Cell-free nucleic acids for the analysis of the human microbiome and its components

Abstract

To provide a method, a device, a composition, and a kit for the analysis of a microbiome in an individual or each component of the microbiome.SOLUTION: A method for determining the presence and the possession rate of a microorganism sequence in a sample of cell-free nucleic acid from a non-microorganism host has: (i) preparing a sample of cell-free nucleic acid from an individual, (ii) performing determination of high throughput sequence of the nucleic acid, (iii) performing bioinformatics analysis and subtracting a host sequence from the analysis, and (iv) determining the presence and the possession rate of a microorganism sequence for microbiome evaluation of the non-microorganism host.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to cell-free nucleic acids for the analysis of the human microbiome and its components.

[0002] Government Rights This invention is based on the findings of the National Institutes of Health (NIH). U.S. Government support under Grant No. RC4AI092673 awarded by the National Institute of Health The U.S. Government has certain rights in this invention. [Background technology]

[0003] The human microbiome is now a critical component of human health. Community-level analyses have been conducted based on age, diet, geographic location, antibiotic resistance, and other factors. Shape the structure of the bacterial and viral components of the microbiome, as well as quality treatment and disease For example, individual microbiomes are altered by infection with pathogenic organisms. This may result in an increased prevalence of the microorganism systemically or in undesirable tissues. The microbiome can also be altered by changes in an individual's immune competence.

[0004] For various purposes, specific microbiome components in individual microbiomes, e.g. Rapid detection of the presence and prevalence of commensal, mutualistic, parasitic, opportunistic and pathogenic organisms It would be desirable to have methods for the analysis of specific as well as global microbiome structure. The present invention provides a method for monitoring microbiome composition in clinical samples. It provides a highly sensitive, rapid and non-invasive method. Summary of the Invention

[0005] Overview of the invention The present invention provides methods, devices and methods for the analysis of the microbiome or individual components thereof in an individual. The present invention provides a method for determining whether a microbiome structure is present in a patient, a composition, and a kit. These are useful in analyzing the immune system, determining the immune competence of an individual, and the like. Specifically, the present invention provides a method for (i) extracting cell-free nucleic acids, i.e., DNA and / or RNA, from an individual. preparing the sample; and (ii) high-throughput sequencing, e.g., about 10 5 ~about 10 9 pieces or more reads, and (iii) bioinformatics A cross-sectional analysis is performed to subtract the host sequence (i.e., human, feline, canine, etc.) from the analysis. (iv) Coverage of sequences mapped to, for example, a microbial reference sequence The presence of microbial sequences is determined by comparing the coverage of the host reference sequence with that of the host. and determining the presence and prevalence of a microorganism in an individual, including determining the presence and prevalence of a microorganism in an individual. This provides a method for

[0006] Subtraction of host sequences is performed by subtracting the reference host sequence from the Identifying microbial sequences and masking microbial or microbial-mimicking sequences present in the reference host genome Similarly, determining the presence of a microbial sequence by comparison with a microbial reference sequence may involve the steps of: Identify microbial sequences and mask host or host-mimicking sequences present in the reference microbial genome. The method may include a step of checking.

[0007] A feature of the present invention is the unbiased analysis of cell-free nucleic acids from an individual. The methods of the present invention generally include: For example, by performing PCR with universal primers or by activating the nucleic acid. Unbiased amplification by ligating an adapter and amplifying with primers specific to the adapter The methods of the present invention are typically carried out in the absence of sequence-specific amplification of the microbial sequence. The advantage of this approach is that the analysis includes all available microbiome sequences. and it is important to identify sequences of interest in complex datasets where host sequences are dominant. This requires bioinformatics analysis.

[0008] Another advantage of the present invention is that it can provide a rapid assessment of an individual's microbiome. For example, the analysis may be performed in less than about 3 days, less than about 2 days, less than 1 day, e.g., less than about 24 hours, less than about 2 Less than 0 hours, Less than approximately 18 hours, Less than approximately 14 hours, Less than approximately 12 hours, Less than approximately 6 hours, Approximately 2 hours The process may be completed in less than about 10 minutes, less than about 30 minutes, less than about 15 minutes, or less than about 1 minute.

[0009] In some embodiments, analysis of cell-free nucleic acid is used to calculate a pathogenicity score. wherein the pathogenicity score is a measure of the pathogenicity of the organism to facilitate interpretation by, for example, a medical practitioner. It is a numerical or alphabetical value that summarizes the overall pathogenicity of the microorganisms present in the microbiome. Different microorganisms can be assigned different scores.

[0010] Analysis of the presence and prevalence of microbial sequences can be used to treat antimicrobial therapies, e.g., antibiotics, antivirals, Response to treatments, including drugs, immunization, and passive immunotherapy, diet, and immunosuppression; clinical trials The information obtained from the analysis can be used to determine the response, infection, etc. to diagnose conditions, to monitor treatment, to select or modify treatment regimens, This approach can be used to improve the quality of treatment and to optimize treatment. Treatment and / or diagnostic regimens may be tailored according to specific data obtained at various time points over time. can be individualized and adapted, thereby obtaining individually tailored regimens. For analysis, patient samples are also available for testing against pathogens during the course of treatment. They can be obtained at any time after exposure, during the course of infection, etc. Presence and prevalence of microbial sequences The analysis may be provided as a report, which may be provided to an individual, a medical professional, or the like.

[0011] In some embodiments, the cell-free nucleic acid is obtained from blood, serum, cerebrospinal fluid, synovial fluid, urine, and The nucleic acid is obtained from a biological sample selected from the group consisting of feces and the like. Extracts from cellular fractions, for example, the serum or plasma fraction of blood, can be used. In embodiments, the nucleic acid is double-stranded DNA, single-stranded DNA, a single-stranded DNA hairpin, a D Consists of NA / RNA hybrids, single-stranded RNA, double-stranded RNA, and RNA hairpins In some embodiments, the nucleic acid is selected from the group consisting of double-stranded DNA, single-stranded DNA, In some embodiments, the nucleic acid is selected from the group consisting of mA and cDNA. In some embodiments, the nucleic acid is circulating cell-free DNA.

[0012] In some embodiments, the method determines the prevalence of a microorganism in a sample. In some embodiments, the method includes quantifying one or more nucleic acids to detect a nucleic acid that exceeds a predetermined threshold. The amount of one or more nucleic acids detected is indicative of infection or a change in prevalence. There is a predetermined threshold value that varies depending on the microorganism. Temporal differences in nucleic acid abundance are indicators of changes in infection, prevalence, and treatment response. become.

[0013] In some embodiments, the present invention provides a computer-readable medium, comprising: ) a high-throughput assay from one or more nucleic acids detected in a sample of cell-free nucleic acid from a subject; (ii) receive the put sequencing data and perform bioinformatics analysis to identify the (iii) subtracting host sequences (i.e., human, feline, canine, etc.) from the analysis, e.g., By comparing the coverage of sequences mapped to the organism reference sequence with the coverage of the host reference sequence. and configuring the computer to perform the steps of determining the presence and prevalence of microbial sequences. The program includes a set of instructions recorded on a computer-readable medium.

[0014] In some embodiments, the present invention provides a method for treating a cancer by performing one or more of the methods described herein. The present invention provides reagents and kits for carrying out the method.

[0015] In some embodiments, analysis of the microbiome, e.g., the virome, e; in vivo viral population) for the evaluation of the immune competence of individuals, especially individual humans. Compositions and methods are provided. In some embodiments of the present invention, immunosuppressive regimens In some embodiments, the individual is treated with, for example, drugs, radiation therapy, etc. In some embodiments, the patient is a transplant recipient treated with an immunosuppressive regimen. In another embodiment, the individual has an autoimmune disease that is being treated with an autoimmune regimen. assesses individuals for immune competence in the absence of an immunosuppressive regimen.

[0016] In some embodiments, measurements are taken from an individual at two or more time points, where the virus Changes in immune load are indicative of changes in immune competence. Individuals can be treated according to their immune competence assessment. For example, in this case, the indications for undesired enhanced immune function in transplant patients are increasing. The undesired reduction in immune function may be treated with a therapeutic agent, e.g., It is treated with antiviral drugs.

[0017] Nucleic acid analysis was performed to identify and quantify non-human cell-free nucleic acids in samples collected from patients. The microbiome components are constructed as described above. The structure of the viral components (virome) allows for prediction of immunocompetence. In such cases, the method may further comprise administering the immunosuppressant prior to, at the initiation of, or during an immunosuppressive regimen. This involves establishing a virome profile during the course of a therapeutic regimen, which is specific to each individual virus. In some embodiments, circulating acellular leukemia (CLE) is used as a measure of changes in CLE. The cytoplasmic DNA is annellovirus DNA.

[0018] In particular, the Anelloviridae virus load is a predictor of immune strength, which may contribute to organ transplantation. Other viruses are predictable, but patients should be aware that they may be more susceptible to such viruses. It is typically treated with antiviral drugs that affect the viral load.

[0019] In some embodiments, the present invention provides a method for detecting a graft from a donor, the method comprising: (i) detecting a graft from a subject receiving a transplant from a donor; providing a sample; (ii) determining the presence or absence of one or more virome nucleic acids; ii) diagnosing or predicting transplant status or outcome based on virome burden; Methods for diagnosing or predicting transplant status or outcome are provided. , transplant status or outcome based on rejection, tolerance, non-rejection, allograft injury, graft function, graft Some studies have shown that pharmacologic immunosuppression may improve survival, chronic graft injury, or titer. In an embodiment, the amount of one or more nucleic acids above a predetermined threshold is an indicator of viral load and immune competence. In some embodiments, the threshold is a threshold that indicates evidence of transplant rejection or other pathology. In some embodiments, the IL-10 expression level is a normative value for a clinically stable post-transplant patient without IL-10. There are predetermined thresholds that vary depending on the outcome or condition of the transplant. Temporal differences in the amount of one or more nucleic acids are indicative of immune competence.

[0020] In any of the embodiments described herein, the graft may be any solid organ, bone, or In some embodiments, the transplant may be a kidney transplant, a heart transplant, or a skin graft. transplantation, liver transplantation, pancreas transplantation, lung transplantation, intestinal transplantation and skin transplantation.

[0021] In some embodiments, the present invention provides a method for treating a cancer by performing one or more of the methods described herein. The present invention provides reagents and kits for carrying out the method.

[0022] All publications and patent applications mentioned herein are to be construed as a separate entity, independent of the individual publication or patent issuer. as if the application were specifically and individually indicated to be incorporated by reference herein. , which is incorporated herein by reference.

[0023] The novel features of the invention are set forth with particularity in the appended claims. A further understanding of the invention and its advantages will be apparent from the following description of exemplary embodiments in which the principles of the invention are utilized. This can be obtained by reference to the following detailed description and the accompanying drawings, which are described below.

[0024] Detailed Description of the Invention Particularly preferred embodiments of the present invention will be described in detail below. Specific examples of preferred embodiments are: Illustrated in the Examples section below.

[0025] Unless otherwise defined, all technical and scientific terms used herein are intended to be limiting of the present invention. It has the same meaning as commonly understood by those skilled in the art. All patents and publications cited herein are incorporated by reference in their entirety.

[0026] When a range of values ​​is given, there shall be a clear distinction between the upper and lower limits of the range, unless the context clearly contradicts this. Each intervening value to one-tenth of the unit of the lower limit unless otherwise specified, and any value within that range It is understood that other stated or intervening values ​​are included in the present invention. The upper and lower limits of a smaller range may independently be included in the smaller range. and subject to any specifically excluded limits within the ranges shown. The same is encompassed by the present invention when the stated range includes one or both of its limits. , ranges excluding either or both of those inclusive limits are also included in the invention.

[0027] In this specification, ranges are expressed by numerical values ​​preceded by the word "about." The term "number" as used herein refers to the exact number that follows it, as well as to the number that is near or close to the number that follows it. Used to indicate the literal correspondence of similar numbers. When determining whether a number is close or approximate, A number, in the context in which it appears, represents the substantial equivalent of the number specifically recited. It can be a number indicating.

[0028] The practice of the present invention may involve any of the techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, and the like, unless otherwise indicated. conventional techniques of biology, cell biology, genomics and recombinant DNA, which are within the skill of the art. Within the skill of the art. Sambrook, Fritsch, and Maniatis ,MOLECULAR CLONING:A LABORATORY MANUAL,2 nd edition(1989);CURRENT PROTOCOLS IN MO LECULAR BIOLOGY (eds. FMAusubel et al. (1987)); MET HODS IN ENZYMOLOGY series (Academic Press, I nc.): PCR 2:A PRACTICAL APPROACH(MJMacP Hamerson, B.D. Hames, and G.R. Taylor (eds., 1995), Ha Rharrow and Lane (eds.) (1988) ANTIBODIES, A LABORATOR Y MANUAL, and ANIMAL CELL CULTURE (RIFre See Shney (ed.) (1987).

[0029] The present invention provides methods, devices and methods for the analysis of the microbiome or individual components thereof in an individual. The present invention provides a method for determining whether a microbiome structure is present in a patient, a composition, and a kit. These are useful in analyzing the immune system, determining the immune competence of an individual, and the like. Thus, the present invention provides a method for determining whether a patient or subject is immunocompetent. As used herein, the terms "individual," "patient," or "subject" refer to humans and other Includes mammals.

[0030] definition As used herein, the terms "diagnosis" or "diagnosing" a condition or outcome refers to the diagnosis of a condition or outcome. predicting or diagnosing a condition or outcome, determining a predisposition to a condition or outcome, to monitor the treatment of a patient, to diagnose a patient's treatment response, condition or outcome, progression, and prognosis of response to specific treatments.

[0031] Microbiota. As used herein, the term microbiota refers to the microorganisms of an individual (usually an individual mammal, e.g., The term "microbiota" refers to the collection of microorganisms present within a given organism (usually a human individual). The microbiota includes pathogenic species; It constitutes the normal flora of certain tissues, such as the skin and oral cavity, but it also occurs in other tissues, such as the blood and lungs. species that are undesirable in the environment; commensal organisms that are found in the absence of disease, etc. One subset of the lobiome is the virome, which is the microbiome Contains the virus component of the virus.

[0032] As used herein, the term "microbiome components" refers to individual strains or species. The component may be a viral component, a bacterial component, a fungal component, or the like.

[0033] In healthy animals, internal tissues such as the brain and muscles are usually relatively free of bacterial species. However, surface tissues, i.e., skin and mucous membranes, are in constant contact with environmental organisms. and is easily colonized by a variety of microbial species. The mixture of organisms known or suspected to be found in humans is called the resident microbiota. It includes various components of the resident microbiota. In addition to the resident microorganisms, pathogenic or opportunistic There are various transient components such as infection. Reference sequences for the following microorganisms are available from, for example, Genba. nk database and are publicly available and known.

[0034] The human gut microbiota is dominated by species found within two bacterial phyla: Bacteria Bacteroidetes and Firmicutes Members of the Actinobacteria (S) make up over 90% of bacterial populations. Actinobacteria (e.g., Bifidobacterium members of the genus Proteobacteria (Proteobacteria) The common species of interest are the prominent or It encompasses less abundant members, including, but not limited to, Bacteroides species Bacteroides thetaiotaomicron, Bacteroides caccae, Bacteroides Bacteroides fragilis, Bacteroides melanino Bacteroides melaninogenicus, Bacteroides Bacteroides oralis, Bacteroides uniformis Bacteroides uniformis, Lactobacillus acillus), Clostridium perfringens (Clostridium perfringens, Clostridium septicum septicum), Clostridium tetani i), Bifidobacterium bifidum (Bifidobacterium bifi dum), Staphylococcus aureus us), Enterococcus faecalis s), Escherichia coli, Salmonella enterica Salmonella enteritidis, Klebsiella species ( Klebsiella sp., Enterobacter spp. sp.), Proteus mirabilis, Pseudomonas Pseudomonas aeruginosa, Peptost Leptococcus species (Peptostreptococcus sp.), Peptococcus Peptococcus sp., Faecalibacterium sp. ibacterium sp., Roseburia sp., Ruminococcus sp., Dorea s p.), Alistipes sp., etc.

[0035] In the skin microbiome, most bacteria fall into four distinct phyla: Actinobacteria, Firmicutes utes, Bacteroidetes and Proteobacteria Proteobacteria. Microorganisms commonly considered to be skin colonizers include coliforms, Actinobacteria (Corynebacteria) Genus Corynebacterium, Propionibacterium Propionibacterium genus, e.g. Propionibacterium acnes (Propion acnes) and Brevibacterium erium], Micrococcus and Staphylococcus Staphylococcus spp. are the most commonly isolated Examples of fungal species that can cause this include Malassezia spp. , which is especially prevalent in sebaceous areas. Demodex mite [For example, Demodex folliculorum ) and Demodex brevis may also be present. Other types of fungi thought to grow on the skin include Debaryomyces yomyces and Cryptococcus spp. As for non-symbionts, burns commonly contain Streptococcus pyogenes (S pyogenes), Enterococcus spp. or Pseudomonas aeruginosa ) and may also be infected by fungi and / or viruses. S. epidermidis is a very common skin commensal, but it It is also the most frequent cause of hospital-acquired infections in indwelling medical devices such as catheters or heart valves. One theory is Nat Rev Microbiol.(2011)Apr;9(4):2 See pp. 44-53.

