagctacca atgactttct tcacagaatt 60ggaaaaaact actttaaagt tcatatggaa ccaaaaaaga gcccgcattg ccaagacaat 120cctaagccaa aagaacaaag ctggaggcat cacactacct gacttcaaac tatactacaa 180ggctacagta accaaaacag catggtactg gtaccaaaac agagatatag accaatggaa 240caggatagag ccct 25481300DNAHomo sapiens 81atcacaaaca agtttctgag aatgcttctg tctagttttt atgggaagat atttcctttt 60tcatcatagg cctcaaagcg ctccaaatgt ccacttccag atagtgcaga aagagtgtct 120caaacctggt atataaaagg gaacattcta ctctgtgact tgaatgaaaa catcacaaag 180cagtttctga gaatgcttcc gtctagattt tatatgaaga tattcccgtt tccaacgaaa 240ccttcaaagc tatccgaata tccacctgca gattctacaa aaagagtgtt tccaaaatgc 30082300DNAHomo sapiens 82tcccgtttcc aacgaaatcc tcaaagctat ccaaatatcc actttcagat tccacaaaaa 60gagtgtttca aaactgctct gtaaaaagaa aggttcatct ctgttagttg aatacacaca 120tcacaaacaa gtttctgaga atgcttctgt ctagttttta tgggaagata tttccttttt 180caacataggc ctcaaagcgc tccaaatgtc cacttccagg tagtgcagaa agagtgtttc 240aaacctgctc tataaaaggg aatattcaac tctgtgactt gaatgcaaac atcacaaagc 30083300DNAHomo sapiens 83aaacctgctc tataaaaggg aatattcaac tctgtgactt gaatgcaaac atcacaaagc 60actttctgag aatgcttccg tctagatttt atatgaagat attcccgttt ccaaggaaat 120cttcctagct atctaaatat caacttgcag attctactaa aggtatgttt ccaaaatgct 180gtatccacac aaaggttcaa ctctgttaat tgaggacata catcgcaaat aagtttctga 240gaatgcttct gtctagattt tatatgaaga tattccgttt tcaccacagg cctgaaagcg 30084300DNAHomo sapiens 84ttttctaccg ttggcctcaa agcgcttgaa gtctccccct gaaaattcca caaaaagtgt 60ttccaatctg ctccgtctaa aggaagcttc aactctgtga gttgaatacc cacaacacaa 120agaagttact gagaattctt ctgtctcgca ttatatgaag aaatcccgtt tccaactaag 180gcctcaaata catccacata tccagttgct gactttacaa actgagtgtt tccaaactgc 240tctatgaaaa gaaaggttaa acactgtgag ttgaacacac acgtaccaaa gtagtttctg 30085300DNAHomo sapiens 85ggaagatatt tcctttttca tcataggcct caaagcgctc caaatgtcca cttccagata 60gtgcagaaag agtgtctcaa acctggtata taaaagggaa cattctactc tgtgacttga 120atgaaaacat cacaaagcag tttctgagaa tgcttccgtc tagattttat atgaagatat 180tcccgtttcc aacgaaacct tcaaagctat ccgaatatcc acctgcagat tctacaaaaa 240gagtgtttcc aaaatgccgt atcaaaacaa aggttcaact ctgttagttg agaacacaca 30086300DNAHomo sapiens 86gatatttcct tttctacatg tggcctaaat gtgcttgaaa tctccatctg caaatatcac 60agaaagagtg tttcacatct gctctgtcta aaggaatgtt caactctgtg agttgaatac 120acacaacaca aagaagttac ggagaattat tctgtctggc attatatgaa gaaatcccgt 180ttccaacgaa ggactcaaag aggtccatat atccacttgc agaatttaca aagagagtgt 240ttccaaactg ctcaatcaaa agaaaggtta aactctgtga attgaacgca cacatcacaa 30087300DNAHomo sapiens 87ccacttgcag attctacaaa aagagtgttt ccaaactgct gtatcaaaag aaaggttcaa 60ctctgttagt tgaggacaca catcacaaat aagtttctga gaatccttct gtctagtttt 120tatgggaaga tatttccttt ttcaccatga gcctgaaagc gctcgaaacg tccacttcca 180gatactacag aaagagtgtt tcaaacctgc tctatgaatg cgaatgttca actctgtgac 240ttaaaagcaa acatcacaaa gaagcttctg agaatgctgc tgtctacttt ttatatgtaa 30088300DNAHomo sapiens 88aagaagtttc tgtgaatgat gctgtctaga ttttataaga agatgtttcc ttttctacca 60taggcctcaa agcgctagaa atctccagct gcaaattcca caaaaagtgt gtttaacatc 120tgctctgtct aaagtaaagt tcagctctgt gagtagaata cacacagcac aaagaagtta 180ctgagacttc ttctgtctaa cattatatga agaaatcccg tttccaacga aggcctcaaa 240gaggtccaaa tatccacttg cagacttgtc agagtgtttc caaactgcac catcaaaaga 30089300DNAHomo sapiens 89tatgaagaaa tcccgtttcc aacgaaggcc tcaaagaggt ccaaatatcc acttgcagac 60attaaaaaca gagtgtttcc aaactgctca gtcaaaagaa aggctaaact cagtgagttg 120aatgcacaca tcacaaagta ctttctgaga atgattctgt ttagtgttta tacgaagata 180tttccttttc tacacttggc ctaaaaacgc ttgaaatctc cacctgcaaa tttcacaaaa 240agagtttttc aaatctgctc tgtctaaaga aaggttcaac tcactgagtt gaatacacac 30090200DNAHomo sapiens 90agaaaggttc atctctgtta gttgaataca cacatcacaa acaagtttct gagaatgctt 60ctgtctagtt tttatgggaa gatatttcct ttttcatcat aggcctcaaa gcgctccaaa 120tgtccacttc cagatagtgc agaaagagtg tctcaaacct ggtatataaa agggaacatt 180ctactctgtg acttgaatga 20091200DNAHomo sapiens 91ccttttctac cgttggcctc aaagcgcttg aagtctcccc ctgaaaattc cacaaaaagt 60gtttccaatc tgctccgtct aaaggaagct tcaactctgt gagttgaata cccacaacac 120aaagaagtta ctgagaattc ttctgtctcg cattatatga agaaatcccg tttccaacta 180aggcctcaaa tacatccaca 20092200DNAHomo sapiens 92tcacaaagca gattctgata atgcttctgt ctaattttta tataaagata ttcctgtttc 60caatgaaatc ctcaaagcta ttcaaatatc cacttgcaga ttatacaaaa accgtgtttc 120aaaactacca aaataaatgt tcaactctgt tctttgagta cactcatcat aaacaagttt 180ctgagaaggc ttctgtcaat 20093200DNAHomo sapiens 93gtttcaaaac tgctctatga aacgaatgtt caaccctgtg acgtgaatgc agacatcaca 60aagcagtttc tgagaatgct tctgtctcga ttttacatga agatattccc gtttccaaag 120aaatcttcaa agttatccaa atatccactt gcagattcta caaaaagagt gtttccaaac 180tgctgtatca aaagaaaggt 20094212DNAHomo sapiens 94gcacacgtat gtttatcgca gcactactca caacaacaaa gacttggaac caacccaaat 60gtccaccaat gatagactgg attaagaaaa tgtggcacat atacaccatg gaatactatg 120cagccataaa aaatgatgag ttcatgtcct ttgtagggac atggatgaag ctggaaacca 180tcattctcag caaactattg caaggacaaa aa 21295270DNAHomo sapiens 95tttttttttg agacggagtc tcgctctgtc gcccaggctg gagtgcagtg gcgcaatctc 60ggctcactgc aagctccgcc tcccgggttc aacgccattc tcctgcctca gcttcccaag 120tagctgggac tacaggcgcc cgccactacg gccggctaat tttttgtatt tttagtagag 180acggggtttc accgttttag ccgggatggt ctcgatctcc tgacctcgtg atccgcccgc 240ctcggcctcc caaagtgctg ggattatagg 27096135DNAHomo sapiens 96catgtgtctt tatagcagca tgatttataa tcctttgggt atatacccag taatgggatg 60gctgggtcaa atggtatttc tagttctaga tccttgagga attgccacac tgtcttccac 120actggttgaa ctagt 13597273DNAHomo sapiens 97tgtgtccatg tgttctcatt gttcgattcc cacctatgag tgagaatatg tggtgtttgg 60ttttttgtcc ttgtgatagt ttgctgagaa tgatggtttc cagcttcacc catgtcccta 120caaaggacat gaactcatca ttttttatgg ctgcatagta ttccatggtg tatatgtgcc 180acattttctt aatccagtct atcattgttg gacatttggg ttggttccaa gtctttgcta 240tgtgaataat gccgcaataa acatacgtgt gca 27398164DNAHomo sapiens 98tcccacagaa taataatggg agaatttgat acgccactgt caacattaga cagatcaacg 60agacagaaag ttaacaagga tatccaggaa ttgaactcag cactgcacca agcagaccta 120atagacatct acagaactct ccacaccaaa tcaacagaat atac 16499102DNAHomo sapiens 99tggctggtac cagttgttcc tttccatgtt tagtgcttcc ttcaggagct cttttagggc 60aggcctggtg gtgacaaaat ctctcagcat ttgcttgtct gt 102100232DNAHomo sapiens 100tttttgttgc gatagtttac tgagaatgat ggtttccaat ttcatccatg tccctacaaa 60ggacatgaac tcatcatttt ttatggctgc atagtattcc atggtgtata tgtgccacat 120tttcttaatc cagtctatca ttgttggaca tttgggttgg ttccaagtct ttgctattgt 180gaataatgcc gcaataaaca tacgtgtgca tgtgtcttta gagcagcatg at 232101133DNAHomo sapiens 101ctctatactt cccttctcgc ttcatttcat tcatttcatc ttccattgct gatacccttt 60cttccagttg atcgcatcgg ctcctgaggc ttctgcattc ttcacgtagt tctcgagcct 120tggttttcag ctc 133102286DNAHomo sapiens 102atggtgtata tgtgccacat tttcttaatc cagtctatca ttgttggaca tttgggttgg 60ttccaaatct ttgctattgt gaataatgcc gcaaaaaaca tacatgtgcg tgtgtcttta 120tagcagcatg atttatattc ctttgggtat atacccagta atgggatggc tgggtcaaat 180ggtatttcta gttctagatc cctgaggaat tgccacactg acttccacaa tggttgaact 240agtttacagt cccaccaaca gtgtaaaagt gttcctattt ctccac 286103231DNAHomo sapiens 103aggcatgggc aaggatttca agactaaaac accaaaagca atggcaacaa aagctaatat 60tgacaaatgg gatctaatta aactaaagag cttctgcaca gcaaaagaaa ctaccatcag 120agtgaacagg caacctacag aatgggagaa aatttttgca atctactcat ctgacaaagg 180gctaatatcc ggaatctaca atgaactcaa acaaatttac aaaaaaaaac c 231104270DNAHomo sapiens 104gtttctgctg agggatctgc tcttagtcta atgggcttcc ctttgagggt aacccgacct 60ttctctttgg ctgcccttaa cattttttcc ttcatttcaa ctttggtgaa tctgacaatt 120atgtgtcttg gagttgctct tctcgaggag tatctttgtg gcattctctg tattttctga 180atctgaatgt tggcctgcct tgctagattg gggaagttct cctggataat atcctgcaga 240gtgttttcca acttggttcc attctcccca 270105246DNAHomo sapiens 105gatcacgagg tcaggagatt gagaccatcc tggctaacac ggtgaaaccc cgtctctact 60aaaaatacaa aaaattagcc aggcgtggtg gcaggcgcct gtagtcccag ctactcggga 120ggctgaggca ggagaatggc atgaacccag gaggcggagc ttgcagtgag ccaagatcgc 180gccactgcat tccagcctga gcgacagact gagactccgt ctcaaaaaaa agaaaaagaa 240aaaaaa 246106241DNAHomo sapiens 106tataaaacat gctgctataa aggcacatgc acatgtatgt ttattgcagc actattcaca 60atagcaaaga cttggaacta acccaaatgt ccatcagtga tatactggat taagaaaacg 120tggcacatat acaccatgga atactatgca gccataaaaa atgatgagtt catgtccttt 180gtagggacat ggatgaaatt ggaaatcatc attctcagta aactatcgca aggacaaaaa 240a 241107233DNAHomo sapiens 107tgtggtctga gagacagttt gttataattt ctgttctttt acatttcctg aggagtgctt 60tacttccaac tatgtggtca attttggaat aggtgtggtg tggtgctgag aagaatgtat 120attctgttga tttggggtgg agagttctgt agatgtctgt taggtccact tggtgcagag 180ctgagttcaa gtcctggata tccttgttaa gcttctgtct catggatctg tct 233108244DNAHomo sapiens 108aaacatacgt gtgcatgtgt ctttatagta gaatgattta taatcctttg ggtatatacc 60cagtaatggg attgctgggt caaatggtat ttctggttcc agatccttga ggaatcaccc 120actgtcttcc acaatggttg aactaattta cactcccacc aacagtgtaa aagcattcct 180gtttctccac atcctctcca gcatctgttg ttttctgact ttttaatgat catcattcta 240actg 244109238DNAHomo sapiens 109attcaacata gtattggaag ttctggccag ggtaattagg caggagaagg aaataaaggg 60tattcaattg ggaaaagagg aagtcaaatt gttcctgttt gcagatgaca tgattgtata 120tctagaaaac cccattgtct cagcccaaaa tctccttaag ctgataagca acttcagcaa 180agtctcagga tacaaaatca atgtacaaaa atcataagca ttcttataca ccaacaac 238110198DNAHomo sapiens 110ggtcaggagt tcgagaccag cctggccaac atggtgaaac cccatctcta ctaaatatac 60aaaaattacc cgggcatggg gacgggtgcc tgtaatccca gctgttcagg aggctgaggc 120aggggaatcg cttgaacccg ggaggcggag gttgcagtga acagagatcg tgtcactgca 180ctccagcctg ggtgatag 198111111DNAHomo sapiens 111gtgtctgtgt agaaagaagt agacatggga gacttttcat tttgttctgt actaagaaaa 60attcttctgc cttgggatcc tgttgatctg tgaccttacc cccaaccctg t 111112160DNAHomo sapiens 112tctttgaaac caacaagaac aaagacacaa cataccagaa tctctgggac acattcaaag 60cagtgtgtag agggaaattt atagcactaa atgcccacaa gagaaagcag gaaagatcca 120aaattgacac cctaacatca caattaaaag aactagaaaa 160113288DNAHomo sapiens 113tctctgtttg tctgttattg gtgtataaga atgcttgtga tttttgcacg ttgattttgt 60atcctgagac tttgctgaag ttgcctatca gcttaaggag attttgggct gagacaatgg 120ggttttctag atatacaatc