[0036] Pathogenic species can be bacteria, viruses, protozoan parasites, or fungal species. Bacteria include Brucella Brucella sp., Treponema sp., Mycobacterium sp., Listeria sp. steria sp.), Legionella sp., Helicobacter Helicobacter sp., Streptococcus sp. tococcus sp), Neisseria sp, Clostridium Clostridium sp., Staphylococcus sp. lococcus sp. or Bacillus sp. , including but not limited to Treponema pallidum dum), Mycobacterium tuberculosis (Mycobacterium tu berculosis), Mycobacterium leprae leprae), Listeria monocytogenes (Listeria monocytog enes), Legionella pneumophila la), Helicobacter pylori, streptococcus aureus Streptococcus pneumoniae, Neisseria meningitis, clostridium Clostridium novyi, Clostridium botulinum Clostridium botulinum, Staphylococcus aureus (Staphylococcus aureus), Bacillus anthracis (Baci Illus anthracis) and others.

[0037] Protozoan parasites include Trichomonas and Toxoplasma plasma), Giardia, Cryptosporidium sporidium, Plasmodium, Leishmania (L eishmania), Trypanosoma, Entamoeba ( Entamoeba, Schistosoma, Filariae ariae, Ascaria, and Fasciola Rarely, but not exclusively, Trichomonas vaginalis vaginalis), Toxoplasma gondii ), Giardia intestinalis, Cryptosporidium parva, plus Plasmodium falciparum, trypanosomiasis Trypanosoma cruzi, Entamoeba histolytica Chika (Entamoeba histolytica), Giardia lamblia (Gi ardia lamblia, Fasciola hepatica tica) and others.

[0038] Viruses that infect humans include, for example, adeno-associated viruses, bat lyssavirus, Australian bat lyssavirus, BK polyomavirus, Nna virus, Barma Forest virus, Bunyamwera virus, Lacrosse Bunyau virus Bunyavirus La Crosse, snowshoe rabbit bunyavirus (Bunyavirus snowshoe hare), Cercopithecine herpesvirus , Chandipura virus, Chikungunya virus, Cosavirus ) A, cowpox virus, coxsackievirus, Crimean-Congo hemorrhagic fever virus, dengue fever virus, Dori virus, Djugbe virus, Dubenhage virus, Eastern equine brain Viruses such as encephalomyocarditis, Ebola, Echovirus, Encephalomyocarditis, and Epstein-Barr Virus, European bat lyssavirus, GB virus C / G hepatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hendra virus Hepatitis E virus, Hepatitis Delta virus, Horsepox virus, Human adenovirus, Human astrovirus, human coronavirus, human cytomegalovirus, human enterovirus virus 68, 70, human herpesvirus 1, human herpesvirus 2, human herpesvirus virus 6, human herpesvirus 7, human herpesvirus 8, human immunodeficiency virus, Human papillomavirus 1, human papillomavirus 2, human papillomavirus 16, 18. Human parainfluenza, human parvovirus B19, human respiratory syncytial virus , human rhinovirus, human SARS coronavirus, human spumaretrovirus, human Human T-lymphotropic virus, human tyrosine kinase virus, influenza A virus, influenza B virus Influenza virus, influenza C virus, Isfahan virus, JC polio encephalitis virus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, jinjin virus, Lagos bat virus, Lake Victoria Marburg virus, Langa Viruses: Lassa virus, Lordsdale virus s), louping disease virus, lymphocytic choriomeningitis virus, Machupo virus, Mayalouy Rus, MERS coronavirus, measles virus, Mengo encephalomyocarditis virus, Merkel cells Polyomavirus, Mokola virus, Molluscum contagiosum virus, Monkeypox virus, Mumps encephalitis virus, Murray Valley encephalitis virus, New York virus, Nipah virus, and Nowo virus ark virus, Onyongnyong virus, Orf virus, Oropausche virus, Pichi Viruses, Poliovirus, Punta Toro Phlebovirus phlebovirus), Puumala virus, rabies virus, Rift Valley fever virus Rus, Rosavirus A, Ross River virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus virus), Salivirus A, sandfly fever, Sicilian Virus, Sapporo virus, Semliki Forest virus, Seoul virus, Simian foamy virus s, Simian virus 5, Sindbis virus, Southampton virus, St. Louis virus Encephalitis virus, tick-borne Powassan virus, Torque tenosynovial virus, Tuscany virus Ukuniemi virus, vaccinia virus, varicella-zoster virus, smallpox virus Venezuelan equine encephalitis virus, vesicular stomatitis virus, Western equine encephalitis virus, WU Polyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, Yellow Fever virus, Zika virus.

[0039] Anelloviridae. The Anelloviridae family consists of non-enveloped circular single-stranded DNA viruses. Three genera of anelloviruses (designated TTV, TTMDV, and TTMV) are infectious to humans. It is known to stain.

[0040] Torque teno virus (TTV) is a non-enveloped single-stranded D virus with a circular negative-sense genome. NA virus. Torque Teno-like minivirus He also characterized a smaller virus, later named Thyroid Toxic Mini Virus (TTMV). A third strain, with a genome size intermediate between that of TTV and TTMV, has been identified. A virus was discovered and named Torque Teno-like midivirus It was later named the Toxic Midi Virus (TTMV). Recent changes in nomenclature include These three anelloviruses that can infect humans are classified as alphaviruses of the Anelloviridae family. Alphatorquevirus (TTV) Betatorquevirus (TTMV) and gamma torque virus (G Currently, it is classified into the genus A. ammatorquevirus (TTMDV). Neroviruses are still "orphan" viruses waiting to be linked to human disease. It is considered to be

[0041] Human anelloviruses are 3.8-3.9 kb for TTV and 3.2 kb for TTMDV. and TTMV differs in genome size, which ranges from 2.8 to 2.9 kb. A distinctive feature of anelloviruses is the extreme diversity found both within and between anellovirus species. They are highly diverse and can show 33% to 50% divergence at the nucleotide level. Despite the diversity of nucleic acid sequences, anelloviruses share a common virion structure and gene. Conserved genome organization, transcriptional profile, and non-coding GC-rich regions confer gene function They share common regions and sequence motifs.

[0042] Anellovirus infections are highly prevalent in the general population. Between 75 and 100% of patients tested are infected with at least one of the three human anelloviruses. Anelloviruses are common in young children. The earliest documented infection occurred within the first month of life. The virus was detected in plasma, serum, peripheral blood mononuclear cells (PBMC), nasopharyngeal aspirate, bone marrow, saliva, and maternal Milk, feces, and various tissues including the thyroid, lymph nodes, lungs, liver, spleen, pancreas, and kidneys It has been found in nearly every body site, fluid, and tissue tested, including tissues. The replication kinetics of Neroviruses is essentially mediated by the inability of these viruses to replicate in culture. Positive-strand TTV DNA, an indicator of local viral replication, is expressed in hepatocytes and bone marrow cells. , has been described in circulating PBMCs.

[0043] Anelloviruses are primarily transmitted via feces, although maternal-fetal and respiratory tract infections have also been reported. It is spread by oral transmission. There are conflicting reports regarding the presence of TTV in cord blood samples. exist.

[0044] Anellovirus reference sequences are available in Genbank, e.g., as follows: Available: Torque teno minivirus 1, Accession: NC 014097.1; Torque tenominivirus 6, accession: NC 014095.1; Torquetenomide Ivirus 2, Accession: NC 014093.1; Torque tenomidi virus 1 ,Accession:NC 009225.1; Torque teno virus 3, Accession: NC 014081.1; Torque teno virus 19, Accession: NC 01407 8.1; Torque tenominivirus 8, Accession: NC 014068.1.

[0045] As used herein, the term "antibiotic" includes all commonly used bacteriostatic and bactericidal Antibacterial antibiotics, including those usually administered orally. Antibiotics include: Included: Aminoglycosides, e.g., amikacin, gentamicin, kanamycin, neomycin isin, streptomycin and tobramycin; cephalosporins, such as Cefama Andole, cefazolin, cephalexin, cephaloglycin, cephaloridine, cepha cephapirin and cephradine; macrolides such as erythromycin and Troleandomycin; penicillins, e.g. penicillin G, amoxicillin, ampicillin carbenicillin, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, feneticillin and ticarcillin; polypeptide antibiotics, such as basit cyclosporin, colistimethate, colistin, polymyxin B; tetracyclines, e.g. Lortetracycline, demeclocycline, doxycycline, methacycline, minocycline cycline, tetracycline, and oxytetracycline; and other miscellaneous antibiotics Substances such as chloramphenicol, clindamycin, cycloserine, lincomycin rifampin, spectinomycin, vancomycin, and viromycin. The antibiotics are listed in "Remington's Pharmaceutical Sciences" es,”16th Ed.,(Mack Pub.Co.,1980),pp.1121 -1178.

[0046] Antiviral Agents. Individuals may receive antiviral therapy. This may include those affected by the therapy. Examples of viral infections that can be treated in this way include: These include: HIV, Bowenoid papulosis, chickenpox, childhood HIV disease, human-bovine Pox, Hepatitis C, Dengue fever, Enterovirus, Epidermodysplasia verruciformis, Erythema infectiosum (5th disease), Buschke-Levenstein giant condyloma acuminata, hand, foot and mouth disease, herpes simplex, Herpes virus 6, shingles, Kaposi's varicelliform rash, measles, milker's nodule, molluscum contagiosum , monkeypox, Orf, infantile roseola, rubella, smallpox, viral hemorrhagic fever, genital warts and non-sexual Vessel warts.

[0047] Antiviral agents include: azidouridine ), anasmycin, amantadine, Bromovinyldeoxusidine, chloro Chlorovinyldeoxusidine, cytalbine (cytarbine), didanosine, deoxynojirimycin deoxyjirimycin, dideoxycytidine idine), dideoxyinosine, dideoxynucleic acid dideoxynucleoside, desciclovir vir), deoxyacyclovir, edoxuidine ( edoxuidine), enviroxime, fiacitabine ( fiacitabine), foscamet, fialuridine ialuridine, fluorothymidine, Floxuridine, hypericin, interleukin -feron, interleukin, isethionate, nevirapine nevirapine, pentamidine, ribavirin ribavirin), rimantadine, citavirdine avirdine, sargramostin, suramin ramin), trichosanthin, tribromothymidine ( tribromothymidine, trichlorothymidine ymidine, vidarabine, zidoviridin ridine, zalcitabine and 3-azido-3-deoxy-2-methylpropional oxythymidine and their analogs, derivatives, pharmaceutically acceptable salts, esters, prodrugs Drugs, codrugs and protected forms.

[0048] As used herein, immunosuppression or immunosuppressive regimen refers to immunosuppression against self-antigens or grafts. The present invention relates to the treatment of an individual, e.g., a transplant recipient, with a substance that reduces the immune response of the host immune system to the immune system. Exemplary immunosuppressive regimens are described in more detail herein.

[0049] The main immunosuppressants include calcineurin, which binds to binding proteins and inhibits calcineurin activity. Sineurin inhibitors include, for example, tacrolimus cyclosporine A, etc. Both cyclosporine and tacrolimus Your levels should be monitored carefully. Initially, your levels should be between 10 and 20 ng. / mL range, but after 3 months, to reduce the risk of nephrotoxicity, Therefore, levels can be kept lower (5-10 ng / mL).

[0050] Adjunctive medications are usually combined with calcineurin inhibitors, such as steroids, azathioprine, and azathioprine, mycophenolate mofetil These include ate mofetil and sirolimus. One protocol involves calcineurin inhibitors in combination with mycophenolate mofetil. The use of adjuvants allows clinicians to achieve adequate immunosuppression while maintaining individual This allows for a reduction in the dose and toxicity of the substance. Several clinical trials have demonstrated the efficacy of azathioprine. Significantly reduced incidence of acute cellular rejection and reduction in one-year treatment failure compared to placebo After showing that mycophenolate mofetil is effective in reducing immunosuppression in kidney transplant recipients, It plays an important role in

[0051] Antibody-based therapies include monoclonal (e.g., muromonab-C D3) or polyclonal antibodies or anti-CD25 antibodies (e.g., basiliximab (b asiliximab, daclizumab) can be used. Antibody-based therapy is effective in treating carcinoma and is administered early (up to 8 weeks) after transplantation. Allows avoidance or dose reduction of urinary inhibitors, possibly reducing the risk of nephrotoxicity The side effect profiles of polyclonal and monoclonal antibodies vary in some patients. Restrict their use in

[0052] As used herein, the term "nucleic acid" refers to a polynucleotide containing two or more nucleotides. It can be DNA or RNA. A "mutant" nucleic acid is one that has been modified (e.g. at least one nucleotide (e.g., deleted, inserted, or substituted, respectively) A polynucleotide having a nucleotide sequence identical to that of the original nucleic acid except that it contains a nucleotide sequence The variant is a nucleotide sequence that is at least about 80% different from the nucleotide sequence of the original nucleic acid. %, 90%, 95% or 99% identical nucleotide sequence.

[0053] Circulating, or cell-free, DNA was first detected in human plasma in 1948 (M andel,P.Metais,P.,CR Acad.Sci.Paris,142 , 241-243 (1948)). Since then, its association with disease has been recognized in several areas. and established (Tong, Y.K.Lo, Y.M., Clin Chim Acta, 3 63, 187-196 (2006)). Research has shown that circulating nucleic acids in the blood Most arise from necrotic or apoptotic cells (Giacona, MB et al., Pan creas, 17, 89-97 (1998)), significant increase in the level of nucleic acids from apoptosis A significant increase is observed in diseases such as cancer (Giacona, MB et al., Pancreatic Cancer, 2014). eas,17,89-97(1998);Fournie, GJ et al., Cancer Lett, 91, 221-227 (1995)). In particular, in the case of cancer, circulating DNA Mutations in cancer genes, microsatellite alterations, and in some cancers, viral genomes The disease is characterized by the presence of DNA or RNA in the plasma that contains the latent sequence associated with the disease. For example, Diehl et al. Recently, quantitative assays for low levels of circulating tumor DNA in total circulating DNA have become clinically applicable. Compared with the standard biomarker used, carcinoembryonic antigen, showed that it can serve as a better marker for detecting cancer (Diehl, F. et al., Proc Natl Acad Sci, 102, 16368-16373(200 5); Diehl, F. et al., Nat Med, 14, 985-990 (2008)). M Aheswaran et al., Epidermal growth factor receptor agonists in lung cancer patients affecting drug treatment. Using genotyping of circulating cells in plasma to detect activating mutations in HIV-1 reported (Maheswaran, S. et al., N Engl J Med, 359, 366-377 (2008)). Collectively, these results highlight the potential for improved detection and treatment of cancer. This study establishes plasma-free circulating DNA as a useful species. It is also useful in healthy patients for fetal diagnosis, in which fetal D circulating in the maternal blood is detected. NA is a marker for sex, Rh (rhesus) D status, fetal aneuploidy, and sex-linked genetic disorders. Fan et al. recently performed a screening test on cell-free DNA extracted from maternal blood samples. We have demonstrated a method for detecting fetal aneuploidy by tumor sequencing. It can replace aggressive and risky techniques such as amniocentesis or chorionic villus sampling. (Fan, HC, Blumenfeld, YJ, Chitkara, U., H udgins, L., Quake, SR, Proc Natl Acad Sci, 105,16266-16271(2008)).

[0054] As used herein, the term "derived" refers to origin or source. and may include naturally occurring, recombinant, unpurified or purified molecules. The nucleic acid derived from the acid may contain, in part or in whole, the original nucleic acid, and may be Nucleic acids derived from a biological sample may be fragments or variants of the nucleic acid. It can be purified.

[0055] The "target nucleic acid" in the method of the present invention is the nucleic acid, DNA or RNA, to be detected. A target nucleic acid derived from an organism is a nucleic acid specific to that organism, having a sequence derived from the sequence of that organism. The target nucleic acid from a pathogen is a specific polynucleotide derived from that pathogen. It means a polynucleotide having a polynucleotide sequence.

[0056] In some embodiments, 1 pg, 5 pg, 10 pg, 20 pg, 30 pg, 40 pg pg, 50pg, 100pg, 200pg, 500pg, 1ng, 5ng, 10ng, 2 0ng, 30ng, 40ng, 50ng, 100ng, 200ng, 500ng, 1μg , 5μg, 10μg, 20μg, 30μg, 40μg, 50μg, 100μg, 200μg In some embodiments, less than 100 μg, 500 μg, or 1 mg of nucleic acid is obtained from the sample for analysis. In the case of 1-5 pg, 5-10 pg, 10-100 pg, 100 pg-1 ng, 1- Nucleic acid analysis samples of 5ng, 5-10ng, 10-100ng, 100ng-1µg Get from.