atgtcatctg caaacaggga caatctgact tcctcttttc 180ctaattgagt accctttatt tccttctcct gcctgattgc cctggccaga acttccaaca 240ctatgttgaa taggagtggt gagagagggc atcgctgtct tctgccag 288114202DNAHomo sapiens 114ttttttgttc ttgtgatagt ttgctgagaa tgatggtttc cagcttcatc catgtcccta 60caaaggacat gaactcatca tttttatggc tgcatagtat tccatggtgt atatgtgcca 120cattttctta atccagtcta tcattgttgg atatttgggt tggttccaag tctttgctat 180tgtcagtaat gtctcaataa ac 202115256DNAHomo sapiens 115atgctggcct cataaaataa gttagggagg attccctctt tttctactga ttggaatagt 60ttcagaagga atggtatcag ctcctccttg tacctctggt aggatccggc tgtgaatcca 120tctggtcctg gacttttttt ggttggtaag ctattaatta ttgcctcaat ttcagagtct 180gttattggtc tattcagaga ttcaacttct tcctgattta gtcttgggag ggtgtatgtg 240tcgaggaatt tatcca 256116233DNAHomo sapiens 116gagagatcca ctgttagtct gatgggcttc catttgtggg taacccgacc tttctctctg 60gctgccctta acattatttc ctcatttcaa ctttgctgaa tctgacaatt atgtgtcttg 120gagttgctct tctcgaggag tatctttgtg gcattctctg tatttcctga atttgattgt 180tggcctgcct tgctagattg gggaagttct cctggataat atcctgcaga gtg 233117140DNAHomo sapiens 117gtcttgctct gtcgcccagg ctggagtgca gtggcgcgat ctcggctcac tgcaagctcc 60gcctcctggg ttcacgccat tctcctgcct cagcctcctg agtagctggg actacaggcg 120tccaccacca cgcccagcta 140118285DNAHomo sapiens 118gtttatattt gggatataaa cccaaaggat tataaatcat gcttctataa agacacatgc 60acacatatgt ttattgtggc actattcacc atagcaaaga cttggaacca acccaaatgt 120ccaacaatga tagactggat taagaaaatg tggcacatat acaccatgga atactatgca 180gccataagaa atgatgagtt catgtccttt gtagggacat ggatgaaatt ggaaatcatc 240attctcagta aagtattgca aggacaaaaa accaacccca aacac 285119292DNAHomo sapiens 119agttatacat tcttctaaat ttttttcaaa gttttcaact tctttgcctt tggtttgaat 60gtcctctcgt agctcagagt aatttgatcg tctgaagcct tcttctctca gctcgtcaaa 120gtcattctcc atccagcttt gttctgttgc tggtgaggaa cttcgttcct ttggaggagg 180agaggcgctc tgcgttttag agtttccagt ttttctgttc tgttttttct ccatctttgt 240ggtttatcta cttttggtct ttgatgatga tgatgtacag atgggttttt gg 292120243DNAHomo sapiens 120gctaaaaact ctcaataaat taggtattga tgggatgtat ctcaaaataa taagagctat 60ttatgacaaa cccacagcca atatcatact gaatgggcaa aaactggaag cattcccttt 120gagaactggc acaagacagg gatgccactc ctattcaaca ctcaccactc ctattcaaca 180tagtgttgta agttctggcc agggcaatca ggcaggagaa agaaataaag ggtattcaac 240tag 243121259DNAHomo sapiens 121ttgttattcc tgagttctag tttggttaca ctgtggtctg agagacagtt tgttataatt 60tctgttcttt tacatttgct gaggagagct ttacttccaa ctatgtggtc agttttggaa 120ctgtccctgt ttgcagatga catgattgta tatctagaaa accccattgt ctcagcccaa 180aatctcctta agctgataag caacttcagc acagtctcag gatacaaaat caatgtacaa 240aaatcacaag cattcttat 259122279DNAHomo sapiens 122aattcggaac tggtaccatt ccttctgaaa ctattccaat taatagaaaa agagggaatc 60ctccctaact cattttaaga ggccagcatc attctgatac caaagccggg cagagacaca 120accaaaaaac agaattttag accaatatcc ttgatgaaca ttgatgcaaa aatcctcaat 180aaaatactgg caaactgaat ccagcagcac atcaaaaagc ttatccacca tgatcaagtg 240ggcttcatcc ctgggacgca aggctggttc aatatacgc 279123272DNAHomo sapiens 123accgctagca agactaataa agaaaaaaag agagaagaat caaatagacg caataaaaaa 60tgataaaggg gataccacca ctgatcccac agaaatacaa actaccatca gagaatacta 120caaacacctc tatgcaaata aactagaaaa tctagaagaa atggataaat tccttgacac 180gtacatgctc ccaagactaa accaggaaga agttgaatct ctgaatagac caataacagg 240agctgaaatt ggggcaataa tcaatagctt ac 272124241DNAHomo sapiens 124tgacatgggt ctcctgaata cagcacactg atgggtcttg actctttatc caatttgcca 60gtctgtgtct tttaattggg gcattcagcc catttacatt taaggttaat attgttatgt 120gtgaatttga tcctgtcatt atgatgttag ctggttattt tgcttgttag ttgatgcagt 180ttcttcctag catctatggt ctttacagtc tggcatattt tgcagtggct ggtactggtt 240g 241125184DNAHomo sapiens 125cctttgtgat gtgtgtgttc aactcacaga gtttaacacc cacaccccaa acacacccaa 60cacaaaggag tttctgagaa tcattctgtc tagtttttct acgaagatat ttccttttct 120actattgacc tcaaagcggc tgaaatctcc acttgcaaat tccacgaaaa gagtgtttca 180agtc 184126278DNAHomo sapiens 126cacccactcc agcatataaa cagaaccaaa gacaaaaacc acatgattat ctcaatggat 60gcagaaaagg cctttgacaa aattcaacaa cccttcatgc taaaaactct caataagtta 120ggtattgatg ggacatattt caaaataata agagctatct atgacaaacc cacagccaat 180atcatactga atgggcaaaa actggaagca tttcctttga aaactggcac aagacaggga 240tgtcctctct taccactctt attcaacata gtaatgga 278127274DNAHomo sapiens 127cctccaagct atccaaatat ccactttcag attccacaaa aagagtgctg ctctgtaata 60agaaaggttc atccctgtta gttgaataca cacatcacaa acaagtttct gagaatgctt 120ctgtctagtt tttatgggaa gatatttcct ttttcaacat aggcctcaaa gcgctccaaa 180cgtccacttc caggtagtgc agaaagagtg tctcaaacct ggtatataac agggaacatt 240ctattctgtg acttgaatga aaacatcaca aagc 274128289DNAHomo sapiens 128taccctttct tccagttgat cgcatcggct cctgaggctt ctgcattctt cacgtagttc 60tcgagccttg gttttcagct ccatcagctc ctttaagcac ttctctgcat tggttattct 120agttataaat tcttctaaat ttttttcaaa gttttcaact tctttgcctt tggtttgaat 180gtcctcccgt agctcagagt aatttgatcg tctgaagcct tcttctctca gctcgtcaaa 240atcattctcc atccagcttt gttccgttgc tggtgaggaa ctgcgttcc 289129227DNAHomo sapiens 129agttggcttc atccctggga tgcatggctg gttcaacata cacaaatcaa taaacgtaat 60ccatcatata aacagaacca aagacaaaaa ccacatgatt atctcaatag atgcagaaaa 120ggcctttgac aaaattcaac aacccttcat gctaaaaact ctcaataaat taggtactga 180tgggacgtgt ctcaaaataa taagagctat ctatgacaaa cccacac 227130283DNAHomo sapiens 130gagtcagaga ggattccctc tttttctgtt gattggaata gtttcagaag gaatggtacc 60agctcctcct tgtacctctg gtagaattcg gctgtgaatc catctggtcc tggactcttt 120ttggttggta agctattgat tattgccaca atttcagatc ctgttattgg tctattcaga 180gattcaactt cttcctggtt tagtcttgag agagtgtaca tgtctagaaa tttatccatt 240tcttctagat tttctagttt atttgcatag aggtatttgt agt 283131277DNAHomo sapiens 131catttattga tttgcatata ttgaaccagc ctttcatccc agggatgaag cccacttgat 60catggtggat aagctttttg atgtgctgct ggattcggtt tgccagtatt ttattgagga 120tttttgcatc aatgttcatc agggatattg ttctaaaatt ctttttgttg tgtctctgcc 180aggctttggt atcaggatga tgctggcctc ataaaatcag ttagggagga ttccctcttt 240ttcagttgat tggaatagtt tcagaaggaa tggtacc 277132246DNAHomo sapiens 132tttggtttgt tttgttacag cattttggaa accttcaaat ctatccaaat atccacctgc 60agatcctaca aaaagagtgt ttccaaaatg ctgtatcaaa acaaaggttc aactctgtta 120gttgagaaca cacatcgcaa ataagtttct gagaatgctt ctgtctagtt tttatttgaa 180gatatttccc ttttcaccac aggcctgaaa gcgcttgaaa cgtccgcttg cagatactac 240aaaagg 246133209DNAHomo sapiens 133gtttgggttg tttgtgcatt gattttgtat gctgagactt tgctgaagtt gcttatcagc 60ttaaggagat tttggcatga agggttgttg aattttgtca aaggcctttt ctgcatctat 120tgagataatc atgtggtttt tgtctttggt tctgtttata tgctggatta catttattaa 180tttgcatata ttgaaccagc cttgcatcc 209134285DNAHomo sapiens 134aactatctct cagaccacag tgcaatcaaa ctagaactca ggattaagaa actcgctcaa 60aaacgctcaa ctacatggaa actgaacaac ctgctcctga atgactactg ggtaaataat 120gaaatgaagg cagaaataaa gattttcttt gaaaccaacg agaacaaaga cacaacatac 180cagaatctct gggacacatt caaagcagta tgtagagaga aatttataga actaaatgcc 240cacaagagaa agcaggaaag atctaaaatt gacaccataa tatca 285135234DNAHomo sapiens 135ggttttttgg tttgaaagtc ctcctgtagc tcgaagtaat ttgatcttct gaagccttct 60tctctcagct tgtcagtcat tctccgtcct gctttgttcc gttgctggtg aggaactgcg 120ttcctttgga ggaggagagg tgctctgctt tttagagttt ccagtttttc tgttctgttt 180tttccccatc tttgtggttt tatgtacttt tggtctttga tgatggtgat gtac 234136270DNAHomo sapiens 136cacacccaac acatcaaaaa gcttatccac catgatcaag tgggcttcat ccctgggatg 60caaggctggt tcaatatacg caaatctata agtgtaatcc agcatacaaa cagaaccaaa 120gacaaaaacc acatgattat ctcaatagat gcagaaaagg cctttgacaa aattcaacaa 180cccttcatgc taaaaactct cagtaaatta ggtattgatg ggatgtatct caaaataata 240agagctattt atgacaaacc cacaccaaac 270137264DNAHomo sapiens 137cctcataagt tggattccta ggtattttgt tctttttgaa gcaattgtga atgggagttt 60actcatgatt tggctctctg tttgtctgtt attggtgtat aggaatgttt gttatttttg 120cacattgatt ttgtatcctg agactttgct gaagttgctt atcagcttaa gaagattttg 180ggctgagacg atggggtttt ctaaatatac aatcatgtca tctgcaaaca gggacaattt 240gagttcctct tttcctaatt gaat 264138195DNAHomo sapiens 138cataaaatga gttagggagg attccctctt tttctattga ttggaatagt ttcagaagga 60atggtaccag ttccttctga aactattcca atcaatagaa aaagagggaa tcctccctaa 120ctcattttat gaggccagca tcattctgat accaaagccg ggcagagaca caaccaaaaa 180agagaatttt agacc 195139205DNAHomo sapiens 139ttggtttggt ttgccagtat tttatgaggc cagcatcatc ctgataccaa agcctggtag 60agacacacca atatccctga tgaacatcaa tgcaaaaatc ctcaataaaa tactggcaaa 120ccgaatccag cagcacatca aaaagcttat ccaccatgat caagtgggct tcatccctgg 180gatgcaaggc tggttcaata tacgc 205140258DNAHomo sapiens 140ctgtttacat gctggattac gtttattgat ttgcgtatgt tgaaccaaag acaaaaacca 60catgattatc tcaatagatg cagaaaaggc ctttgacaaa tttcaacagc ccttcatgct 120aaaaactctc aataaattag gtattgatgg gacgtatctc aaaataataa aagctatcta 180tgacaaaccc acagccaata tcatactgaa tgggcaaaaa ctggaagcat tccctttgaa 240aactggcaca agaaaggg 258141289DNAHomo sapiens 141gctcggagta gtttgatcgt ctgaagcctt cctctctcag ctcgtcaaaa tcattcacca 60tccagctttg ttccgttgct ggtgaggaac tgcgctcctt tggaggagga gaggcgctct 120gctttttaga gtttccagtt tttctgttct gttttttccc catctttgtg gttttatcta 180cttttggtct ttgatgatgg tgatgtacag atgggttttt ggtgtggatg tcctttctgt 240ttgttagttt tccttctaac agacaggacc ctcagctgca ggtctgtac 289142202DNAHomo sapiens 142tttttgttgt gtctctgcct ggctttggta tcagaatgat gctggcctca taaaatgagt 60tagggaggac tccctctttt tctgttgatt ggaatagttt cagaaggaat ggtaccagct 120cctccttgta cctctggtag aattcggctg tgaatccatc tggtcctgga ctctttttat 180gaggccagca tcattctgat ac 202143104DNAHomo sapiens 143ggtctggagt ggacctccaa aaagggtctg gagtggacct ccagcaaact ccaacagacc 60tgcagctgag ggtcctgact gttagaagga aaactaacaa acag 104144200DNAHomo sapiens 144tacttagata accacagcct aagaattcat tagcttttaa aacaatcttg cttcacactt 60gacttacatt gccttcacaa tcaacaaaag tcttttggtt taaatgattt ttcaagtcct 120tttcaatgct gtgatattat tgttatctcc tccacgttct ttgtttaaac tattggtctg 180ccgggcgcgg tggctcacac 200145200DNAHomo sapiens 145ggatggaatg cccaacaaat acttagattt atgggtttgg aacccaggag atagatctca 60gtaaatgata aagagttgag agtcattggc ctgtagattc attcatctac tcattcagta 120aatattcact tactaacgtg ctttgtctca ggcattaagt atacagtggt aaattaaaac 180attattttag ttttcacaaa 200146150DNAHomo sapiens 146gcagcctcta ctattcctta ctttattata cccacccatt ttcctcactt ctatcatttg 60cctggtccat gtaggccatc tgagtttaag atctctggca tacagtttat ctgggaagat 120aagctacaca taccacacac tccacatatc 150147200DNAHomo sapiens 147tctgcgcttc aaatgccact ttgtttactc gctgtggtca tccttttctg taaatactgt 60gtcagcatta acagcctcct ctatggctac ctagcaggtc acttagtaaa ctatctttta 120gtaacaacta catagaatga atactcagga gccttaattc aatttacctg cataaagaaa 180ctattcacta ttacaacaca 200148200DNAHomo sapiens 148cagtcctaaa gagggagaca ttattacttt ccccattcgt agaatgagta aactgagttt 60taagtaggtt aaatggtttg tacaggatta cagaattaga gaatgaaaga accacatggc 120aaacccatgt