[0057] In some embodiments, the methods described herein involve the use of a microorganism of interest. To detect and / or quantify nucleic acid sequences corresponding to the microbiome of an organism. The methods described herein are used to , 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10, 000, 20,000, 50,000, 100,000, 200,000, 300,00 0, 400,000, 500,000, 600,000, 700,000, 800,00 0, 900,000, 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 10 9 One or more sequence reads may be analyzed.

[0058] In some embodiments, the methods described herein involve, for example, the removal of a microorganism from a microorganism. Gene expression can be determined by determining the presence of mRNA in relation to DNA from the microorganism. In some embodiments, the present invention is used to detect and / or quantify The methods described herein provide highly discriminatory quantitative analysis of multiple genes. The methods described in the book are at least 1, 2, 3, 4, 5, 10, 20, 50, 100 , 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000 , 50,000, 100,000 or more distinct target nucleic acid expression; Can be quantified.

[0059] The sample containing cell-free nucleic acid is obtained from a subject. Such subjects may be humans, livestock, or For example, the animal may be a cow, chicken, pig, horse, rabbit, dog, cat, goat, etc. In some embodiments, the cells used in the present invention are obtained from a patient. Examples of samples include whole blood, sweat, tears, saliva, ear fluid, sputum, lymph, bone marrow suspension, lymph fluid, urine, saliva, semen, vaginal fluid, cerebrospinal fluid, brain fluid, ascites, milk, respiratory tract, intestinal tract or genitourinary tract duct secretions, washings of tissues or organs (e.g., lungs), or tissues of the breast, lungs, intestines, skin, or fetus Cell-free fractions of tissue extracted from organs such as the cervix, prostate, pancreas, heart, liver, and stomach Such samples may be collected by centrifugation, elutriation, density gradient separation, apheresis, etc. , affinity selection, panning, FACS, centrifugation in Hypaque Once the sample is obtained, it can be used directly or frozen. or can be maintained for short periods in suitable media.

[0060] Blood samples can be obtained using any technique known in the art (e.g., syringe or Blood samples may be optionally pretreated or Samples such as blood samples may be stored for 4 weeks or 2 weeks from the time of sample acquisition. Week, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 6 hours, 3 hours, 2 hours Within 1 hour or 1 hour, or longer if frozen, The subject may be analyzed in any of the methods and systems described in If a sample from the subject (e.g., a blood sample) is to be obtained, the amount will depend on the size and This may vary depending on the conditions being screened. Also available in 10ml, 5ml, 1ml, 0.5ml, 250, 200, 150, 100, 50, 4 Obtain 0, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 μL samples In some embodiments, the sample contains 1 to 50, 2 to 40, 3 to 30, or 4 to 20 μL. In some embodiments, 5, 10, 15, 20, 25, 30, 35 samples are obtained. , 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 1 Obtain a minimum of 100 μL of sample.

[0061] The cell-free fraction is preferably serum or plasma. The term "cell-free fraction" is a fraction of a biological sample that is substantially free of cells. As used herein, the term "substantially cell-free" refers to a concentration of approximately 20,000 cells / ml. less than about 2,000 cells / ml, preferably less than about 200 cells / ml , most preferably containing less than about 20 cells / ml. In contrast to some prior art methods, genomic DNA is excluded from the cell-free sample. Typically, it contains about 50% to about 90% of the nucleic acids present in a sample.

[0062] The method of the present invention may further comprise preparing a cell-free fraction from the biological sample. Cell fractions can be prepared using conventional techniques known in the art. For example, blood samples can be prepared using conventional techniques. The cell-free fraction of the pellet is stirred for about 3 to 30 minutes, preferably for about 3 to 15 minutes, more preferably for about 3 to 15 minutes. 10 minutes, most preferably about 3 to 5 minutes, at about 200 to 20,000 g, preferably about 20 0 to 10,000 g, more preferably about 200 to 5,000 g, most preferably about 350 It can be obtained by centrifuging a blood sample at a low speed of ∼4,500 g. The sample is separated into cells and their fragments from the cell-free fraction containing soluble DNA or RNA. To achieve this, ultrafiltration can be used. Typically, ultrafiltration is performed using a 0.22 μm membrane filter. This is done using a filter.

[0063] The methods of the present invention further include concentrating (or enriching) target nucleic acids in the cell-free fraction of a biological sample. The target nucleic acid can be isolated using conventional techniques known in the art, for example, by Solid-phase absorption in the presence of high salt concentrations, followed by organic extraction with phenol-chloroform Precipitation with ethanol or isopropyl alcohol, or high salt concentrations or 70-8 It can be concentrated by direct precipitation in the presence of 0% ethanol or isopropyl alcohol. The enriched target nucleic acid is at least about 2, 5, 10, 20, or more times more concentrated than that in the cell-free fraction. The target nucleic acid, whether concentrated or not, can be concentrated by 100 times. , can be used for amplification according to the methods of the present invention.

[0064] In some embodiments, the present invention provides methods for diagnosing or predicting transplant rejection. The term "transplant rejection" includes both acute and chronic transplant rejection. "AR" is a condition caused by the immune system of a tissue transplant recipient when the transplanted tissue is immunologically foreign. Acute rejection is characterized by infiltration of the transplanted tissue by the recipient's immune cells. The immune cells then perform their effector function and destroy the transplanted tissue. Onset is rapid, generally occurring within a few weeks of transplantation in humans. Sexual rejection can be prevented by immunosuppressive drugs, such as rapamycin, cyclosporine A, and anti-CD40L monoclonal antibodies. This can be inhibited or suppressed by antibodies or other methods.

[0065] "Chronic transplant rejection or CR" generally occurs in humans after successful immunosuppression of acute rejection. Fibrosis occurs within months to years of transplantation. Fibrosis is a chronic rejection syndrome in all types of organ transplants. Chronic rejection is typically characterized by a range of events characteristic of individual organs. For example, in lung transplantation, such disorders may include airway fibroproliferative destruction of the lungs (bronchiolitis obliterans), heart transplants or transplants of cardiac tissue, e.g. For example, in valve replacement, such disorders include fibroatherosclerosis and renal In transplantation, such disorders include obstructive nephropathy, nephrosclerosis, and tubulointerstitial nephropathy. In liver transplantation, such disorders include vanishing bile duct syndrome. Chronic rejection is a condition characterized by immunosuppression. Immunosuppressant-related ischemic injury, graft denervation, hyperlipidemia, and hypertension are also prominent. It can be characterized.

[0066] In some embodiments, the present invention further provides a method for treating a subject who has undergone a transplant, e.g., an allogeneic transplant. The present invention also includes a method for determining the effectiveness of an immunosuppressive regimen for the treatment of a cancer.

[0067] Certain embodiments of the present invention provide methods for predicting graft survival in a transplanted subject. The present invention provides a method for determining whether a graft will survive or be lost in a transplant patient or subject. In one embodiment, the present invention provides a method for diagnosing or predicting long-term implantation. "Long-term graft survival" refers to the presence or absence of one or more of the following: Despite the occurrence of acute rejection episodes up to now, at least In one embodiment, graft survival is at least about 5 years. The results are determined for patients who have had at least one acute rejection episode. Embodiments provide methods for determining or predicting graft survival after acute rejection. In certain embodiments, transplantation therapy, e.g., immunosuppressive therapy (immunosuppressive therapy is known in the art). In yet another embodiment, acute rejection is determined or predicted. Provides a method for determining the class and / or severity (not just its presence) of .

[0068] As is known in the transplantation art, transplanted organs, tissues, or cells may be allogeneic or xenogeneic. Thus, the graft can be an allograft or a xenograft. The characteristics of the graft tolerance phenotype detected or identified by the method are those that are associated with immunosuppressive therapy. that is, it is a phenotype that occurs without immunosuppressive therapy in a host ( Therefore, it is present in the host (in the absence of immunosuppressive drugs). The graft can be any solid organ or skin graft. Examples of organ transplants that can be analyzed include, but are not limited to, kidney transplants, pancreas transplants, etc. , liver transplantation, heart transplantation, lung transplantation, intestinal transplantation, pancreas after kidney transplantation, and simultaneous pancreas-kidney transplantation Included.

[0069] Microbiome detection and analysis The methods of the present invention include high-throughput sequencing of a cell-free nucleic acid sample from an individual; and Subsequent bioinformatic analysis to determine the presence and prevalence of microbiome sequences tic analysis, where the sequences are compared with normal microorganisms in the indigenous organism, e.g., gut, skin, etc. They can be from the biome or non-indigenous, e.g., opportunistic, pathogenic The analysis can be done on the complete microbiome or on its entirety. Components in, e.g., viromes, bacterial microbiomes, fungal microbiomes, protozoan parasites Examples of nucleic acids include, but are not limited to, bispecific nucleic acids. stranded DNA, single-stranded DNA, single-stranded DNA hairpin, DNA / RNA hybrid, RNA (e.g., mRNA or miRNA) and RNA hairpins. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. For example, cell-free RNA and DNA are present in human plasma.

[0070] Genotyping of microbiome nucleic acids and / or detection of microbiome-specific nucleic acids Detection, identification and / or quantification generally involves an initial step of amplification of the sample. However, there may be cases where sufficient cell-free nucleic acid is available and can be sequenced directly. If the nucleic acid is RNA, the amplification step may be preceded by a reverse transcription step to convert the RNA to DNA. A transcriptase reaction can be carried out. Preferably, the amplification is unbiased, i.e., the amplification primer The primer is a universal primer or an adapter ligated to the nucleic acid to be analyzed. The amplification primers are specific for the adapter. Examples of PCR techniques include, but are not limited to: However, there are some limitations, such as hot-start PCR, nested PCR, and in situ polony (pol monomy PCR, in situ rolling circle amplification (RCA), bridging (br These include idge, picotiter PCR, and emulsion PCR. Other suitable amplification methods include ligase chain reaction (LCR), transcription amplification, and self-sustaining amplification. Array replication, selective amplification of target polynucleotide sequences, consensus sequence priming (con Sensus sequence primed polymerase chain reaction (CP-PCR) ), arbitrarily primed polymerase chain reaction ( AP-PCR), degenerate oligonucleotide primed PCR (DOP-PCR) and nuclear Includes nucleic acid-based sequence amplification (NABSA), used to amplify specific polymorphic loci Other amplification methods that can be used include those described in U.S. Patent Nos. 5,242,794 and 5,494,810. , including those described in Nos. 4,988,617 and 6,582,938 do.

[0071] After amplification, the amplified nucleic acids are sequenced. Sequencing can be accomplished using high-throughput systems. Some of the high-throughput systems allow for the detection of their incorporation into growing chains. Detection of sequencing nucleotides immediately or upon incorporation (i.e., real-time or In some cases, high throughput is possible. The number of sequencing runs per hour is at least 1,000, at least 5,000, or at least 10,000, at least 20,000, at least 30,000, at least 40,0 00, at least 50,000, at least 100,000 or at least 500,0 00 sequence reads, where each read contains at least 50 At least 60, at least 70, at least 80, at least 90, at least 100, The sequence is at least 120 or at least 150 bases. The nucleic acid, genomic DNA, cDNA derived from RNA transcripts, or RNA is used as a template. This can be done using

[0072] In some embodiments, high throughput sequencing is performed using Helicos Bio Sciences Corporation(Cambridge,Massachus) Technologies available through the ETTS, such as Single Molecule Sequencing by Synthesis (SMS) Molecule Sequencing by Synthesis (SMSS) SMSS allows for whole genome sequencing without the need for a pre-amplification step. This is unique because it reduces nonlinearities and distortions in nucleic acid measurements. SMSS is disclosed in U.S. Published Application Nos. 2006002471 and 20060024678 , 20060012793, 20060012784 and 20050100932 is described in detail.

[0073] In some embodiments, high-throughput sequencing is performed using 454 Lifesciences. Available from ences, Inc. (Branford, Connecticut) Techniques such as the use of Pico Titer Plate devices The device includes a CCD camera within the device that records the sequence generated by the sequencing reaction. A fiber optic plate (fiber optic plate) that transmits the chemiluminescent signal This use of fiber optics involves the transfer of at least 20 million base pairs in 4.5 hours. This allows detection within

[0074] Bead amplification followed by fiber optics Methods for using detection are described by Marguiles, M. et al., "Genome sequencing" ncing in microfabricated high-density pr icolitre reactors”,Nature,doi:10.1038 / na ture03959, and U.S. Published Application Nos. 20020012930 and 200300 58629, 20030100102, 20030148344, 2004024816 1, 20050079510, 20050124022 and 20060078909 It is written.

[0075] In some embodiments, clonal single molecule arrays Molecule Array (Solexa, Inc.) or reversible terminator Sequencing-by-synthesis using ter chemistry High-throughput sequencing is performed using sequencing-by-synthesis (SBS). The technology is disclosed in U.S. Patent Nos. 6,969,488, 6,897,023, and 6,833,2 46, No. 6,787,308 and U.S. Published Application No. 200401061 30, 2 0030064398, 20030022207 and Constans,A,,The Partially described in Scientist 2003,17(13):36.

[0076] In some embodiments of this aspect, high-throughput sequencing of RNA or DNA is performed. The decision was made to use AnyDot. chips (Genovoxx, Germany). AnyDot. chips can be used to measure biological processes, such as miRNA expression or Enables monitoring of allelic variability (SNP detection). In particular, AnyDot chips AnyDot allows for 10- to 50-fold enhancement of nucleotide fluorescent signal detection. The chip and its method of use are described in International Publication Nos. WO 02088382 and WO 030 20968, WO 0303 1947, WO 2005044836, PCTEP 0 5105657, PCMEP 05105655 and German Patent Application No. DE 10 1 49 786, DE 102 14 395, DE 103 56 837, DE 10 2004 009 704, DE 10 2004 025 696, DE 10 2004 025 746, DE 10 2004 025 694, DE 10 2 004 025 695, DE 10 2004 025 744, DE 10 200 4 025 745 and DE 10 2005 012 301 There are.

[0077] Another high-throughput sequencing system is described in Venter, J. et al., Science 16 February 2001; Adams, M. et al., Science 24 March 2000; and MJ, Levene et al., Science 299:682-686 , January 2003; and U.S. Published Application Nos. 20030044781 and 2006 / 0078937. Taken together, such systems include: The polymerization reaction measured on the nucleic acid molecule produces a target with multiple bases by the addition of bases over time. The method also involves sequencing a target nucleic acid molecule, i.e., sequencing the nucleic acid on the template nucleic acid molecule to be sequenced. The activity of the polymerase is followed in real time. Which bases are incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerase? By identifying which sequences are present along the target nucleic acid molecule, it is possible to deduce the sequence. The target moves to a suitable position for extension of the oligonucleotide primer at the active site. A polymerase on a target nucleic acid molecule complex is provided. A plurality of label types are attached near the active site. Nucleotide analogs are provided, and each distinguishable type of nucleotide analog is a target The growing nucleic acid strand is complementary to a different nucleotide in the target nucleic acid sequence. elongation by using a polymerase to add nucleotide analogs to the nucleic acid chain wherein the nucleotide analogue added is a nucleotide analogue of the target nucleic acid at the active site. The nucleic acid sequence added to the oligonucleotide primer as a result of the polymerization process is complementary to the nucleic acid sequence. Identifying nucleotide analogs. Providing labeled nucleotide analogs to polymerize growing nucleic acid chains. The process of identifying the added nucleotide analogue is repeated to further extend the nucleic acid chain. , determining the sequence of the target nucleic acid.

[0078] In some embodiments, shotgun sequencing is performed. In this method, DNA is randomly broken into many small fragments and then sequenced using chain termination. This fragmentation and sequencing is carried out in several rounds to obtain reads. This gives multiple overlapping reads for the target DNA. ,using the overlapping ends of different reads to join them into a continuous strand.

[0079] In some embodiments, the present invention provides for the detection and quantification of microbial sequences by sequencing. In this case, it is possible to estimate the detection sensitivity. There are two sensitivity components: (i) the number of molecules analyzed (sequencing depth) and (ii) the sequence. Error rate of the sequencing process. For sequencing depth, frequent variations in inter-individual A reasonable estimate is that there is about one base difference per 1000. Genome analyzers such as the Illumina Genome Analyzer The sequencer reads lengths greater than 36 base pairs. Although this may be variable depending on the body condition, 90% should be used as a baseline estimate. With this fraction of donor DNA, approximately 10 molecules out of 10 analyzed are One of them is likely a microorganism. Genome Analyzer It is possible to obtain approximately 10 million molecules per analytical channel, and There are eight analytical channels per instrument run. Therefore, one sample per channel. Loading one tube provides information about the state of the microbiome and the microbial Approximately 10 that can be identified 6 It should be possible to detect individual molecules. The degree of accuracy is simply achieved by sequencing a larger number of molecules, i.e., a larger number of channels. This can be achieved by using:

[0080] The sequencing error rate also affects the sensitivity of this technique. The fixed error rate varies between platforms but is between 0.5 and 1.5%. This imposes a potential limitation on sensitivity of 6 to 0.50%. However, Helicos BioSc iences (Harris, TD et al., Science, 320, 106-109 ( As demonstrated by [End Page 2008], sample templates were resequenced multiple times. By doing so, it is possible to systematically reduce the sequencing error rate. A single application would reduce the expected error rate.