agtttgtgcc aaatttatac tcttctgtgt catagcggtc cttgattgtg 180aggttcattc aaagccagca 200149230DNAHomo sapiens 149ctaactctct ttgtaggtca ctaaggacag cttatccacc atgatcaagt gggcttcatc 60actgggatgc aaggctggtt caacatacaa aaatcaataa atgtaatcca gcatataaac 120agaaccaaag acaaaaacca catgattacc tcaatagatg cagaaaaggc ctttgacaaa 180attcaacaac ccttcatgct aaaaactctc aataaattag gtattgatgg 230150266DNAHomo sapiens 150gatattggct gtgggtttgt catagatagc tcttattatt ttgagatatg tcccatcaat 60acctaattta ttgagagttt ttagcatgaa gggttgttga attttgtcaa aggctttttc 120tgcatctatt gagataatca tgtggttttt gtctttggct ctgtttatat gctggattac 180atttattgac ttgtgtatat tgaaccagcc ttgcatccca gggatgcaag gctggttcaa 240tatacacaag tcaataaatg taatcc 266151172DNAHomo sapiens 151gtttggttgt ttgttgaacc agccttgcag cacatcaaaa agcttatcca ccatgatcaa 60gtgggcttca tccctgagat gcaaggctgg ttcaatatac gcaaatcaat aaatgtaatc 120cagcatataa acagagccaa agacaaaaac cacatgatta tctcaatcga tg 172152234DNAHomo sapiens 152cctcaacaca taccctctcc caagactaaa ccaggaagaa gttgaatctc tgaatagacc 60aataacagga tctgaaattg tggcaataat caatagctta ccaaccaaaa agagtccagg 120accagatgga ttcacagccg aattctacca gaggtacaag gaggaactgg taccatttct 180tctgaaacta ttccaatcaa atagacgcaa tcaaaccacc gatcccacaa aaac 234153214DNAHomo sapiens 153atattgttat gtgtgaattt gatcctgtca ttatgatgtt agctggtgat tctcccaaga 60ctaaaccagg aagaagttga atctctgaat agaccaataa caggctctga aattgtggca 120ataatcaata gtttaccaac caaaaagagt ccaggaccag atggattcac agccgaattc 180taccagaggt acaaggagga actggtacca ttcc 214154228DNAHomo sapiens 154gcatataaac agaaccaaag acaaaaacca catgattatc tcaatagagg cagaaaaggc 60ctttgacaaa attcaacaac ccttcatgct aaaaactctc aataaattag gtattgatgg 120gacgtatctc aaaataataa gagctatgca tgacacaccc acagccaata tcatactgaa 180tgggcaaaaa ctggaagcat tccctttgaa aactggcaca acccaacc 228155283DNAHomo sapiens 155gtatgatgct ggcctcataa aatgagtcag ggagaatttc ctctttttct attgattgga 60atagtttcag agggaatggt accaattcct ctttgtacct ctggtagaat tcagctgtga 120atccatctgg tcctggactc tttttggttg gtaagctatt gattattgcc acaatttcag 180ctcctgttat tggtctattc agagattcaa cttcttcctg gtttagtctt gggagagtgt 240atgtgtcgag gaatttatcc atttcttcta gattttctag ttt 283156255DNAHomo sapiens 156ctatttattg gaatagtttc agaaggaatg gtaccagttc ctccttgtac ctctggtaga 60attcggctgt gaatccatct ggtcctggac tctttttggt tggtaaacta ttgattattg 120ccacaatttc agctcctgtt attggtctat tcagagattc aactaaactc agattcctgg 180tttagtcttg ggagagtgta tgtgtcgagg aatttatcca tttcttctag attttctagt 240ttatttgcgt agagg 255157281DNAHomo sapiens 157atacacaaat caataaatgt aatccagcat ataaacagag ccaaagacaa aaaccacatg 60attatctcaa tagatgcaga aaggcctttg acaaaattca acaacccttc atgctaaaaa 120ctctcaataa attaggtatt gatgggacgt atttcaaaat aataagagct atctatgaca 180aacccacagc caatatcata ctgaatgggc aaaaactgga agcattccct ttgaaaactg 240gcacaagaca gggatgccct ctctcaacac atattcaaaa t 281158221DNAHomo sapiens 158gatacatccc atcaatacct aatttattga gagtttttag catgaagggc tgttgaattt 60tgtcaaaggc cttttctgca tctattgaga taatcatgtg gtttttgtct ttggttctgt 120ttatatgctg gattacgttt attgatttgc gtatgttgaa ccaggcttgc atcccaggga 180tgaagcctgg ttcaacatac gcaaatcaat aaacgtaatc c 221159250DNAHomo sapiens 159gccttacaag agctcctgaa ggaagcacta aacatggaaa ggaacaaccg gtaccagccg 60ctgtaaaatc atgccaaaat gtaaagacca tcgagactag gaagaaactg catcaactaa 120cgagcaaaat aaccagctaa catcataatg acaggatcaa attcacacat aacaatttaa 180atgtaaatgg actaaatgct ccaattaaaa gacacagact ggcaaattgg ataaagagtc 240aagacctatc 250160272DNAHomo sapiens 160gtcccttttt tcaaagggaa tgcttccagt ttttgcccat tcagtatgat attggctgtg 60ggtttgtcat agatagttct tattattttg aaatacgtcc catcaatacc taatttattg 120agagttttta gcatgaaggg ttgttgaatt ttgtcaaagg ctttttctgc atctattgag 180ataatcatgt ggtttttgtc tttggctctg tttatatgct ggattacatt tattgatttg 240catatattga accagagaca aaaaccacaa cc 272161269DNAHomo sapiens 161actacaaaca cctctacgca aataaactag aaaatctaga agaaatggat acatacactc 60tcccaagact aaaccaggaa gaagttgaat ctctgaatag accaataaca ggctctgaaa 120ttgaggcaat aattaatagc ttaccaacca aaaaaagtcc aggaccagat ggattcacag 180ccgaattcta ccagaggcac aaggaggagc tggtaccatt ccttctgaaa ctattccaat 240caatagaaaa agagggaatc ctccttaac 269162229DNAHomo sapiens 162gggtttgttg gtaacctgac ctttttaaag gcagccagag aaaaaggtca ggttacccac 60aaaaggaagc ccatcagact aactgctgat ctctcggcag aaactctaca agccagaaga 120gagtaggggc caatattcaa cattcttaaa gaaaagaatt ttcaacccag aatttcatat 180ccagccaaac taagcttcat aagtgaagga gaaataaaat actttacag 229163275DNAHomo sapiens 163ggtcaaaatg aaaaaaaaaa tgttaatggc agccagagag aaaggtcggg ttaccctcaa 60agggaagccc atcagactaa cagtggatct cttggcagaa accctacaag ccagaagaga 120gtgggggcca atattcaaca ttcttaaaga caagaatttt caacccagaa tttcatatcc 180agccaaacta agcttcataa gtgaaggaga aataaaatcc tttacagaca agcaaatgct 240gagagatttt gtcaccacca ggcctgccct aaaag 275164292DNAHomo sapiens 164gagcaactct tctcctccaa aggaacgcag ctcctcacca gcaacagaac aaagctggac 60ggagaatgac tttgacgagc tgagagaaga aggcttcaga cgatcgaatt actctgagct 120acgggaggac attcaaacca aaggcagaga agttgaaaac tttgaaaaaa atttagaaga 180atgtataact agaataacca atacagagaa gtgcttaaag gagctgatgg agctgaaaac 240caaggctcaa gaactacgtg aagaatgcag aagcctcagg agccgatgcg at 292165225DNAHomo sapiens 165gctttgtctt gtgggcattt agtgctataa atttccctct acacactgct ttgaatgcgt 60cccagagatt ctggtatgtt gtgtctctgt tgtaattgtc cctgtttgca gacgacatga 120ttgtttatct

agaaaacccc atcgtctcag cccaaaatct ccttaagctg ataagcaact 180tcagcaaagt ctcaggatac aaaatcaatg tacaaaaatc acaag 225166239DNAHomo sapiens 166cagcttatct ctcagcagaa acactacaag ccagaagaga gtaggggcca atattcaaca 60ttcttaatga aaagaatttt caacccagaa tttcatatcc agccaaacta agcttcataa 120gtgaaggaga aataaaatcc tttacagaca agcaaatgct gagagatttt gtcaccacca 180ggcctgccct aaaagagctc ctgaaggaag cactaaacat agaaaggaac aactggtac 239167255DNAHomo sapiens 167acttccccaa tctagcaagg caggccaaca ttcagattca ggaagtacag agaacgccac 60aaagatactc ctcgagaaga gcaactccaa gacacataat tgtcagattc accaaagttg 120aaatgaagga aaaaatgtta agggcagcca gagacaaagg tcggattacc cacaaaaaga 180agcccatcag actaacagcg gatctcttgg cagaaactct acaagccaga agagagtggg 240gaccaatact caaca 255168244DNAHomo sapiens 168atacatccca tcaataccta atttattgag agtttttagc atgaagggtt gttgaatttt 60gtcaaaggcc ttttctgcat ctattgagat aatcatgtgg tttttgtctt tggttctgtt 120tatatgctgg attacattta ttgatttgtg aatattgaac cagccttgca tcccagggat 180gaagcccacc tgatcatggt ggacaagctt tttgatgtgc tgctggattc agtcccacac 240aaac 244169255DNAHomo sapiens 169atgtaatcca gcatataaac agaaccaaag acaaaaacca catgattatc tcaatagatg 60cagaaaaggc ctttgacaaa attcaacaac ccttcatgct aaaaactctc aataaattag 120gtattgatgg gacgtatctc aaaatactaa gagctaccta tgacaaaccc acagccaata 180tcacactgaa tgggcaaaaa ctggtagcat tctctttgaa aactggcatg tgacagggat 240gccctctctc accac 255170292DNAHomo sapiens 170ttctcacaga gggggatttg gcagggtcat aggacaatag tggagggaag gtcagcagat 60aaacaagtga acaaaggtct ctggttttcc taggcagagg accctgcggc cttccgcagt 120gtttgtgtcc ctggatactt gagattaggg agtggtgatg actcttaacg agcatgctgc 180cttcaagcat ctgtttaaca aagcacatct tgcaccgccc ttaatccatt taaccctgag 240tggacacagc acatgtttca gagagcatag ggctgggggc aaggtcatag at 292171253DNAHomo sapiens 171tgatcgttct ggccagaact tccaacacta tgtggaatag gagtggtgag agagggcatc 60cctgtcttgt gccagttttc aaagggaatg cttccagttt ttgcccattc agtatgatat 120tggctgtggg tttgtcatag atagctctta ttattttgag atacgtccca tcaataccta 180atttattgag agtttttagc atgaaatgtt gttgaatttt gtcaaaggcc ttttctgcgt 240ctattgagag tca 253172208DNAHomo sapiens 172ccctcttttt ctattgattg gaatagtttc agaaggaatg gtaccagttc ctccttgtac 60ctctggtaga attcggctgt gaatccatct ggtcctggac tctttttggt tggtaagcta 120ttattgccac aatttcagat cctgttattg gtcgacatga ttgtatatct agaaaacccc 180attgtctcag cccaaagtct ccttaagc 208173247DNAHomo sapiens 173ggttttgttt ttctagttcc tttaattgtg atgttagggt gtcaattttg gatctttcct 60gcttaaccag gaagaagttg aatctctgaa tagaccaata acaggctctg aaattgtggc 120aataatcaat agcttaccaa ccaaaaagag tccaggacca gatggattca cagccgaatt 180ctaccagagg tacaaggagg aactggtacc attccttctg aaactattcc aatcaataga 240taaagag 247174200DNAHomo sapiens 174ctttacacag agcagatttg aaacactctt tttgtggaat ttgcaagtgg agatttcaag 60cgatttgatg ccaacagtag aaaaggaaat atcttcaaat aaaaactaga cagaatcatt 120ctcagaaact actttgtgat gtgtgccttc aactcacaga gtttaacctt tcttttctta 180caaacagtgt gtttctaaac 200175201DNAHomo sapiens 175ggtctccggt tttcctaggc agaggaccct gcggccttcc ggccttccgc agtgtttgtg 60tccctgggta cttgagatta gggagtggtg atgactctta acgagcatgc tgccttcaag 120catctgttta acaaagcaca tcttgcaccg cccttaatcc atttaaccct gagtggacac 180agcacatgat tcagagagca c 201176268DNAHomo sapiens 176ctgtaggttg cctgttcact ctgatggtaa cttcttttgc tgtgcagaag ctctttagtt 60taattagatc ccatttgtca attttggctt ttgttgccat tgcttttggt gttttagtca 120tgaagccctt gcccatgcct atgtcctgaa tggtattgcc taggttttct tctaggtttt 180aggtttaaca tttaagtctt taatccatct tgaattaatt tttgtataag gtgtaaggaa 240gggatccagt ttcagctttc tacatatg 268177188DNAHomo sapiens 177ataaagggat ggaggaagat ctaccaagca aatggaaaac aaaaaaaggc aggggttgca 60atcctagtct ctgataaaac agactttaaa ccaacaaaaa ccaaacacac ccaacacagg 120agcaccaaga ttcataaagc aagtcctgag tgacctataa agagacttag actcccacac 180aataataa 188178251DNAHomo sapiens 178gccatatgta gaaagctgaa actggatccc ttccttacac cttatacaaa aatcaattca 60agatggatta aagacttaaa cgttagacct aaaatcataa aaaccctaga agaaaaccta 120ggctaattta ccattcagga cataggcatg ggcaaggact tcatgtccaa aacaccaaaa 180gcaatggcaa caaaagacaa aattgacaaa tgggatctaa ttaaactcaa gagcttctgc 240acaaaaaaac c 251179189DNAHomo sapiens 179tttggtttgt gtgtgatttt tgcacattga ttttgtatcc tgagactttg ctgaagttgc 60ctatcagctt aaagagattt tgggccgaga cgatggggtt tcctagatat acaatcatgt 120catctgcaaa cagggacaat ttgacttcct cttttcctaa ttgaataccc tttatttcct 180tctcctgcc 189180200DNAHomo sapiens 180tccatgtccc tacaaaggac atgaactcat cattttttat ggctgcatag tattccatgg 60tgtatatgtg ccacattttc ttaatccagt ctatcattgt tggacatttg ggttggttcc 120aagtctttgc tattgtgaat agtgccgcaa taaacatatg tgtgcatgtg tctttatagc 180agcatgattt atagtccttt 200181200DNAHomo sapiens 181gcatcaactg aacgcaaatc agccacttta attaagctaa gcccttacta gaccaatggg 60acttaaaccc acaaacactt agttaacagc taagcaccct aatcaactgg cttcaatcta 120cttctcccgc cgccgggaaa aaaggcggga gaagccccgg caggtttgaa gctgcttctt 180cgaatttgca attcaatatg 200182151DNAHomo sapiens 182gtgtgtgtgt gtgtgtgtgt gtgtgtgtat ctatgtatgt acgtatgtat gtatgtatgt 60gagtgagatg ggtttcgggg ttctatcatg ttgcccacgc tggtctcgaa ctcctgtcct 120caagcaatcc gcctgcctgc ctcggccgcc c 15118320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 183aaacgtccgc ttgcagatac 2018421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 184agtatgctgc tgtgtacgtt t 2118521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 185tttgaggctt tcgttggaaa c 2118621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 186tcagctaaca gaggtggatc t 2118720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 187cttgtggcct tcgttggaaa 2018823DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 188gatagctgtg aagatttcgt tgg 2318921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 189tcagctaaca gaggtggatc t 2119022DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 190gtggagaaca cacatcacaa tc 2219122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 191ccctacaagc tagaaagaag ca 2219222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 192atgacgtatg cactcaccta ac 2219323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 193ttgtatctgg atgtggacat ttg 2319423DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 194gacagaatca ttcccacaaa ctg 2319524DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 195gaattctcag tagcttcttt gtgt 2419620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 196attcccgttt ccaacgaagg 2019725DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 197cctttcttat gatacagcag tttgg 2519827DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 198tctgtctagc atagtatgaa gaaatcc 2719922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 199gtaaagtctg caagtggatg tt 2220019DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 200ctccaccata gccctcaaa 1920123DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 201tgagaatgct tctgtctaga gtt 2320222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 202ggaatgttca actctgtgac tt 2220319DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 203ccctgtcttg tgccagttt 1920423DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 204aaagagacaa agaaggccat tac 2320520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 205aaagacacag actggcaaat 2020626DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 206cagtagagga tataactgcc cataaa 2620723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 207tgtgtaaagg atcgttcaac tct 2320820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 208tctcttgcca ctgccaatta 2020924DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 209gaataatgcc gcaataaaca tacg 2421022DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 210tctgcaagtg gatattcaga cc 2221122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 211cccattatta acgtgtggga gt 2221223DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 212tggaaacact ctgtctgtaa agt 2321318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 213tctgcgatgt gtgcgttc 1821422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 214gtctctttgt aggtcactca gg 2221526DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 215ccattattat tgtttgggcg tctaag 2621618DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 216tggtgtgtgc gttcaact 1821722DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 217tttatgaatc tgggtgctcc tg 2221823DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 218tttgtctagc tttgaggatt tcg 2321924DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 219gggaattctt ctgtctagcc ttat 2422022DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 220gttcaaccat tgtggaagac ag 2222120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 221gacttcaaag cggctgaaat 2022225DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 222cccattatta ttgtgtggga gtcta 2522320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 223gatgattcca ttcgggtcca 2022424DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 224gagttgaata cacacaacac aagg 2422522DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 225cgtttctgag aatgcttctg tc 2222623DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 226tcgaatggac tcgaatggaa taa 2322721DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 227acattccgtt tcagagagca g 2122827DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 228caacaactct tcatgctata aactctc 2722921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 229tccatccgat gatgattcca t 2123023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 230atgattctgt ctggtttctt cct 2323120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 231tcaacaccac cttcttcgac 2023222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 232gtggatattc ggacctcttt ga 2223326DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 233atttggatac ataggtatgg tctgag 2623418DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 234acagaacagg ctcctcta 1823521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 235ccaacggaac aagttaccct a 2123622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 236gttacaatcg gcatcaacca ac 2223728DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 237gtagatgtgg tttgactatt tctgtatg 2823821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 238attgccacaa ctaacctcct c 2123917DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 239agccaggcaa ccttcta 1724023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 240gctgtgaaga tttcgttgga aac 2324122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 241cctttgtact gacagagcag tt 2224220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 242gcacacatca caaagcagtt 2024328DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 243ctatgagttg aatggaaata tccgaaag 2824421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 244attgaactca aagcggctga a 2124521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 245agcgtttcaa acctctctag g 2124625DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 246ttctgagaat gcttctgtct agatt 2524725DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 247gtagaatctg caagtggata tttgg 2524822DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 248aaaggcaatg ttcaactctg tg 2224924DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 249gatattcccg tttccaacga aatc 2425027DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 250gtctagagtt tatatgaaga caatccc 2725122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 251gtgtaaagga tcgttcagct ct 2225219DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 252gttgaacgca cacatcaca 1925322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 253tgtgttcgtt caactcacag ag 2225424DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 254cctccaagct atccaaatat cctc 2425525DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 255ctcagaaact tctttgtgat gtgtg 2525623DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 256tttctactat tgacctcaaa gcg 2325722DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer

257acaattggat aattggaacc ct 2225825DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 258tcttagaatc ctatttgtga tgtgc 2525922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 259acttgtttgt gatgtgtgaa ct 2226021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 260ggccagaact tccaacacta t 2126122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 261tttacgtctg attgtgtgtt cc 2226225DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 262ttaggatagt tagctcttct tgttg 2526323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 263aggttctgag aatgcttctc ttt 2326422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 264ctttgaggat ttcgtgggaa ac 2226521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 265aatctgggtg ctcctgtatt g 2126622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 266tggttccaag tctttgctat tg 2226722DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 267ggatcgttca actctgtcag tt 2226821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 268aaatgtctgt ctgggcatga t 2126922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 269ctccacttgc aaattccaca aa 2227027DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 270tctgtctagc agaatatgaa gaaatcc 2727122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 271ggatagagtc aagacccatc ag 2227225DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 272ggttgcaatc ctagtctctg ataaa 2527322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 273tctgcaagtg gatattcaga cc 2227418DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 274cctggccagc aacaaaga 1827527DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 275gaagatattt cctttctcac catagac 2727622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 276agagcagtta ggaaacactc tg 2227724DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 277ccagtctatt attgatgggc attt 2427820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 278cctttgtgat gtgtgcgttc 2027921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 279aatctgggtg ctcctgtatt g 2128022DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 280tggaatcaac atcaaacgga aa 2228124DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 281tcttcataga gcagtttgga aaga 2428223DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 282ctctcaaagg gaatgttcaa ctc 2328323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 283tcgatgatga tcacactgga ttt 2328422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 284tctgtgatgt ctgcattcaa gt 2228521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 285ggccagaact tccaacacta t 2128623DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 286ggaataatcc atggactcga atg 2328723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 287acaaacagag tgtttccaaa ctg 2328825DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 288ttgatatcat agctcagacc atacc 2528920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 289gcgcttgaaa tctccacttg 2029020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 290tattgcttcc gtggagtgtg 2029121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 291acccaaactg ggattagata c 2129221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 292ctggattact ccggtctgaa c 2129319DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 293ggtgagggag gttgaagtg 1929422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 294ggctaggagg gtgttgatta tt 2229520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 295ggtgtgcctt gtggtaagaa 2029622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 296agaagcttat gttgtttatg cg 2229722DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 297gggtggagag ttctgtagat gt 2229825DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 298tccaattctg tgaagaaagt cattg 2529923DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 299acctaggaat ccaacttaca agg 2330017DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 300accttgggca gtatggc 1730124DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 301acataatgtt ggaagttcta gcca 2430225DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 302tccaattctg tgaagaaagt cattg 2530324DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 303gttcttccat ttgtttgtct cctc 2430428DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 304cacaataata atgggagact ttaacacc 2830523DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 305atctgagagc ctgtttgtta tga 2330621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 306cccatcaagc taccaatgac t 2130722DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 307tggtatcagt accatgctgt tt 2230827DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 308tgtagccttg tagtatagtt tgaagtc 2730924DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 309aatggaagaa cattccatac tcgt 2431020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 310tctctcacca ctcccattca 2031121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 311agcaacttca gcaaagtctc a 2131220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 312ggtacaagta ccatgctgtt 2031323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 313ttgtatcctg agactttgct gaa 2331420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 314tgattgcact gtggtctgag 2031525DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 315cactgtcaac attagacaga tcaac 2531625DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 316acagagatat agaccaatgg aacag 2531723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 317aatggaagaa cattccatgc tca 2331821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 318atttcgttga gcagtggttt g 2131927DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 319ggtaatttat agattcaatg ccatccc 2732024DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 320agtttctgag aatgcttctg tcta 2432123DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 321caacgaaatc ctcaaagcta tcc 2332223DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 322agggaatatt caactctgtg act 2332320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 323ttctaccgtt ggcctcaaag 2032420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 324tcatcatagg cctcaaagcg 2032525DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 325tttctacatg tggcctaaat gtgct 2532622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 326aaagagtgtt tccaaactgc tg 2232724DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 327gaagtttctg tgaatgatgc tgtc 2432821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 328gaaatcccgt ttccaacgaa g 2132926DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 329ggttcatctc tgttagttga atacac 2633020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 330tttctaccgt tggcctcaaa 2033124DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 331tcacaaagca gattctgata atgc 2433224DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 332ctgctctatg aaacgaatgt tcaa 2433323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 333caggagcacc cagattcata aag 2333425DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 334aaatacctag gaatccacct tacaa 2533525DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 335tccaattctg tgaagaaagt cattg 2533624DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 336gcattcttat acaccaacaa caga 2433724DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 337ttgtatcctg agactttgct gaag 2433823DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 338ctcccattca caattgctac aaa 2333925DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 339atcacaagca ttcttataca ccaac 2534021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 340tttgcaatag gtgtggtgat g 2134124DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 341tcaacattag acagatcaac gaga 2434221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 342ggtaccagta ccatgctgtt t 2134323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 343gctaccaatg actttcttca cag 2334422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 344atggaagaac attccatgct ca 2234525DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 345tccaattctg tgaagaaagt cattg 2534624DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 346ttgtatcctg agactttgct gaag 2434721DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 347cttgaagagg tccttcacgt c 2134822DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 348gctcatggat aggaagaatc aa 2234921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 349aagcattccc tttgaaagct g 2135023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 350agacagatca acgagacaga aag 2335124DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 351ctgagttcta gtttgattgc actg 2435222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 352gtgtaaggaa gggatccagt tt 2235325DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 353tccaattctg tgaagaaagt cattg 2535422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 354tcacaagcat tcctatacac ca 2235522DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 355gctctatcct gttccattgg tc 2235622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 356tttgtagaat ctgcaggtgg at 2235724DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 357ctttgtgatg tttgcattca agtc 2435818DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 358ttcaggcctg tggtgaaa 1835922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 359actactttgg tacgtgtgtg tt 2236023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 360tgtgtgttct caactaacag agt 2336122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 361ttgtgatgtg tgcgttcaat tc 2236222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 362aagtagacag cagcattctc ag 2236321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 363gatggtgcag tttggaaaca c 2136425DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 364ctcagtgagt tgaacctttc tttag 2536523DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 365gtcacagagt agaatgttcc ctt 2336620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 366tgaggcctta gttggaaacg 2036725DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 367atgatgagtg tactcaaaga acaga 2536823DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 368ttgatacagc agtttggaaa cac 2336920DNAArtificial