[0081] After sequencing, the sequence dataset is processed in a data processor for bioinformatics analysis. Subtract host sequences (i.e., human, cat, dog, etc.) from the analysis. and, for example, the coverage of sequences mapped to the microbial reference sequence. ge) to the coverage of the host reference sequence to determine the presence and prevalence of microbial sequences. Subtracting the host sequence identifies the reference host sequence and determines the sequence present in the reference host genome. A step of masking the microbial sequence or microbial mimic sequence may also be included. Determining the presence of a microbial sequence by comparison to a reference microbial genome identifies a reference microbial sequence. The method may include masking any host sequences or host-mimicking sequences present within the nucleic acid.

[0082] Check the quality of the sequence and remove any remaining sequencer-specific nucleotides (adapter sequences). Removes overlapping paired end reads and merges them to generate better reads with fewer read errors. The data set may optionally be cleaned to obtain a higher quality consensus sequence. Repeated sequences are identified as having the same start site and length, and overlaps are can be removed from the analysis.

[0083] A key feature of the present invention is the subtraction of human sequences from the analysis. Because it is biased, the predominant sequence in the sample will be the host sequence. The process can be optimized in several ways to improve its speed and accuracy. For example, this can be done by performing multiple subtractions, where the initial alignment is a coarse filter. is set to filter (i.e., use a fast aligner), and An additional alignment is performed with a finer filter (i.e., a sensitive aligner).

[0084] The read database is bioinformatically filtered to subtract host DNA. First, the human reference genome (including, but not limited to, the Genbank hg19 reference sequence) Each sequence is aligned to the best fit in the human reference sequence. Humans were bioinformatically removed from the analysis. Therefore, the sequence is positively identified.

[0085] The reference human sequence is located in a genome that is not well represented in the reference database. Concentrations with high hit rates, including but not limited to highly repetitive sequences, The optimization can also be achieved by adding a tag. If a database (e.g., the entire NCBI NT database) is used, access Of the unaligned reads, a significant proportion will ultimately be human at a later stage in the pathway. It is recognized that these leads are identified earlier in the analysis. This can be done by constructing an extended human reference body. A human sequence database other than the reference (e.g., It was generated by identifying human contigs in the NCBI NT database. These contigs are then added to the human reference to provide a more comprehensive reference set. Furthermore, newly constructed human contigs from the cohort study further masked the human-derived reads. It can be used as follows.

[0086] Regions of the human genome reference sequence that contain non-human sequences, e.g., within the genome of the reference sample Integrated viral and bacterial sequences can be masked. For example, Epstein-Barley Approximately 80% of the EBV genome is integrated within hg19.

[0087] The sequence reads identified as non-human were then compared to a nucleotide database of microbial reference sequences. The database contains microorganisms known to be associated with the host. Selection can be made for biological sequences (e.g., human commensal and pathogenic microorganisms).

[0088] The microbial database can be optimized to mask or remove contaminating sequences. For example, many public database entries contain artifact sequences that are not derived from microorganisms, e.g. It is recognized that it contains primer sequences, host sequences, and other contaminants. It is recommended to perform multiple alignments on the database. When aligned, regions showing irregularities in read coverage are considered artifacts. The detection of such irregular coverage can be achieved using various metrics, e.g. For example, the coverage of a particular nucleotide and the average coverage of all contigs in which this nucleotide is found Generally, the ratio of the average coverage of the reference sequence to the average coverage of the reference sequence is about 5 times, about 10 times, or about 10 times the average coverage of the reference sequence. Sequences that are expressed as greater than 25, 50, or 100 times are artifacts. Alternatively, if the total coverage of a contig is given, the per-base likelihood of the coverage can be calculated. A binomial test can be applied to obtain the probability of a match. Removal of contaminating sequences from the sample allows for accurate identification of the microorganism. It is an advantage of the method of the present invention that the database can be improved by To refine the database before clinical use, the database was divided into 1, 10, 20, and 5 0, and can be aligned with 100 or more samples.

[0089] Each high-confidence read is matched against multiple organisms in a given microbial database. Based on this possible mapping redundancy, bioabundance can be accurately estimated. To ensure accurate attribution, an algorithm is used to select the most likely Calculate the high biomass (e.g., Lindner et al., Nucl. Acids Res. (2 (See 013)41(1):e10. For example, The GRAMMy or GASic algorithms are used to calculate the most likely organisms. These data provide information about the presence of microorganisms in cell-free nucleic acid samples. do.

[0090] Alignment and assignment to host sequences or microbial sequences is art-recognized. For example, a 50 nt read can be generated by the method described in 1 or less mismatch, 2 or less mismatch, 3 or less mismatch, 4 or less mismatch Matches a given genome if there are matches, 5 or fewer mismatches, etc. Commercially available algorithms are used for alignment and identification. Non-limiting examples of such alignment algorithms include: The bowtie2 program (Johns Hopkins University) For example, depending on the desired alignment speed, an end-to-end mode can be selected. A pre-set option for the end mode can be selected.

[0091] Very fast: Same as: -D5-R1-N0-L22-iS,0,2.50 Fast: Same as:-D10-R2-N0-L22-iS,0,2.50 High Sensitivity: Same as: -D15-R2-L22-iS,1,1.15 Very sensitive: same as: -D20-R3-N0-L20-iS,1,0.50 In other alignment algorithms or software packages, equivalent settings may be It can be used.

[0092] These leads to the organism (i.e., host or microbiome component) are then analyzed. Sum the attributions and use them to estimate the number of reads attributed to each organism in a given sample. (for determining the prevalence of an organism in a cell-free nucleic acid sample). The counts are normalized for the size of the genome to yield a coverage calculation for the organism. To account for differences in sequencing depth between samples, the normalized coverage for each microorganism was calculated. Compare with the host sequence coverage in the same sample.

[0093] The final determination is based on the microbial prevalence and microbial data represented by the sequences in the sample. These data can be optionally integrated and displayed for immediate visualization, e.g. For example, in the form of a report provided to an individual or healthcare provider, or displayed or hyperlinked to The coverage estimates are based on the metadata from the samples. can be consolidated and sorted into tables and figures for each sample or cohort of samples. do.

[0094] Optionally, the filtered host sequences can be used for other purposes, such as personalized medicine. For example, certain SNPs in the human genome can help physicians identify a given patient's drug sensitivity. Human-derived sequences may allow the incorporation of viruses (e.g., EBV) into the host genome. , HPV, polyomavirus) integration. Alternatively, it may have synergistic clinical applications. (e.g., cell-free tumor DNA may be highly susceptible to infection due to chemotherapy) In some patients, to monitor cancer progression in parallel with monitoring infection. may be used).

[0095] In some embodiments, analysis of cell-free nucleic acid is used to calculate a pathogenicity score. wherein the pathogenicity score is a percentage of the pathogenicity score for ease of interpretation by, for example, a medical practitioner. It is a numerical or alphabetical value that summarizes the overall pathogenicity of a substance. The microorganisms present can be assigned different scores depending on the microorganism. A "score" is a combination of a number of different factors, typically ranging from 0 to 1, 0 to 10, or is an arbitrary unit ranging from 0 to 100 representing all observed values ​​for the microbiome of interest. The pathogenicity score is provided as a percentile based on the specific parameters and their The weights of the parameters in are used to fit a function to the observed disease severity, for example. The importance of various parameters and criteria can be established experimentally or by It can be determined manually by

[0096] Factors important in calculating the pathogenicity score include, but are not limited to, the reference subject or the abundance of the microorganism in a subject group (e.g., a study population) compared with the human lead The abundance of microorganisms calculated by the number of reads compared, known infected, known uninfected individuals, etc. Specific mutations found within the genome of a microorganism may contribute to the virulence, pathogenesis, and survival associated with the microorganism. It is possible to refer to databases such as pathogenicity and antibiotic resistance, and It includes but is not limited to SNPs, indels, plasmids, etc. Co-occurrence of certain microorganisms, including but not limited to: (e.g., detection of mRNA) can be important for the pathogenicity score, e.g., if the microorganism It is rich in information such as whether it is actively replicating or latent. Geographical features (where geography is indicators of exposure to the microorganism of interest), e.g., the host's travel history, interactions with infected individuals, It may also include actions.

[0097] Also provided are reagents and kits thereof for carrying out one or more of the above methods. The reagents and kits thereof can vary widely. Reagents of interest include those for use in the preparation of the above. These include reagents specifically designed for: (i) microbiome and individual profiling; (ii) identification of the microbiome profile and (ii) the Detection and / or quantification of one or more nucleic acids from the microbiome in the sample. The kits are subjected to nucleic acid extraction using the methods described herein, e.g., PCR and sequencing. and / or reagents necessary for performing nucleic acid detection. The kit may further comprise a data analysis kit. It is possible to include a software package for It is possible to include a reference profile for comparison, in particular a reference profile optimized as described above. The kit may include a reference database containing reagents, such as buffers and and H2O.

[0098] Such kits demonstrate or establish the activity and / or benefits of the composition, and and / or information about dosage, administration, side effects, drug interactions, or other information useful to your healthcare provider. such as supporting scientific references, package inserts, clinical trial results and / or summaries thereof. Such kits may also include instructions for accessing the database. Such information may also include information from various studies, for example, experimental animal studies, including in vivo models. The results of this study may be based on studies using human subjects and on studies based on human clinical trials. The kits described in this document are available to healthcare providers, including doctors, nurses, pharmacists, and prescribing staff. The kit may be provided, sold, and / or promoted. In some cases, they may be sold directly to consumers.

[0099] Any of the above methods may be implemented by computer-executable logic recorded on a computer-readable medium. For example, the computer program may be The system may perform some or all of the following functions: (i) control the isolation of nucleic acids from a sample; (ii) preamplifying nucleic acids from the sample; and (iii) detecting specific regions in the sample. (iv) amplifying, sequencing, and aligning the microbial sequences in the sample; (v) obtaining data regarding the presence or prevalence of microorganisms detected in the sample; (vi) comparing to a predetermined threshold; and (vi) determining infection, microbiome health, immunocompetence status, or outcome. (vi) indicating sample status with respect to infection, microbiome health, immune competence, etc. vinegar.

[0100] The computer executable logic may be implemented on a personal computer, a network server, a workstation, or any other device. workstation or other computer platform (currently developed or Any computer can be any of a variety of types of general-purpose computer, such as a In some embodiments, the method may be implemented in a computer-implemented manner. Computer executable logic (including program code) stored in a computer-readable medium a computer program including a computer usable medium having a software program The computer-executable logic is executed by a processor. may cause the processor to perform the functions described herein. Some functions are primarily implemented in hardware, e.g., using hardware state machines. The hardware state machines used to perform the functions described herein are The implementation of Shin will be apparent to those skilled in the relevant art.

[0101] The program involves profiling the microbiome and individuals and / or accessing data reflecting the quantification of one or more nucleic acids from the circulating microbiome of a subject; This may provide a method for assessing the microbial status of an individual.

[0102] In one embodiment, the computer that executes the computer logic of the present invention is a digital The digital input device may also include a digital input device, such as a scanner. For example, presence or prevalence.

[0103] In some embodiments, the present invention provides a computer-readable medium, which comprises: i) receiving data from one or more nucleic acids detected in a sample; and (ii) a microphone. The computer is configured to perform the step of diagnosing or predicting a condition based on the quantification of the lobiome. The computer readable medium includes a set of instructions recorded on the computer readable medium such that:

[0104] It also provides a database of microbial reference sequences and a database of human reference sequences. Such a database would typically include the optimized data set described above.

[0105] In some embodiments, the methods of the present invention provide a status of an individual with respect to an infection. In some such embodiments, the microbial infection is a pathogen, wherein the pathogen sequence The presence of is indicative of a clinically relevant infection. In another embodiment, the prevalence is It is an indicator of microbial load, where a predetermined level is an indicator of clinical relevance. In some such embodiments, the individual is receiving antimicrobial therapy, e.g., antibiotics, passive or or are being treated with active immunotherapy, antiviral agents, or the like, or are being considered for such treatment. Individuals can be tested before, during and after treatment.

[0106] Microbial infection can also be indicated by a burden of commensal organisms, where blood The level of commensal organisms in the fluid sample is an indicator of gut health (e.g., gut luminal disruption).

[0107] Comparison of microbial RNA can be performed alone or in conjunction with microbial DNA; Here, an excess of RNA relative to the microbial sequence (e.g., about 5x the coverage of microbial DNA, 1 0x, 15x, 20x, 25x) are indicative of an active infection. The microorganisms analyzed in this way are those capable of latent infection, e.g., herpes viruses. These include influenza and hepatitis viruses.

[0108] In other embodiments, one is interested in a global estimate of the microbiome, in which case: The relative presence or prevalence of classes of microorganisms is of interest. Treatments such as statins, antibiotics, and immunosuppressants can affect the overall health of the microbiome. It is known in the art that the composition of the microbiome can be affected by the There is interest in determining this.

[0109] In some embodiments, to monitor the effectiveness of antimicrobial therapy, and a temporal change in the abundance of one or more nucleic acids from the microbiome to select a treatment; Differences can be used, for example, to determine the amount of one or more nucleic acids from the microbiome before and after treatment. A post-treatment reduction in one or more nucleic acids from the microbiome can be measured after the treatment. The amount of one or more nucleic acids from the microbiome may also indicate a successful treatment. , for example, can be used to select between treatments of different intensities.

[0110] In one aspect, the present invention provides a method for the treatment of immune deficiency in a subject receiving an immunosuppressive regimen. The present invention provides a method for diagnosing or predicting transplantation status or outcome after immunosuppression. A sample is taken from the patient and the presence or absence of one or more microbiomes containing virome nucleic acids is determined. In some embodiments, the sample is blood, plasma, Serum or urine. The proportion and / or amount of microbial nucleic acid is monitored over time. This increase in ratio can be used to determine immune competence. The amount can be determined by any suitable method known in the art, including those described herein. For example, this can be done by sequencing, nucleic acid arrays or PCR).

[0111] In some embodiments, one or more microorganisms in a sample from an immunosuppressed recipient are The amount of lobiome nucleic acid is used to determine the transplant status or outcome. In some embodiments, the methods further comprise determining one or more nucleic acids from the microbiome. In some embodiments, the method includes quantifying one or more nucleic acids from a donor sample. The amount is determined as a percentage of the total nucleic acid in the sample. For the analysis, the amount of one or more nucleic acids from the donor sample is calculated as a ratio to the total nucleic acid in the sample. In some embodiments, the amount of one or more nucleic acids from the donor sample is determined. The nucleic acid sequence is determined as a ratio or proportion of one or more reference nucleic acids in the pool. the amount of one or more nucleic acids from the nucleic acid sequence is determined to be 10% of the total nucleic acids in the sample; or , the amount of one or more nucleic acids from the microbiome is compared to the total nucleic acids in the sample at a ratio of 1:10 Additionally, the amount of one or more nucleic acids from the microbiome can be a reference, such as β-globin. It may be determined that the ratio is 10% or 1:10 of the genes. The amount of one or more nucleic acids from the microbiome can be determined as a concentration. The amount of one or more nucleic acids from the sample can be determined to be 1 μg / mL.

[0112] In some embodiments, one or more nucleic acids from the microbiome exceed a predetermined threshold. The amount of erythrocyte segregation (E1) is indicative of immunocompetent status, e.g., those showing no evidence of transplant rejection or other pathology. Normative values ​​for clinically stable patients can be determined. An increase in the amount of one or more nucleic acids from the microbiome below the normative value indicated a stable result. On the other hand, microbiome analysis above the normative values ​​for clinically stable post-transplant patients may be possible. The amount of one or more nucleic acids from could be indicative of enhanced immune competence and risk of transplant rejection.

[0113] In some embodiments, different predetermined thresholds indicate different transplant outcomes or conditions. For example, as noted above, there are differences in the mitochondria that exceed the normative values ​​for clinically stable post-transplant patients. An increase in the amount of one or more nucleic acids from the chromosome may be a sign of a post-transplant condition such as transplant rejection or transplant injury. However, clinically stable post-transplant patients may show changes in their condition or outcome. Amounts of one or more nucleic acids from the microbiome above a normative value and below a predetermined threshold level An increase could indicate a condition less serious than transplant rejection, such as a viral infection. An increase in the amount of one or more nucleic acids from the microbiome above a high threshold may indicate transplant rejection. It will be.