SequenceDescription of Artificial Sequence Synthetic primer 369gcacacgtat gtttatcgca 2037018DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 370agacggagtc tcgctctg 1837125DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 371catgtgtctt tatagcagca tgatt 2537223DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 372gtgtccatgt gttctcattg ttc 2337325DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 373tcccacagaa taataatggg agaat 2537421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 374tggtaccagt tgttcctttc c 2137525DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 375ttgttgcgat agtttactga gaatg 2537623DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 376ctctatactt cccttctcgc ttc 2337725DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 377atccagtcta tcattgttgg acatt 2537818DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 378aggcatgggc aaggattt 1837920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 379tgctgaggga tctgctctta 2038021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 380gatcacgagg tcaggagatt g 2138121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 381ctgctataaa ggcacatgca c 2138222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 382gtggtctgag agacagtttg tt 2238321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 383acatacgtgt gcatgtgtct t 2138424DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 384tcaacatagt attggaagtt ctgg 2438519DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 385ggtcaggagt tcgagacca 1938623DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 386gtgtctgtgt agaaagaagt aga 2338723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 387gaaaccaaca agaacaaaga cac 2338828DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 388ctctgtttgt ctgttattgg tgtataag 2838924DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 389ttgttcttgt gatagtttgc tgag 2439024DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 390ataagttagg gaggattccc tctt 2439125DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 391gagagatcca ctgttagtct gatgg 2539217DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 392caggctggag tgcagtg 1739322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 393ttgggatata aacccaaagg at 2239422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 394tcaacttctt tgcctttggt tt 2239525DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 395aggtattgat gggatgtatc tcaaa 2539625DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 396attcctgagt tctagtttgg ttaca 2539724DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 397ctggtaccat tccttctgaa acta 2439822DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 398accgctagca agactaataa ag 2239921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 399tgacatgggt ctcctgaata c 2140022DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 400cctttgtgat gtgtgtgttc aa 2240122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 401cacccactcc agcatataaa ca 2240223DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 402cctccaagct atccaaatat cca 2340322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 403taccctttct tccagttgat cg 2240419DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 404ggatgcatgg ctggttcaa 1940525DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 405tgttgattgg aatagtttca gaagg 2540623DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 406tgcatatatt gaaccagcct ttc 2340728DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 407ggaaaccttc aaatctatcc aaatatcc 2840822DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 408tgggttgttt gtgcattgat tt 2240921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 409ctctcagacc acagtgcaat c 2141022DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 410tggtttgaaa gtcctcctgt ag 2241120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 411ccatgatcaa gtgggcttca 2041223DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 412gaagcaattg tgaatgggag ttt 2341322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 413gagttaggga ggattccctc tt 2241422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 414ttggtttggt ttgccagtat tt 2241525DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 415ctgtttacat gctggattac gttta 2541622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 416ctcggagtag tttgatcgtc tg 2241720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 417ctctgcctgg ctttggtatc 2041818DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 418ctggagtgga cctccaaa 1841924DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 419acttagataa ccacagccta agaa 2442022DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 420ggatggaatg cccaacaaat ac 2242124DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 421gcagcctcta ctattcctta cttt 2442223DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 422gcttcaaatg ccactttgtt tac 2342325DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 423cagtcctaaa gagggagaca ttatt 2542421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 424ttgtccttgc aatagtttgc t 2142522DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 425cctataatcc cagcactttg gg 2242622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 426tagttcaacc agtgtggaag ac 2242721DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 427cacgtatgtt tattgcggca t 2142822DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 428tctgttgatt tggtgtggag ag 2242921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 429agacaagcaa atgctgagag a 2143021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 430tgctgctcta aagacacatg c 2143120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 431aaccaaggct cgagaactac 2043221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 432tttacactgt tggtgggact g 2143325DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 433ttgtttgagt tcattgtaga ttccg 2543422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 434ggagaatgga accaagttgg aa 2243519DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 435acggagtctc agtctgtcg 1943623DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 436gtccttgcga tagtttactg aga 2343722DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 437gacagatcca tgagacagaa gc 2243821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 438aaacaacaga tgctggagag g 2143922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 439gtatcctgag actttgctga ag 2244017DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 440caggctggag tgcagtg 1744120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 441gggtaaggtc acagatcaac 2044222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 442aattgtgatg ttagggtgtc aa 2244318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 443ctggcagaag acagcgat 1844426DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 444gagacattac tgacaatagc aaagac 2644525DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 445tggataaatt cctcgacaca tacac 2544621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 446caatctagca aggcaggcca a 2144717DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 447tggtggacgc ctgtagt 1744825DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 448tttgtccttg caatacttta ctgag 2544923DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 449aacccatctg tacatcatca tca 2345023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 450ccctttattt ctttctcctg cct 2345122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 451gtatcctgag actgtgctga ag 2245221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 452gcgtatattg aaccagcctt g 2145319DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 453cccaatttca gctcctgtt 1945418DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 454agtaccagcc actgcaaa 1845520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 455cgtggaattt gcaagtggag 2045626DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 456gaataagagt ggtaagagag gacatc 2645725DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 457tcacagaata gaatgttccc tgtta 2545818DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 458aacgcagttc ctcaccag 1845924DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 459gtgtgggttt gtcatagata gctc 2446025DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 460ctagacatgt acactctctc aagac 2546125DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 461ggtaccattc cttctgaaac tattc 2546220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 462tatctgcaag cggacgtttc 2046321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 463ggatgcaagg ctggttcaat a 2146425DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 464ttagatcttt cctgctttct cttgt 2546523DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 465catcaccatc atcaaagacc aaa 2346625DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 466tttggtgtgg gtttgtcata aatag 2546722DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 467aggaactcaa attgtccctg tt 2246817DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 468gtctctgccc ggctttg 1746923DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 469gtatattgaa ccagccttgc atc 2347019DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 470ccctttcttg tgccagttt 1947120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 471tgagggtcct gtctgttaga 2047222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 472agaatgatgc tggcctcata aa 2247319DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 473taacagtcag gaccctcag 1947420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 474cccggcagac caatagttta 2047524DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 475accactgtat acttaatgcc tgag 2447622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 476gtggagtgtg tggtatgtgt ag 2247724DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 477aggtaaattg aattaaggct cctg 2447821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 478tcacaatcaa ggaccgctat g 21