[0114] In some embodiments, the amount of one or more nucleic acids from the microbiome is determined over time. Differences are indicative of immune competence, e.g., determining the abundance of one or more nucleic acids from the microbiome. To do this, transplant patients can be monitored over time. If the amount of erythrocyte sediment decreases over time and then returns to normal, this is not as serious as transplant rejection. On the other hand, a persistent decrease in the amount of one or more nucleic acids from the microbiome may indicate a Serious conditions could be present, such as lack of effective immunosuppression and transplant rejection.

[0115] In some embodiments, the amount of one or more nucleic acids from the microbiome is determined over time. The difference is in the use of immunosuppressive therapy to monitor its effectiveness or to select immunosuppressive therapy. For example, the amount of one or more nucleic acids from the microbiome can be used to determine whether or not a particular nucleic acid is present in an immunosuppressive treatment. The post-treatment reduction of one or more nucleic acids from the microbiome can be determined before and after It may be shown that the treatment is successful in preventing immune rejection. The amount of nucleic acid in the target gene may be used to select between immunosuppressive treatments, e.g., between immunosuppressive treatments of different strengths. For example, smaller amounts of one or more nucleic acids from the microbiome can be used to This may indicate the need for very strong immunosuppression. Higher amounts of nucleic acid may indicate that less potent immunosuppression can be used.

[0116] The present invention provides a highly sensitive and specific method. In some embodiments, the transplant condition or the methods described herein for diagnosing or predicting outcome are at least 50 %, 60%, 70%, 80%, 90%, 95% or 100% sensitivity. In embodiments, the methods described herein have a sensitivity of at least 50%. In some embodiments, the methods described herein achieve a sensitivity of at least 78%. In some embodiments, the methods described herein provide a 70% to 100% In some embodiments, the specificity of the antibody is about 100%. The method has a specificity of about 80% to about 100%. The method described therein has a specificity of about 90% to about 100%. In this case, the method described herein has a specificity of about 100%.

[0117] The present invention relates to individuals who are being treated with immunosuppressive regimens, antimicrobial agents, etc. The present invention provides a non-invasive diagnostic for an individual, including a human body, the diagnostic using cell-free DNA or This is based on monitoring the sequence of RNA. For example, an individual may It is noted herein that the viral load varies depending on the immune competence of the individual. The preferred viruses for monitoring immune competence are anelloviruses. In this case, viral load is shown herein to correlate with the immune competence of the individual.

[0118] In some embodiments, the present invention provides a method for detecting viral particles that are normally free in plasma or from viral particles. for detecting and / or quantifying circulating nucleic acids in the body, or for assessing infection, immune competence, transplantation status, Methods, devices, compositions and methods for the diagnosis, prognosis, detection and / or treatment of conditions or outcomes Provide a kit.

[0119] In some specific embodiments, the present invention provides a method for determining the risk of HIV infection in transplant patients by virome analysis. This provides an approach for the non-invasive detection of immune competence in the presence of DNA from other foreign sources. This avoids the potential problem of microchimerism from organ transplants and allows for whole organ recipients without gender considerations. In some embodiments, genetic phenotypes are used to identify individuals with viromes that are universal to the individual. This approach is independent of the gender of the donor and recipient. This allows for reliable identification of sequences that can be made in a relevant manner.

[0120] After immunosuppressive regimens (e.g., in combination with transplantation, treatment of autoimmune diseases), blood Bodily fluids such as may be collected from a patient and analyzed for markers. Examples of bodily fluids include, but are not limited to: Although not typical, smears, sputum, biopsies, secretions, cerebrospinal fluid, bile, blood, and lymph , saliva, and urine. The detection, identification, and / or quantification of virome sequences is , real-time PCR, chips, high-throughput detection of circulating nucleic acids (e.g., cell-free DNA) Quantitative sequencing is performed using standard shotgun sequencing and other methods known in the art (as described herein). Viral load can be monitored over time, and this An increase in the ratio can be used to determine the status or outcome of immunocompetence.

[0121] In any of the embodiments described herein, the graft may be any solid organ (e.g., a solid organ). The tissue samples that can be analyzed by the methods described herein can be tissue samples (e.g., tissue samples), or skin grafts. Examples of organ transplants include, but are not limited to, kidney transplants, pancreas transplants, liver transplants, and heart transplants. These include transplants of the pancreas after kidney transplantation, lung transplantation, intestinal transplantation, and simultaneous pancreas-kidney transplantation.

[0122] In some embodiments, the methods of the present invention involve analysis at the individual level or of patient populations. in, for example, clinical trials, to assess the efficacy of therapies for the treatment of diseases, including infections. Such embodiments are typically used in determining the risk of a patient or group of patients. This includes comparisons of two time points between treatments, treatment regimens, or patients receiving treatment. As a result of the disease challenge, the patient's condition is expected to differ between the two time points.

[0123] Examples of such embodiment forms include, but are not limited to, microbiological The method may include analyzing the ohm, wherein the first time point is a time point at which the diagnosis is made. is a treatment-naive patient, and a second or additional time point is a patient receiving a candidate therapeutic agent or regimen. The patient was treated with

[0124] In another embodiment, the first time point is a time point at which a candidate therapeutic agent or regimen results in: For example, diagnosed patients in disease remission confirmed by current clinical criteria. The first or additional time points are treated with a candidate therapeutic agent or regimen and challenged with disease-causing factors. patients who have been vaccinated (e.g., in the case of vaccines).

[0125] In such clinical trial formats, each set of time points may be used for a single patient, a group of patients, e.g., a cohort. As known in the art, the data may correspond to a population, or a mixture of individual and population data. Such clinical trial formats also include additional control data, such as placebo and disease-free groups. The types of studies of interest are crossover, randomized, double-blind, placebo-controlled, and parallel-group studies. It is also possible to test the efficacy of drugs. Trials: A Methodologic Perspective Secon d Edition, S. Piantadosi, Wiley-Interscience e;2005,ISBN-13:978-0471727811;and Design and Analysis of Clinical Trials:Concept s and Methodologies, S. Chow and J. Liu, Wiley -Interscience;2003;ISBN-13:978-047124985 6, each of which is specifically incorporated herein by reference. I want to be. [Brief explanation of the drawings]

[0126] [Figure 1] Study design, read statistics, and lineage distribution. A. Immunosuppression reduces the risk of transplant rejection but increases the risk of infection. B. Study design. 656 plasma samples were collected, cell-free DNA purified, and sequenced to an average depth of 1.2 Gbp / sample. C. Number of samples collected as a function of time for different patient groups in the study. D. Treatment protocols for patients in the study cohort; all patients are treated with maintenance immunosuppression (tacrolimus-based (TAC) for adult heart and lung transplant recipients and cyclosporine (CYC) for pediatric patients). CMV-positive (donor or recipient, CMV+) transplant recipients are treated with the anti-CMV prophylactic agent valganciclovir (VAL). Mean tacrolimus levels measured in the blood of transplant recipients treated with a TAC-based protocol (dashed lines are real, solid lines are windowed mean filters). E. Percentage of reads remaining after filtering of low-quality and duplicate reads (average 86%, left) and after removal of human and low-complexity reads (average 2%, right). F. Relative genome abundance at various levels of taxonomic classification after removal of human reads (average of all samples from all organ transplant recipients (n=656)). [Figure 2]Relative viral genome abundance as a function of drug dose and comparison with healthy controls. A. Mean virome composition for patients treated with the immunosuppressant tacrolimus (47 patients, 380 samples) as a function of antiviral drug dose (valganciclovir) and tacrolimus concentration measured in the blood. To account for the delayed effect of drug dose on virome composition, data on drug dose were subjected to window average filtering (window size 45 days; see Figure 1C). When patients received low doses of immunosuppressants and antivirals, Herpesviridae and Caudovirales predominated in the virome. Conversely, when patients received high doses of these drugs, Anelloviruses predominated in the virome. B. Comparison of virome composition corresponding to healthy reference individuals (n=9), samples from day 1 post-transplant with low drug exposure (n=13), and samples corresponding to high drug exposure (tacrolimus >9 ng / ml, valganciclovir >600 mg, n=68). The virome structure measured for the day 1 sample (I) and a set of healthy individuals (H) differs from the anellovirus-dominated distribution measured for the sample corresponding to the high drug dose (D). Pie charts show mean proportions, and boxplots show p-values ​​based on the Mann-Whitney test. C. Bray-Curtis beta diversity for all samples, patients with the same transplant type (heart or lung), subjects, patients treated with similar drug doses (tacrolimus levels ±0.5 ng / ml, valganciclovir ±50 mg), and samples collected from the same subjects within a one-month period. [Figure 3]A. Temporal variation in microbiome composition after transplantation. Relative abundance of dsDNA and ssDNA viruses (averaged across all samples) for various time periods. The relative abundance of ssDNA viruses increases rapidly after the initiation of post-transplant drug therapy. A reverse trend is observed after 6 months. B. Viral genome abundance at the family and order level of taxonomic classification for various time periods. The proportion of Anelloviruses increases rapidly in the first few months after transplantation. During that same period, the proportions of Herpesviridae, Caudovirales, and Adenoviridae decrease. A reverse trend is observed after 6 months. C. Temporal variation in the relative abundance of bacterial phyla. Compared to viral abundance, the representation of various bacterial phyla remains relatively constant over the observed post-transplant period. D. Shannon entropy as a measure of within-sample alpha diversity for bacterial and viral genera as a function of time (data grouped by 1-month time periods). [Figure 4] Virome composition and total viral load in the absence and presence of antiviral prophylaxis. A. Absolute viral load as a function of time, measured as viral genome copies per human genome copy detected by sequencing. Boxplots are shown for various time periods, with the center of the period indicated on the x-axis. For all patient classes, total viral load increases in the first weeks after transplant (black line is a sigmoidal fit, change in load is 7.4±3). B. Viral load and composition for CMV+ cases treated with both immunosuppressants and antivirals (78 patients, 543 samples). C. Viral load and composition for CMV- / - cases treated with immunosuppressants only (12 patients, 75 samples). [Figure 5]Lower anelloviral load in patients with graft rejection. A. Time dependence of anelloviral load in the subgroup of patients with severe rejection episodes (biopsy grade >2R / 3A; red data, 20 patients, 177 time points) and in the subgroup of patients without a non-severe rejection post-transplant course (blue data, 40 patients, 285 time points). Boxplots for various time periods are shown, with the center of the period indicated on the x-axis. The solid line is a cubic spline (smoothing parameter 0.75). The inset illustrates the expected inverse association between the occurrence of rejection and infection and immune competence. B. Anelloviral load relative to the average load measured for all samples at the same time point. The time-normalized load of non-rejecting patients (N=208) is compared with the load measured for patients with mild rejection events (biopsy grade 1R; N=102) and severe rejection episodes (biopsy grade >2R / 3A; N=22). The p-value reflects the probability that the median viral load will be higher for the subgroup with greater rejection risk. The p-value is calculated by randomly sampling the population with the greater amount of measurement points. For N-fold random sampling, p = sum(median(Arej) > median(Anon-rej)) / N), where N = 10, and Arej and Anon-rej are the relative viral loads for the greater and lesser risk populations of rejection and non-rejection, respectively. C. Test of the performance of relative aneroviral load in classifying patients as non-rejection vs. severe rejection receiver operating characteristic curves (area under the curve = 0.72). [Figure 6]Effect of read length on genome size and hit statistics, qPCR assays, and the measured relative abundance of species at various levels of taxonomic classification. (A) Distribution of genome sizes in a reference database containing 1401 viral genomes, 32 fungal genomes, and 1980 bacterial genomes. (B) Distribution of unique blast hits per million unique molecules sequenced (mean number of hits is shown on the x-axis). C. Distribution of genome equivalents (infectious agent / diploid human) for viruses, bacteria, and fungi (mean number of genome equivalents is shown on the x-axis). (D) Comparison of sequencing hits found per million total reads sequenced to the number of viral copies detected using qPCR. For qPCR assays, DNA was purified from 1 ml of plasma and eluted in a volume of 100 μl. (E) Measurement of CMV and parvovirus load for selected cases. The highest CMV virus (genome equivalents, virus / human diploid, GE) load measured across all samples corresponded to two cases of clinically diagnosed disseminated CMV infection (a and b, shaded areas indicate the time window of clinical diagnosis, * indicates time of death). (c) shows the time trace of a pediatric patient with CMV viremia. Parvovirus was detected in one pediatric heart transplant patient shortly after transplantation (d). * (F) Effect of read length on the measured relative abundance of species at various levels of taxonomic classification (n = 52). Spearman sample-to-sample correlation, r, and p-value, p, (two-sample Mann-Whitney U test) for abundance of the most abundant node extracted from the 50 and 100 bp datasets: r=0.80, p=0.8 (a), r=0.86, p=0.4 (b), r=0.92, p=0.6 (c), r=0.84, p=0.5 (d), r=0.7, p=0.28 (e), r=0.99, p=1 (f). [Figure 7]Mean drug doses and measured levels and the effect of drug dose on virome composition in adult heart and lung transplant patients after transplant. (A-C) Mean doses of valganciclovir and prednisone administered (A and C) and measured levels of tacrolimus in the blood (B) in adult heart and lung transplant patients for this study. (D) Compared to the viral component, the composition of the bacterial component of the microbiome is relatively insensitive to antivirals and immunosuppressants. (E) Virome composition as a function of dose of anti-CMV drug (valganciclovir) and immunosuppressant (prednisone). [Figure 8] Temporal variation in the bacterial component of the microbiome after transplantation. (A) Relative abundance of bacterial phyla as a function of time. (B) Relative abundance of bacterial genera as a function of time. [Figure 9] Virome composition and total viral load for different patient classes (A and B). Viral load and composition for CMV-positive adult heart (A), adult lung (B), and pediatric heart (C) transplant recipients treated with both immunosuppressants and antivirals. [Figure 10] CMV infection-induced allograft injury. A. Correlation (P value; Mann-Whitney U test) between clinical reports of CMV (human herpesvirus 5, HHV-5) infection from specific body fluids (BAL and serum) and donor organ cfdDNA signals matched to clinical trial date. B. P value for the correlation between clinical diagnosis of infection and cell-free DNA levels for infections with a clinically positive test result above 1 (dashed line indicates Bonferroni-corrected significance threshold). C. ROC curve (AUC = 0.91) testing the performance of CMV-derived cell-free DNA levels in CMV-positive and CMV-negative patients. [Figure 11]Infectome monitoring. A. Clinical testing frequency compared to viral infection events detected in sequencing. B. Time series data for patients with positive test results (red arrows) for specific infections compared to untested patients. (1) Adenovirus signal in L78, showing enhanced clinical positivity compared to untested patient (L34). (2) Polyomavirus signal in L69, showing one positive test result compared to a persistent signal in untested patient (L57). (3) Three herpesvirus infections (HHV-4, 5, and 8) in L58, showing both positive (red) and negative (black) test results for CMV (HHV-5), highlighted. (4) Microsporidia signal in I6, showing four positive test results compared to the signal observed in L78, who had symptoms of microsporidiosis but was untested. Data are logarithms of genome equivalents for humans, where a value of zero is replaced by the detection limit of the assay (the number of genome equivalents that matched a single sequence read assigned to the target genome).

[0127] Example Temporal response of the human virome to immunosuppression and antiviral therapy The viral component of the microbiome, i.e., the human virome, remains relatively understudied. (Wylie et al. (2012) Transl Res 160, 283-290 ), little is known about the effects of immunomodulatory and antiviral therapy on virome composition. The healthy gut virome remains remarkably stable over time (Reyes et al. (2010) Nature 466, 334-338), and the relationship between diet and virome composition. Although correlations have been found, the main source of variability is subject-to-subject differences. (Minot et al. (2011). Genome Research 21, 1616- 1625) has already been shown.

[0128] Immunosuppressive therapy significantly reduces the risk of graft rejection in organ transplants, but does not significantly reduce the risk of infection. Increases the recipient's susceptibility to viral pathogens, especially herpesviruses. Infection with cytomegalovirus (CMV) occurs frequently and increases the risk of graft failure in recipients. Therefore, organ transplant recipients should receive antiviral prophylaxis against CMV or It is often treated preemptively.

[0129] The inverse correlation between the level of immunosuppression and the risk of infection and rejection results in a narrow therapeutic window. Currently available methods for the diagnosis of infection and rejection are Numerous legal restrictions make post-transplant care even more difficult. Diagnosis of rejection is often by observer. This is due to the variability caused by the disease, the high cost and the discomfort of invasive biopsies. Symptoms of infection disappear after immunosuppression and are readily detectable by currently used diagnostic methods, such as antigen detection and P CR-based molecular tests are target-specific and therefore do not provide a priori hypotheses regarding the source of infection. Diagnosis of infection is challenging given that the diagnosis is based on the presence of HIV.