Claims

1. A method comprising administering to a human identified as having sepsis a therapeutically effective amount of at least one agent used to treat sepsis, wherein the human is identified as having sepsis by analyzing a biological sample isolated from the human for over-representation or under-representation of at least one polynucleotide relative to an internal reference region, wherein the at least one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148 and wherein the over-representation or under-representation of the at least one polynucleotide in the biological sample is indicative of sepsis, thereby identifying the human as having sepsis.

2. The method of claim 1, wherein the at least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 over-represented or under-represented relative to the internal reference region is at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, or at least twenty-four of the polynucleotides comprising any one of SEQ ID NOs: 1-57 or 94-148.

3. The method of claim 1, wherein the biological sample is blood, a product derived from blood, or a fraction derived from blood.

4. The method of claim 1, wherein the internal reference region comprises at least one polynucleotide comprising at least one of SEQ ID NOs: 59, 61, or 68.

5. The method of claim 1, wherein detecting the over-representation or under-representation of the at least one polynucleotide relative to an internal reference region comprises at least one of a polymerase chain reaction (PCR)-based detection method, a hybridization-based method, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), solid-phase enzyme immunoassay (EIA), mass spectrometry, or microarray analysis.

6. The method of claim 5, wherein the PCR-based detection method is performed using at least one primer pair, wherein each primer pair of the at least one primer pair is specific for any one of SEQ ID NOs: 1-57 or 94-148, and a primer pair specific for at least one of SEQ ID NOs: 59, 61, or 68.

7. The method of claim 6, wherein the primer pair specific for any one of SEQ ID NOs: 1-57 or 94-148 and the primer pair specific for at least one of SEQ ID NOs: 59, 61, or 68 is any one of the primer pairs presented in Tables 1-3.

8. The method of claim 7, further comprising sequencing amplification products corresponding to any one of SEQ ID NOs: 1-57 or 94-148 at least one of SEQ ID NOs: 59, 61, or 68 generated by the PCR-based detection method.

9. The method of claim 1, wherein the at least one polynucleotides comprise circulating nucleic acids.

10. The method of claim 1, wherein the at least one agent used to treat sepsis comprises at least one of an antibiotic, anti-fungal agent, anti-viral agent, anti-parasitic agent, or fluids suitable for intravenous administration.

11. A primer comprising a manmade nucleotide sequence that binds specifically to a polynucleotide comprising any one of the SEQ ID NOs: listed in Table 4 and at least one manmade tag conjugated thereto, wherein the manmade nucleotide sequence is any one of the polynucleotide primer sequences listed in Tables 1-3 or a variant thereof.

12. The primer of claim 11, wherein the variant of any one of the polynucleotide primer sequences listed in Tables 1-3 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polynucleotide sequences listed in Table 4.

13. The primer of claim 11, wherein the manmade tag is a detectable marker.

14. The primer of claim 11, the primer consisting essentially of or consisting of a manmade nucleotide sequence that binds specifically to a polynucleotide of any one of the SEQ ID NOs: listed in Table 4 and at least one manmade tag conjugated thereto, wherein the manmade nucleotide sequence is any one of the polynucleotide primer sequences listed in Tables 1-3 or a variant thereof and at least one manmade tag conjugated thereto, wherein the manmade nucleotide sequence is any one of the polynucleotide primer sequences listed in Tables 1-3 or a variant thereof.

15. The polynucleotide primer sequence of claim 14, wherein the variant of any one of the polynucleotide primer sequences listed in Tables 1-3 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polynucleotide primer sequences listed in Tables 1-3.

16. The primer of claim 14, wherein the manmade tag is a detectable marker.

17. A kit for detecting sepsis in a human comprising at least one primer pair of claim 11 and instructions for use thereof.

18. The kit of claim 17, wherein the at least one primer pair comprises four primer pairs and wherein each primer pair of the four primer pairs specifically amplifies a different polynucleotide comprising any one of the SEQ ID NOs: listed in Table 4.