[0130] A final complication is the patient-to-patient variability in sensitivity to immunosuppressive drugs. Variability can lead to over- and under-immunosuppression, increasing the risk of infection or rejection, respectively. It can increase.

[0131] There are few intrinsic methods for measuring immune system health, and the relationship between immune function and the microbiome The relationship between the viral components of organ transplant recipients is not fully understood. Immunosuppressive and antiviral, providing a means for immunomodulatory effects on the human virome The present inventors have studied a cohort of organ transplant recipients and have reported that they are treated with post-transplant therapy in combination with drugs. To investigate drug-virome interactions in a cohort of patients (656 samples, 96 patients), and that antiviral and immunosuppressant drugs have a strong influence on the structure of the virome in plasma. To find out whether the effects of plasma sequencing on cell-free DNA are related to the The present inventors have observed a significant change in the virome composition at the start of treatment, The bacterial burden increases with immunosuppression, whereas the bacterial component of the microbiome is largely unaffected. The data reveals that the human virome, the state of the immune system, and drugs remain unknown. Willow provides insight into the relationship between the effects of physical therapy and predicts immune competence. This brings about potential uses for the system.

[0132] In this study, we investigate drug-microbiome interactions after organ transplantation. To investigate this, we sequenced cell-free DNA circulating in plasma. Prevalence of infection in heart and lung transplant recipients receiving a combination of flu- and antiviral prophylaxis. The present inventors have investigated the role of immunosuppressants and antivirals in the microbiome. We found that the structure of the soluble components of the bacteria is strongly affected by the soluble components, but not by the bacterial components. The virome composition of different individuals converges to a similar drug-determining state, resulting in a potent Composition dynamics have been observed at the initiation of drug therapy. Viruses, especially anelloviruses, can affect immune function. Because this utilizes a reduction in viral load, the total viral load increases significantly in response to therapy. The authors concluded that measurement of anelloviral load allows stratification of rejecting and non-rejecting recipients. This indicates that

[0133] 656 plasma samples were collected from 96 solid organ recipients (41 adult hearts, 24 Cell-free DNA was purified from plasma and collected longitudinally from 100,000 pediatric hearts and 31,000 adult lungs. A total of 820 gigabases (Gbp) of sequencing data was obtained (average 1.2 5 Gbp / sample) (Illumina HiSeq, 1 × 50 bp reads, Figure 1B Organ transplant recipients were consecutively enrolled in the study for at least 2 years, and the recipients Samples were collected from the donor at fixed time points after transplantation, with the highest sample collection occurring in the first month after transplantation. Figure 1C shows the frequency of peritoneal thrombosis as a function of time after transplant for various patient classes. The number of samples analyzed is indicated.

[0134] Patients in this cohort received antiviral prophylaxis and immunosuppression as part of standardized post-transplant therapy. Maintenance immunosuppression was administered to adult heart and human transplant recipients (Figure 1D). , based on tacrolimus, and mycophenolate mofetil and prednisone Pediatric patients were treated with cyclosporine-based anti-rejection therapy. Transplant recipients (and / or previous CMV infection in the recipient and / or donor) should be monitored for antiviral therapy. The recipients were treated with antiviral prophylaxis and the CMV-negative recipients were not. The plan includes high doses of immunosuppressants and antivirals for the first few months after transplant, followed by The dose is gradually reduced as the risk of rejection and infection decreases. The narrow therapeutic window and the large interpatient variability in the pharmacokinetics of tacrolimus In consideration of this, tacrolimus concentrations should be measured directly in the blood and the target drug level should be maintained. Figure 1D shows the tacrolimus-treated patients' blood levels of tacrolimus. The mean levels of rolimus are shown, and the drug treatment protocol design is shown.

[0135] DNA sequence analysis. Computerized subtraction of human sequences. After genomic DNA subtraction, microbiome-derived sequences were identified. , duplicates and low-quality reads were removed, and the remaining reads were aligned to the human reference genome [build )hg19(BWA (Li and Durbin, 2009), see Methods)] Then, unmapped reads were collected and low-complexity reads were removed. Figure 1E shows the results after applying overlap and quality filters. Distribution of remaining read fraction (average 86%) and the remaining fraction after subtraction of human reads cloth (average 2%).

[0136] To identify infectious agents, the remaining high-quality, unique, non-human reads are analyzed using BL AST was used to identify viruses (n=1401), bacteria (n=1980), and fungi (n= 32) Match against the genome reference database (downloaded from NCBI, Figure 6A). 0.12% of unique sequencing reads matched at least one of the target genomes. We aligned the sequences identified by the sequencing-based approach (Fig. 6B, C). To validate the positive hits, the target (Herpesvirus 4) identified by sequencing was Quantitative PCR (qPCR) assays targeting a subset of viruses (e.g., 5, 6, and parvoviruses) We found a significant difference between the number of viruses measured by sequencing and qPCR. A quantitative agreement was found.

[0137] The inventors further demonstrated that the sensitivity of sequencing assays for the detection of herpesviruses is significantly improved by qPC Therefore, we found that the R measurement is equivalent to the more efficient R measurement available for sequencing assays. The large capture cross-section (compared to the complete target genome and the PCR amplicon target region) Due to limited efficiency in library preparation and library undersampling This is sufficient to overcome the signal reduction in sequencing caused by The highest CMV load measured using whole-sample sequencing in This corresponded to two adult heart transplant patients with disseminated CMV infection who were treated (see Figure 6E). (I want to be).

[0138] Potential contamination in reagents used for DNA extraction and sequencing library preparation To test this, we performed two control experiments. First, we used a known template (Lamb da gDNA, Pacbio Part no: 001-119-535) Samples were prepared and DNA purified for sequencing using the workflow described above (II). umina Miseq, 3.4 million and 3.5 million reads). Excluding lambda-derived sequences. The remaining sequences (0.4%) were aligned with the BLAST reference database. No evidence was found for the various infectious agents considered in this study, The present inventors have identified Enterobacteriaceae bacteria family (phylum Proteobacteria), mainly E. coli li) and enterobacterial phages (<1%). In a second control, we found that the nuclear Sequencing samples were prepared from enzyme-free water. The sequence was used in conjunction with the pool to perform sequencing, and only a limited number of sequences (15 in total) were collected. This was then mapped to the genomes of two bacterial species, again as discussed below. No evidence of an infectious agent was found.

[0139] We used sequence similarity data obtained by BLAST to perform maximum likelihood estimation of species relative abundance. Using Grammy, a tool that uses sex data, we We investigated the plasma microbiome composition at various levels. GRAMMy was used for lead assignment. This approach takes into account the ambiguity in the reference data and the differences in target genome size. Note that the database only allows for the assessment of abundance of species for which genomic data are available. It should be noted that Figure 1F shows the relative abundance of species at various levels of taxonomic classification ( (average of all samples). We found that viruses (73%), bacteria (25%) and fungi We found that viruses were more abundant than bacteria (2%) (Figure 1F, panel a). ssDNA viruses account for a larger proportion (72%) than dsDNA viruses (28%). The present inventors have found that seven different virus families have been identified (present >0.75%), with one predominant family, Anellovirida e) accounted for 68% of the total population (Fig. 1F, panel b). The majority (97%) belong to the genus Alphatorquevirus. The Alpha Torque genus is composed of the Torque teno viruses (Figure 1F, panel c). The genus Torqueteno-Virotype (TTV) is associated with 14 different Torqueteno-Virotypes. Polyomavirus infections are widespread in human populations. Polyomavirus DNAemia is widespread in the first year after solid organ transplantation. Polyomavirus-derived sequences were identified in 7 of the 36 patients in this cohort. Found in 5 samples (11%): BK (41%), JC (27%), TS (4%), WU polyomavirus (6%), SV40 (6%) and the recently discovered H Evidence for the presence of pyV6 (13%) ( Schowalter et al., 2010 ) was found ( (Figure 1F, panel e). Among bacteria, Proteobacteria ) (36%), Firmicutes (50%), Actinobacteria Actinobacteria (10%), Bacteroidetes (Bacterioides detes) (4%) was the most abundantly represented phylum (lineage) in the sample (Figure 1F, panel f).

[0140] Examine the potential inaccurate attribution of the relatively short reads (50 bp) available for this study To this end, we conducted a longer-term analysis of the data collected for a subset of samples (n=55). Read length based on paired-end reads (2 × 100 bp) We investigated the dependence of abundance assessment on the number of nucleotides. Abundance estimates based on 0 bp reads are based on all of the taxonomic classifications described herein. We found that the levels were similar (Fig. 6F).

[0141] Virome composition sensitivity to drug treatment. Available clinical data on drug treatment. Here, we analyzed drug-microbiome interactions using Data on adult heart and lung transplant patients treated with a Mus-based anti-rejection protocol A study (47 patients and 380 observations) showed that cyclosporine-treated Pediatric patients and those transitioning from tacrolimus to cyclosporine immunosuppression due to drug intolerance issues Patients who were switched were excluded. Data on measured levels of tacrolimus in various medications were collected from individual patient records. The mean composition of the samples corresponding to the dose level was extracted. To account for the delayed effect of drug synthesis, drug level and dose data were analyzed using a sliding window average. The images were filtered with an equalization filter (see Figure 1C and Figure 7A-C; window size 45 days).

[0142] We demonstrate that the structure of the viral component of the microbiome is a sensitive function of drug dose. (47 patients, 380 samples, Figure 2A). As discussed in [1], the structure of the bacterial component of the microbiome is not significantly altered by drug therapy. (Figure 7D). In cases where the virus was transmitted, the Herpesviridae and Caudovirales were predominant in the virome. In contrast, high doses of immunosuppressants and antivirals were associated with anorexia. This resulted in a virome structure dominated by the family Mycobacterium (up to 94% occupancy at high drug levels). Viral prophylaxis is intended to prevent CMV disease, but other herpes viruses may also be affected by the drug. are sensitive, and therefore higher doses of valganciclovir may be effective against a lower proportion of It is not surprising that viruses of the order Rupesvirales are brought into the picture. The observation that viruses exploit the suppression of the host immune system is consistent with various observations from the literature. That is, the occurrence of Anelloviridae viruses is thought to be a contributing factor in the progression of HIV patients to AIDS. and that the total burden of anellovirus TTV increases after liver transplantation. Furthermore, the prevalence of Anelloviridae viruses in pediatric patients with fever has been shown to be An increase in prevalence has recently been reported.

[0143] Next, the virome composition measured in organ transplant recipients was compared with that measured in patients with immunosuppressive or antiviral drugs. Antiviral-naive healthy individuals (n=9, with available sequencing data from previous studies) The composition of healthy subjects is compared to that observed in the control group (data), where the healthy composition is compared to that corresponding to minimal drug exposure. The data were collected in organ transplant recipients at the start of drug therapy (postoperative day 1, n=13). composition, and transplant recipients exposed to high drug levels (significant time after transplant procedure) After a while, tacrolimus > 9ng / ml, valganciclovir > 600 mg, n=68) We compare the composition measured for healthy reference samples and minimal drug exposure. We found similar virome compositions for the corresponding samples (Fig. 2B). The composition of the healthy reference and minimal drug exposure samples was measured relative to the high drug exposure samples This is different from the anellovirus-dominated composition.

[0144] Tacrolimus-based immunosuppressive therapy was administered during the first 3 days after transplantation, as part of induction therapy (antibody therapy). The patient is then complemented with thymocyte globulin, daclizumab, or basiliximab, and the patient is then further treated with Patients will receive the corticosteroid prednisone throughout post-transplant treatment. The time-administration profiles of donisone and tacrolimus are similar (at the start of therapy (Figures 7A-C). Therefore, the data in Figure 2A were The combined effect of prednisone and tacrolimus on virome composition is shown. Analysis of the differential effects of valganciclovir and valganciclovir (Figure 7E) was consistent with the results observed in Figure 2A. The results show the same trend as those reported previously, with higher prednisone doses associated with anelloviral , resulting in a larger representation. Finally, we They note that a proportion of patients were not treated with antiviral drugs. Corresponding data are presented below for antiviral and immunosuppressant effects on virome composition. This allowed us to further elucidate the differential effects of the inhibitors.

[0145] Partitioning Microbiome Diversity. We partition the bacterial and viral components of the microbiome. For both bacteria and viruses, intrasubject diversity was higher than intersubject diversity. The diversity (Bray-Curtis beta diversity, phylum-level bacterial composition, family and Viruses at the level of the genotype, Figure 2C). Patients by transplant type, heart or lung, or age Splitting the data did not reduce diversity. Within subjects, samples collected within a one-month period For the samples, diversity was again lower for both bacteria and viruses. For viruses rather than bacteria, we used similar drug doses (tacrolimus) Samples collected at serum levels of ±0.5 ng / ml and valganciclovir ±50 mg When comparing the pools, we found that the diversity was lower. Combined with the population-average sensitivity to the same drug administration, we found that We found that the virome compositions of selected patients converged to a similar state.

[0146] Dynamic response of virome to changes in drug dose. A strong temporal response of the virome to the drug dose was observed, consistent with the sensitivity of the virome composition to the drug dose. Figure 3A shows the time dependence of the relative genome abundance of ssDNA and dsDNA viruses. (Data from all patient groups and samples, n=656) The virus rate increased rapidly in the first month after transplantation, then showed a reverse trend after 6 months. Figure 3B shows the time-dependent relative abundance of the most abundant viruses classified at the family and order level. and provide further details on the dynamics of virome composition (from the entire patient population and samples). (Data from n=656) The dsDNA ratios were from the Caudovirales, Adenoviridae, and Polyviridae families. The virus consists of the genoviruses Oomaviridae and Herpesviridae, which together account for 1% of the total virus count in the first week after transplantation. ssDNA viruses account for 5% of the early virome. The Adenoviridae, Caudal The proportion of cases accounted for by the order Virales and the family Herpesviridae decreased significantly in the first few months. because these virotoypes may be effectively targeted by antiviral prophylactic drugs. In contrast, the relative abundance of Anelloviridae viruses increases rapidly. Because these virotoypes largely escape targeting by antiviral drugs and affect the patient's immunity, This is because it takes advantage of the decreased immune capacity (up to 84% at 4.5-6 months). The opposite trend was observed 6 months after the treatment, indicating that the antiviral This is consistent with the reduction in rhesus and immunosuppressants.

[0147] Compared to the viral component, the bacterial component of a microorganism remains relatively stable over time, which These are observations made at the phylum, order, and genus taxonomic levels (Fig. 3C, n = 656 and Figure 3D shows the intra-sample alfa concentrations of bacterial and viral genera as a function of time. The Shannon entropy indicates the diversity of the bacteria (590 bacterial genera, 168 The observed diversity of virus genera decreased at the start of treatment (first 1.05±0.5 in month 1 to 0.31±0.33 in months 4-5, p<<10 - 6 , Mann-Whitney U test), while bacterial alpha diversity increased during the course of post-transplantation therapy. remained relatively unchanged (from 2.2±1.14 in Month 1 to 2. 6±0.85, p=0.1, Mann-Whitney U test).

[0148] Increase in total viral load at the start of post-transplant therapy. Effect of therapeutic agents on total viral load. To gain insight into this, we compared the coverage of viral targets against the coverage of the human genome. Normalizing genome coverage yields absolute coverage of all viruses relative to the human genome copy number. Genome abundance was extracted. For all patients in this study, transplant type (heart or lung) or Increased total viral load was observed at the start of treatment, regardless of age (adult or pediatric) (Figure 4A) (Loading change, 7.4 ± 3, sigmoidal fit, black line). Relative abundance data Combined with the above, total viral load data are available for patients treated simultaneously with antivirals and immunosuppressants. For patients with HIV-positive HIV, the net reduction in herpesviridae load during the first 3 months after transplantation was and a net increase in Anelloviridae load.

[0149] Therefore, the data suggest that combinations of antivirals and immunosuppressants against various virions may be beneficial. The data also show a reduction in total adenoviridae load. This indicates that Adenoviridae virus replication is associated with valgancicloviral replication, consistent with previous studies. Figure 4B summarizes the data for all transplants. However, the same trend is observed when stratified by different transplant types [Adult Heart Transplant Recipe ents (n = 268, Figure 9A), adult lung transplant recipients (n = 166, Figure 8B), and pediatric patients treated with cyclosporine rather than tacrolimus (n = 99, Figure 9C)].

[0150] All patients in the study cohort received both antiviral and immunosuppressant medications Both donor and recipient had a previous CMV antibody test result. In transplant recipients without evidence of MV infection, the risk of complications from antiviral prophylaxis is emerging. The risk of CMV infection was deemed to be greater than the potential risk of subsequent CMV infection. They are not treated with antiviral prophylaxis; therefore, these patients are treated with immunosuppressants alone. Figure 9C shows the time-dependent viral load and composition of CMV-negative cases (n=75). The net effect of immunosuppressive therapy alone is to reduce the number of viruses, including herpesviridae and adenoviridae. The increase in all virions, including HIV-1, HIV-2, and HIV-1, is due to the increased prevalence of HIV-1. Tapering of immunosuppression leads to a decrease in the total viral load.

[0151] Lower anelloviral loads in patients with transplant rejection episodes. Immunosuppression Correlation of anelloviral load with the degree of immunosuppression (see Figure 2A and Figure 4) and immunosuppression Given the association between immunogenicity and risk of rejection, we have developed a method for comparing rejecting and non-rejecting transplant recipes. We asked whether anelloviral load could be used to classify ent. Figure 5A shows Anelloviruses measured in rejecting and non-rejecting patients as a function of time after transplantation The burden is shown in Table 1, where patients have at least one biopsy-determined moderate or severe One rejection episode, biopsy grade > If the patient has 2R / 3A, the patient is classified as rejected (red (Color; 20 patients, 177 data points). Non-rejecting patients showed a significant improvement over the entire post-transplant course. corresponds to patients who are not diagnosed as having moderate or severe graft damage (blue; biopsy grade <2R / 3A, 40 patients, 285 data points).

[0152] Figure 5A shows that the anellovirus load was significantly higher in rejecting animals at almost all time points. We next investigated the anellovirus load for patients at the time of rejection. The load was directly compared to that measured for patients in the absence of rejection. Considering the time dependence of virus load (Figure 5A), we performed the same test on all samples at the same time point. The anelloviral load was calculated relative to the average load measured for each group. Patients with rejection events (biopsy grade 1R, N=102) and those with severe rejection episodes Patients (biopsy grade > Non-rejection patients compared with the load measured for 2R / 3A (N=22) The figure shows the time-normalized loads for participants (N=208). The p-values ​​are significant for patients with a high risk of rejection. It is calculated by randomly sampling the population with the larger amount of points. p = sum(median(A rej )>Median(A non-rej )) / N, where N=10 4 and A rej oh Yobi A non-rej are the larger and smaller values ​​for rejection and non-rejection, respectively. The relative viral load for the risk population (p=0.011, p=0.0002 and p=0.036).

[0153] These observations suggest that the risk of rejection and the occurrence of infection are inversely related to the patient's immune competence. This is consistent with the view that rejection patients The lower viral load observed for these patients was due to the fact that they were receiving the same immunosuppressive protocol. This subgroup of patients showed a higher level of immunocompetence, even when treated with It is known that there is inter-patient variability in sensitivity to immune suppression. Lack of predictability in immunosuppression is a significant risk factor in transplantation. Current commercial assays used to measure predict acute rejection or significant infection Therefore, it is not known whether it can replace or complement existing assays. It will be important to develop methods for the direct measurement of immune function in organ transplant recipients. The total anellovirus load recorded in the sera could serve as a surrogate marker. shows the receiver operating characteristics and the relative anelloviral load in the non-rejecting and rejecting patient categories The performance of the sigma-based ...

[0154] By sequencing cell-free DNA in the recipient's plasma, the inventors were able to Drug-microbiome interactions were studied after organ transplantation. The data were based on human virions in plasma. It tells us a lot about the basic structure of the drug and how it influences pharmacology. respond to pharmacological perturbations They also demonstrate the role of the composition of the bacterial component of the microbiome in immune suppression. These data suggest a relative insensitivity to HER2. These data will inform the design and optimization of post-transplant treatment protocols. For example, they are useful in tapering antiviral prophylaxis from initial high doses. This indicates that CMV DNA load leads to the resurgence of the Herpesviridae fraction. It has already been shown to predict disease recurrence and rejection, allowing patients to choose longer-term preventative therapy. The question arises as to whether the government will benefit from this.

[0155] The significant increase in abundance of Anelloviridae viruses upon immunosuppression also merits further investigation. Anelloviruses are ubiquitous in the human population and, although not pathogenic, Viruses are currently being investigated as potential cofactors in carcinogenesis. The potential for immunosuppression by Anelloviridae viruses is particularly significant given the increased incidence of cancer seen in The susceptibility of organ transplants to the Anelloviridae virus makes them a valuable tool for studying their properties. This would be an ideal situation. Observation of the burden of immunosuppression was performed while these patients received the prescribed levels of immunosuppressant according to the protocol. Even if the immunosuppression was still present, it would be an indicator of inadequate immunosuppression in this patient subgroup. This involves directly measuring the level of a patient's immune function in addition to measuring circulating drug levels. These findings suggest that it would be useful to design assays that would enable transplant recipients The total anellovirus load identified in the blood of such individuals is related to their immunosuppression. It can serve as one of the markers of the overall condition of the body.

[0156] High-throughput DNA sequencing is hypothesis-free for infection It is useful in diagnosis. Infections occur frequently in transplantation and in immunosuppressed individuals. Considering the difficulty of diagnosing the disease, and sequence analysis, the presence of circulating donor Considering that quantification of derived human DNA may provide further information regarding the health of the graft, This approach is particularly suitable for transplantation. Subtractive methods to eliminate host DNA and enrich for viral and microbial DNA It may be useful to develop a subtractive method.

[0157] Experimental Method Clinical sample collection: Patients were admitted to Stanford University Hospital. al(SUH) or Lucile Packard Children's Hosp ital (LPCH) and if they were recipients of multiple organ transplants This study was funded by the Stanford University Institute Approved by the National Review Board (Protocol # 17666) Enrollment began in March 2010. Details of patient recruitment and post-transplant treatment of patients are available at See the detailed Experimental Methods section for details.

[0158] Plasma processing and DNA extraction: Plasma was processed as previously described (Fan et al., 200 8) was extracted from whole blood samples within 3 hours of sample collection and stored at -80°C. If required for analysis, plasma samples were thawed and analyzed using the QIAamp Circulating Nucleic Acid Kit (Circu 0. using a lating Nucleic Acid Kit (Qiagen). Circulating DNA was immediately extracted from 5 ml of plasma.

[0159] Sequencing library preparation and sequencing: Standard Illumina index NEBNext DNA for Illumina with adapters (purchased from IDT) Use the Library Prep Master Mix Set or A microfluidics-based automated library preparation platform (Mondria n ST, Ovation SP Ultralow Library System) Sequencing libraries were prepared from purified plasma DNA from patients. lent 2100 Bioanalyzer(High sensitivity D The samples were characterized using a NA kit and quantified by qPCR. The sequences were part of a larger sequencing run and sequenced over a 22-month period. An average of six samples were sequenced.

[0160] Post-transplant monitoring and clinical sample collection. This analysis is used to monitor acute and chronic disease after thoracic organ transplantation. and Donor-Derived Cell-Free DNA Assays for the Diagnosis of Chronic Rejection and Allograft Failure National Institutes of Health to study clinical utility Previously funded by the National Institutes of Health (RC4 AI092673). This corresponds to a substudy of a prospective cohort study. Stanford University H hospital (SUH) or Lucile Packard Children's We enrolled patients who underwent heart or lung transplants at the LPCH Hospital and If the patient was a recipient of a multiorgan transplant or if they underwent SUH or LPCH after transplantation They were excluded if they were followed up at an outside institution. University Institutional Review Board (Pro The study was approved by the National Institute of Clinical Trials and Clinical Oncology (NIH) under the Protocol #17666, and enrollment began in March 2010.

[0161] Post-transplant treatment protocol, detailed for adult heart transplant recipients. Post-transplant immunosuppression is 500 mg of ruprednisolone was administered immediately after surgery, followed by 125 mg every 8 hours for 3 doses. The study consisted of administering 1 mg / kg of antithymocyte globulin (rATG). Maintenance immunosuppression consisted of prednisone 2 mg / kg / day on postoperative days 1, 2, and 3. 0 mg twice daily, tapered to <0.1 mg / kg / day by the sixth postoperative month, and The plan consisted of further tapering if endomyocardial biopsy showed no evidence of cellular rejection. Clolimus administration was started on the first day after surgery, 10-15 ng / ml from month 0 to month 6, and 6 By December, the level was 7-10ng / ml, and thereafter the level remained at 5-10ng / ml. The dose was further adjusted to allow for a longer treatment time. Mycophenolate mofetil was administered every day from the first day after surgery. Start with 1,000 mg twice daily, and adjust as needed to address leukopenia. was carried out.

[0162] All patients were screened on postoperative day 1 unless both donor and recipient were CMV negative. 5 mg / kg (IV) of gansik starting at 100 mg / kg / day and adjusted hourly according to renal function. Recipients received standard CMV (antiviral) prophylaxis consisting of ribovir. Valganciclovir 900 mg twice daily for 2 weeks if oral medication is tolerated The patient was placed on a daily dose of 900 mg for the first 6 months after surgery, after which the patient was placed on a daily dose of 900 mg. The patient was administered 450 mg daily until the 12th postoperative month, at which point antiviral prophylaxis was initiated. In the event of leukopenia, the dose of valganciclovir was reduced. CMV in allografts - recipients also received CMV hyperimmune globulin 150 mg / kg IV within 72 hours of transplantation and 100 mg / kg IV the second 4, 6, and 8 weeks, and 50 mg / kg at 12 and 16 weeks post-transplant.

[0163] CMV - CMV in allografts - Recipients were on antiviral prophylaxis until May 2012. These recipients were then treated with acyclovir 400 mg daily. Antifungal prophylaxis consisted of daily antibiotics for the first 3 months after transplantation. The treatment consisted of administering 300 mg of laconazole to treat Pneumocystis jirovecii (pneumocyst). Prophylaxis against Mystis jiroveci infection is with trimethoprim / sulfamethoxa. The treatment consisted of daily administration of 80 mg of TMP component. Infection prophylaxis is continued indefinitely, and patients intolerant to TMP-SMX should receive atovaquone (at ovaquone, dapsone, or inhaled pentamidine midine).

[0164] All heart transplant recipients were randomly assigned to receive a cardiac transplant at scheduled post-transplant intervals (weekly in the first month, weekly until the third month). Every other week until the 6th month, then every month until the 9th, 12th, 16th, 20th and 24th month) Endomyocardial biopsy to monitor for acute cellular rejection Biopsies were graded according to the ISHLT 2004 revised grading scale (0, 1R, 2R, 3R). (29) Blood samples were collected from heart transplant recipients at the following time points after transplantation: Weeks 2, 4, and 6; Weeks 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 16, and 20 and 24 months. Blood samples were also collected from some heart transplant recipients on the first day after transplant. If blood sampling and endomyocardial biopsy were performed on the same day, the time of the biopsy procedure Care was taken to ensure that blood was collected beforehand.

[0165] Pediatric heart transplant recipient. Induction immunosuppression was initially administered with daclizumab. ab) 1 mg / kg IV every 2 weeks for a total of 5 doses, From August 1 year, basiliximab 10 mg was administered on days 0 and 4 after surgery. The recipient was also immediately switched to pulse methylprednisolone. Patients were treated with 10 mg / kg IV every 8 hours for 3 doses, followed by prednisolone. were treated with nisone 0.5 mg / kg twice daily for the first 14 days after transplantation; Corticosteroids were then tapered during the first year after transplantation in the absence of acute rejection.

[0166] Calcineurin inhibition was primarily observed at 300-350 ng / ml during the first 3 months after transplantation. , 275-325ng / ml in April-June, 250-300ng / ml in July-December l, and cyclosporine at a target level of 200-250 after the 12th month post-transplant. Patients intolerant to cyclosporine were treated with tacrolimus. For prevention and surveillance of potential infections, endomyocardial biopsy The protocol was similar to that for adult heart transplant recipients.

[0167] Lung transplant recipient. Post-transplant immunosuppression is methylprednisolone 500-1000mg mg immediately after surgery, followed by 0.5 mg / kg IV twice daily. Basiliximab 20 mg I on days 0 and 4 was administered for induction immunosuppression. Maintenance immunosuppression consisted of methylprednisolone 0.5 mg / kg IV postoperatively. Administered twice daily on days 0-3, followed by prednisone 0.5 mg / kg every day until 30 days. 0.1 mg / kg (daily dose) every 2-3 months from the 6th to 12th month after transplantation. Tacrolimus administration was initiated on postoperative day 0 and continued until 12-15ng / ml from January to June, 10-15ng / ml from June to December, and After this, the dose was adjusted to maintain a level of 5-10 ng / ml. Administration of mofetil was initiated at 500 mg twice daily from postoperative day 0 to treat leukopenia. Dose adjustments were made as needed to address the patient's needs. Antiviral, antifungal, and PCP prophylaxis were successful. This was similar to that in the human heart transplant cohort.

[0168] All lung transplant recipients were randomly assigned to receive a 24-month follow-up period at 1.5, 3, 6, 12, 18, and 24 months after transplantation. Patients were monitored for acute cellular rejection by transbronchial biopsy protocol. Biopsies were performed when clinically indicated based on the results of pulmonary function tests. Blood samples were collected from lung transplant recipients for research purposes at the following intervals: on day 1 after transplantation; 2 times, 2 times on the 2nd day and 1 time on the 3rd day, then 1 and 2 weeks, and 1.5, 2 , 3, 4.5, 6, 9, 12, 18 and 24 months. Per protocol and clinically indicated Blood samples were taken before the biopsy was performed.

[0169] Workflow for identifying pathogen-derived sequences. C-based utilities (C-bas) Strict duplicates were removed using the fastq.cpp utility. stx package (fastq quality filter -Q33 -q21 - Use the quality filter that is part of p50 to filter low quality The remaining reads were then aligned to the human reference genome build hg using BWA. Aligned to 19(bwaaln - q25). samtools(samt Use the tools view -f4) to collect unmapped reads and Low-complexity reads using eqclean (seqclean -l 40 -c 1) The reads were then compared to selected viral, bacterial and fungal reference genomes and Total reference body (ncbi Align it against the one downloaded with fungi Ta.

[0170] Figure 6A shows the genome size distribution. The following parameters were used for BLAST alignment: The data used was: reward = 1, penalty = 3. Word Size=1 2, Gap open = 5, Gap extend = 2, e value = 104, Perc_identity = 90, Culling limit limit) = 2. Blast hits with alignment lengths shorter than 45 were removed. For a subset of samples, longer reads are available (23 100b p, n=55). To test the robustness of the genome abundance estimates, we performed compositional measurements. The length dependence of the reads was investigated. Here, the reads were 40, 50, 65, 80 and 100 bp. Adjust the length (Fastx trimmer) and analyzed using the workflow described above. Here, bl with alignment lengths shorter than 37, 45, 59, 72 and 80 bp at hits were removed for 40, 50, 65, 80 and 100 bp reads, respectively Genome abundance estimation. Relative genome abundance estimation was calculated using GRAMMy. The model uses BLAST-derived nucleic acid sequence similarity data to estimate the relative abundance of species in a sample. GRAMMy performs maximum likelihood estimation of the BLAST alignment matrix (E-score, Hits were filtered by alignment length and percent identity to compare the relative positions of the candidate reference genome. Considers ambiguity in read assignment and target genome size in pair abundance assessment. mmy was invoked with the following parameters: python grammy rd t.py;python grammy pre.py -q ''40,40,1'' Input set (input set); python grammy em.py -b 5 -t 0.0001 -n 100 input.mtx;grammy p ost.py input.est setinput.btp.

[0171] Strain-level abundance estimates can be combined to obtain abundance at higher taxonomic levels of abundance. To do this, we used a custom script. Here, we use Taxtastic to A minimal taxonomy for the reference database was constructed.

[0172] Absolute viral load quantification. To quantify the infectious agent load in a sample, The results of the last hit were collected and analyzed using a custom script (Bioperl) for each link. The best hits were selected for the sequence. Figure 6B shows the results of the 1 million unique sequences sequenced. The distribution of the number of unique viral, bacterial, and fungal blast hits per offspring is shown in Figure 6. C is the number of viral, bacterial, and bacterial genome copies compared to the number of human genome copies present in the sample. The genome coverage of infectious agents is normalized to the human genome coverage. It has been transformed.

[0173] qPCR validation of sequencing results for selected viral targets. Human herpesvirus Standard qPCR kits for quantification of viruses 4, 5, and 6 and parvoviruses (Pri merDesign, genesig) on ​​a subset of cell-free DNA samples. The sequencing results were verified. qPCR was performed on cfDNA eluted in buffer (50 mM [pH 8.1–8.2]). The plasma extraction and PCR experiments were performed in different facilities. A no-template control was used to verify that the PCR reagents were included. The relative number of blast hits per million reads obtained was determined using qPCR. The concentration of viral genome copies determined is compared to the concentration of viral genome copies determined.

[0174] No-template control. A no-template control experiment was performed. The sequencing library was nuclease-free. The library was prepared from water (S01001, Nugen). Seven additional sample libraries (without The DNA was prepared with 100% human DNA (100% human DNA). To ensure the formation of dense clusters, the samples were compared with samples unrelated to the study. A sample unrelated to the study collected 16 million reads, The no-template control library was constructed using two species in the reference database [Methanochalcodocus japonicus]. Nashui (methanocalcodoccus janaschii) (9 hits) and Bacillus subtillis (5 hits) Only 15 reads were found that were mapped to the human-related sequences. No evidence was found, indicating that sample-to-sample contamination was low.

[0175] Example 2 Clinical monitoring of the microbiome The reads mapped to the CMV genome were analyzed using the method described in Example 1. In samples that were clinically positive for infection, CMV presence was quantified. An increase in the presence of-9 , Mann-Whitney U test, Figure 10C ). The level of CMV-derived DNA in our samples was 0.91 AUC for CMV. This data is consistent with clinical reports. Using the same sequence data, CMV surveillance was shown to be a predictor of rejection. This indicates that the monitoring can be carried out in parallel with the other We investigated whether viral infections could be similarly monitored.

[0176] We investigated the role of well-characterized pathogenic viruses and oncoviruses (Figure 11A ) and the symbiotic Torque Teno virus (TTV, genus Alpha Torquevirus ). This is consistent with previous observations of an association between immunosuppression and TTV abundance. The frequency of clinical trials for these viruses varies considerably, with CMV (human herpesvirus 5, HHV-5 (n=1082 studies in our cohort) compared with other pathogens The inventors compared the frequency of clinical screening with that of the 2011 study. Evaluate the incidence of infection (the number of samples in which a given virus is detected by sequencing) CMV was the most frequently screened (335 samples) but was not sequenced. The incidence determined by (detected in 22 samples) was polyomaviruses (clinically tested in four and one cases, respectively; Figure 1 This was similar to the case of other pathogens not routinely screened, including 1A).

[0177] Adenovirus is a community-acquired infection that can cause graft loss in lung transplant recipients. Adenoviruses are respiratory infectious agents that pose a particular risk to pediatric patients. A sample from one pediatric patient (L78, Figure 11B panel 1) tested positive for This patient also had the highest adenovirus-derived DNA load in the entire cohort. Studies are typically limited to pediatric lung transplant patients and therefore not clinically screened. In several other adult transplant patients (e.g., L34, Figure 11B, panel 1) who did not have a persistent A significant adenoviral load was also observed.

[0178] Polyomaviruses are a major cause of allograft rejection after kidney transplantation, usually It is not included in post-lung transplant surveillance. We have not tested for this pathogen in two patients. We detected polyomavirus in two patients (L57 and L15, Figure 11B panel 2). In both cases, clinical records revealed persistent symptoms that may have resulted from polyomavirus infection. The patient presented with persistent renal failure.

[0179] In a final example of the benefits of broad, hypothesis-free screening for infection, we have demonstrated that Human herpesvirus (HH), an oncovirus that can cause complications after organ transplantation, We investigated a patient who showed a high burden of V)8 (Figure 11B, panel 3). This patient (L58) Two other herpesviruses (HHV-4) have the potential to stimulate HHV-8 reactivation. Positive test results were obtained for HHV-a and HHV-5. Post-transplant monitoring for HHV-8. While sequencing is only recommended in certain clinical situations, the use of sequencing is not without its use. This allows identification of the virus in unsuspected cases that would otherwise go undetected.

[0180] Clinical monitoring of the microbiome. In addition to viruses measured in serum, other Fungal or bacterial infections detected in body fluids [Klebsiella detected by urine culture] Klebsiella pneumonia infection (ROC=0.98) The present study also examines the correlation between acellular measurements and fungal infections detected in BAL. The authors observed that the characteristics of bacterial and fungal interactions are related to the type of infection and the organism in question. The inventors found that the IL-14 receptor agonist was sensitive to both bodily fluids, which are more closely associated with blood. We observed better performance in terms of the signal intensity and also a lower sensitivity to background signals. For example, the most commonly cultured bacterial infection (Pseudomonas domonas) in cell-free assays in over 80% of our patient samples. This is the highest level of detection, having been detected in only 6% of our patient samples. This was in stark contrast to the commonly detected viral pathogenic species (CMV).

[0181] This is a distinction between commensal infections (including Pseudomonas) that are part of the normal flora and exclusively pathogenic infections (including Pseudomonas). This highlights the important difference between non-symbiotic infections with lower background signals. This difference is due to the difference in the number of symbionts compared to non-symbionts (AUC = 0.91 for CMV). Commonly cultured commensal infections [e.g., Pseudomonas aeruginosa (P. aeruginosa)] AUC = 0.66 and 0 for Bacillus inosa and E. coli, respectively. This may explain the differences in sensitivity and specificity measured for

[62] . In this case, the clinical problem is not presence or absence, but presence or absence in the wrong body area. It is non-existent.

[0182] In our cohort, we found that immunosuppressed patients with enteric infections We also detected cell-free DNA derived from microsporidia, a non-symbiotic fungus that can cause ulcers. in patient L78 (Figure 11B panel 4), who presented with typical symptoms of microsporidiosis. Persistent microsporidia load was measured. Adenovirus infection (L78, Figure 11B panel 1) was the suspected cause, although the results of endoscopy and sigmoidoscopy were inconclusive. In addition, fecal samples tested negative for C. diff and adenovirus. Based on our sequencing data, microsporidiosis is the most likely explanation for the patient's symptoms. This is a possible explanation, because the microsporidia signal measured in this patient was Similar to the case of patient I6 from an unrelated cohort who tested positive for larvae Because it is.

[0183] Circulating cell-free DNA, with over 10 billion fragments per ml of plasma, is essential for the human physiology. It is an information-rich window into the dynamics of genome transplantation. Cancer diagnosis and cancer treatment monitoring using Gravity Transfer Dynamics (GTD) rapidly expanding fields in genetic prenatal diagnosis and monitoring of cardiac transplant rejection. In this study, the inventors have demonstrated that the poor survival rate and allograft rejection Lung transplantation is a particularly challenging type of solid organ transplant limited by imprecise and invasive testing for The principles of GTD were applied to transplantation.

[0184] Lung transplant recipients with allograft infection and acute rejection may present clinically with similar symptoms. Therefore, we have extended the scope of GTD to the monitoring of infectious diseases. First, cfDNA derived from CMV, a major cause of post-transplant graft damage, and clinical trial results The inventors further demonstrated a strong correlation between positive clinical trial results and associated symptoms. Adenovirus in patients with similar microbial cfDNA levels compared with patients with Numerous untested pathogens, including undiagnosed cases of flu, polyomavirus, HHV-8, and microsporidia The study demonstrated the potential for hypothesis-free infection monitoring. These examples demonstrate the effectiveness of sequencing-based broad-spectrum infection monitoring compared to pathogen-specific testing. This approach may aid in determining the outbreak and source of infection. In the case of transplants, there is a high incidence of infection, rejection and irritation. Infections may occur simultaneously, making it difficult to distinguish between symptoms of infection and rejection. However, the approach may be particularly important in the case of such transplants.

[0185] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions will now be apparent to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby. In one aspect, the present invention provides the following. [Item 1] 1. A method for determining the presence and prevalence of a microbial sequence in a sample of cell-free nucleic acid from a non-microbial host, comprising: (i) providing a sample of cell-free nucleic acid from an individual; (ii) performing high-throughput sequencing of the nucleic acid; (iii) performing bioinformatics analysis and subtracting host sequences from the analysis; (iv) determining the presence and prevalence of microbial sequences for microbiome assessment of the non-microbial host. [Item 2] 2. The method of item 1, wherein the presence and prevalence of multiple microorganisms is determined. [Item 3] 2. The method of item 1, wherein high-throughput sequencing is performed on a nucleic acid sample amplified by an unbiased method. [Item 4] At least 10 6 2. The method of item 1, wherein sequence reads are obtained. [Item 5] 2. The method of claim 1, wherein step (iv) comprises comparing the coverage of the sequence mapped to the microbial reference sequence with the coverage of the host reference sequence. [Item 6] 2. The method of claim 1, wherein step (iii) comprises identifying a reference host sequence and masking microbial or microbial-mimicking sequences present in the reference host genome. [Item 7] The method of item 1, wherein step (iii) comprises identifying a reference microbial sequence and masking host sequences or host-mimicking sequences present in the reference microbial genome. [Item 8] 2. The method of item 1, wherein the presence of one or more pathogenic microorganisms is confirmed. [Item 9] 2. The method of item 1, wherein the analysis is performed at two or more time points. [Item 10] 2. The method of item 1, wherein the amount of the one or more microbial nucleic acids is indicative of an infection status or treatment outcome. [Item 11] 11. The method of item 10, wherein the amount of said one or more nucleic acids above a predetermined threshold is indicative of an infectious state or treatment outcome. [Item 12] 2. The method of item 1, wherein the sample is selected from the group consisting of blood, serum, urine and feces. [Item 13] 2. The method of item 1, wherein the nucleic acid is selected from the group consisting of double-stranded DNA, single-stranded DNA, single-stranded DNA hairpin, DNA / RNA hybrid, single-stranded RNA, double-stranded RNA and RNA hairpin. [Item 14] 2. The method of item 1, wherein the nucleic acid is selected from the group consisting of double-stranded DNA, single-stranded DNA and cDNA. [Item 15] 10. The method of claim 1, further comprising providing an assessment of the microbiome to the individual. [Item 16] Item 17. The method of item 1, wherein assessment of the microbiome results in a determination of response to treatment. 2. The method of item 1, wherein assessment of the microbiome provides a measure of human physiology. [Item 18] 2. The method of item 1, wherein the assessment of the microbiome is used to calculate a pathogenicity score for the microorganisms present in the non-microbial host. [Item 19] 1. A computer-readable medium, comprising: (i) receiving high-throughput data from one or more cell-free nucleic acids detected in a sample from a subject; (iii) performing bioinformatics analysis and subtracting host sequences from the analysis; (iv) a computer-readable medium comprising a set of instructions recorded on the computer-readable medium for causing a computer to perform the steps of determining the presence and prevalence of a microbial sequence. [Item 20] Preparing a sample from a subject; determining the presence or absence of one or more microbiome nucleic acids in the sample; A method of assessing immune competence in an individual, comprising assessing immune competence based on the presence of one or more microbiome nucleic acids as described above. [Item 21] 21. The method of item 20, wherein the virome components of the microbiome are analyzed. [Item 22] 22. The method of item 21, wherein temporal differences in the amounts of the one or more virome nucleic acids are indicative of immunocompetence status. [Item 23] 22. The method of item 21, comprising quantifying the viral load in the individual. [Item 24] 24. The method of item 23, wherein the virome is analyzed for anellovirus viral load. [Item 25] 21. The method of item 20, wherein the individual is undergoing an immunosuppressive regimen. [Item 26] 21. The method of item 20, wherein the individual has undergone a transplant. [Item 27] 27. The method of item 26, wherein the transplant is selected from the group consisting of bone marrow transplant, kidney transplant, heart transplant, liver transplant, pancreas transplant, lung transplant, intestine transplant and skin transplant. [Item 28] 21. The method of item 20, wherein the nucleic acid is circulating cell-free DNA. [Item 29] 21. The method of item 20, wherein the presence or absence of the one or more nucleic acids is determined by a method selected from the group consisting of sequencing, nucleic acid array, or PCR. [Item 30] The method of item 20, wherein the amount of the one or more nucleic acids is indicative of transplant status or outcome. 31. The method of item 30, wherein the amount of said one or more nucleic acids above a predetermined threshold is indicative of transplant status or outcome. [Item 32] 31. The method of item 30, wherein the threshold value is a normative value for clinically stable post-transplant patients who show no evidence of transplant rejection or other pathology. [Item 33] 31. The method of item 30, wherein there are predetermined thresholds that vary depending on the outcome or condition of the transplant. [Item 34] 31. The method of item 30, further comprising treating the individual following assessment of immune competence.

Claims

1. (a) providing a sample comprising cell-free nucleic acid, the sample being obtained from a subject at risk for a respiratory infection, the sample being selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, and synovial fluid; (b) treating the cell-free nucleic acid in the sample by unbiased amplification to produce amplified cell-free nucleic acid; (c) performing high-throughput sequencing of the amplified cell-free nucleic acid to generate sequence reads that include sequence reads from at least one microorganism associated with the respiratory infection; (d) detecting the at least one microorganism associated with a respiratory infection by subtracting host sequences from the generated sequence reads and then assigning microorganism sequence reads to a microorganism reference sequence.

1. A method for detecting microorganisms associated with respiratory infections, comprising:

2. 10. The method of claim 1, wherein the at least one microorganism associated with a respiratory infection comprises a virus, a bacterium, a fungal species, a protozoan parasite, or a combination thereof.

3. 10. The method of claim 1, wherein said detecting said at least one microorganism associated with a respiratory infection determines the presence and prevalence of said at least one microorganism associated with a respiratory infection.

4. 10. The method of claim 1, further comprising, prior to step (c), ligating an adapter to the cell-free nucleic acid to produce adapted nucleic acid from the at least one microorganism associated with a respiratory infection.

5. 5. The method of claim 4, wherein step (c) comprises sequencing the adapted nucleic acid from the at least one microorganism associated with a respiratory infection.

6. 10. The method of claim 1, wherein the cell-free nucleic acid comprises DNA.

7. 10. The method of claim 1, wherein the cell-free nucleic acid comprises RNA.

8. 10. The method of claim 1, wherein the cell-free nucleic acid comprises cell-free DNA and cell-free RNA.

9. 10. The method of claim 1, further comprising identifying said at least one said microorganism to the strain or species level.

10. 10. The method of claim 1, further comprising determining the amount of nucleic acid from the at least one microorganism associated with a respiratory infection.

11. 11. The method of claim 10, wherein the amount of nucleic acid from the at least one microorganism associated with a respiratory infection is indicative of the infectious status of the subject.

12. The method of claim 1, further comprising providing the subject or the subject's healthcare provider with an assessment of the presence of the at least one microorganism associated with a respiratory infection.

13. 10. The method of claim 1, further comprising amplifying cell-free nucleic acid from said at least one microorganism associated with a respiratory infection by unbiased amplification prior to step (c), wherein said cell-free nucleic acid comprises DNA.

14. 2. The method of claim 1, wherein the unbiased amplification comprises ligating an adapter to the nucleic acid and amplifying with a primer specific to the adapter.

15. 2. The method of claim 1, wherein the unbiased amplification comprises polymerase chain reaction (PCR) using universal primers.

16. The method of claim 1 , wherein the sample is a plasma sample.

17. 10. The method of claim 1, wherein step (c) generates at least 10,000,000 sequence reads.

18. 10. The method of claim 1, wherein the at least one microorganism associated with a respiratory infection comprises an adenovirus.

19. 10. The method of claim 1, wherein the at least one microorganism associated with a respiratory infection comprises human respiratory syncytial virus, cytomegalovirus (CMV), human rhinovirus, human influenza virus, or human SARS coronavirus.

20. The method of claim 1, further comprising preparing a second sample obtained from the subject at a second time point and repeating steps (b) to (d) on the second sample.

21. 10. The method of claim 1, wherein the at least one microorganism associated with a respiratory infection comprises a virus.

22. 10. The method of claim 1, wherein the at least one microorganism associated with a respiratory infection comprises a bacterium.

23. 10. The method of claim 1, wherein the at least one microorganism associated with a respiratory infection comprises a fungal species.

24. 10. The method of claim 1, wherein the at least one microorganism associated with a respiratory infection comprises a protozoan parasite.

25. 2. The method of claim 1, wherein the sequence reads are 50 bp sequence reads.

26. 10. The method of claim 1, wherein the subject at risk for respiratory infection is an organ transplant recipient.

27. 27. The method of claim 26, wherein the organ transplant recipient is a bone marrow transplant recipient.

28. 10. The method of claim 1, wherein the subject at risk for respiratory infection is immunosuppressed.

29. 1. A computer readable medium for identifying the presence or prevalence of a cell-free microbial sequence in a sample containing cell-free nucleic acid, the sample being obtained from a subject at risk for a respiratory infection, the sample being selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, and synovial fluid, the computer readable medium comprising a set of instructions recorded thereon, the set of instructions, when executed by a processor, comprising the steps of: (a) performing a bioinformatics analysis on high-throughput sequencing data, wherein the high-throughput sequencing data is derived from amplified cell-free nucleic acids, the amplified cell-free nucleic acids being generated by unbiased amplification of cell-free nucleic acids in a sample comprising said cell-free nucleic acids, and wherein the bioinformatics analysis subtracts non-microbial host sequences from the high-throughput sequencing data; (b) identifying the presence or prevalence of the cell-free microbial sequences to assess the presence or prevalence of individual strains or species of bacteria, protozoan parasites, or fungal species within components of the microbiome of a non-microbial host.

30. A computer readable medium having a set of instructions recorded thereon, the set of instructions, when executed by a processor, performing the steps of: (a) receiving high-throughput sequencing data from one or more nucleic acids detected in a sample comprising cell-free nucleic acid from a subject, the sample being obtained from a subject at risk for respiratory infection, the sample being selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, and synovial fluid, and the high-throughput sequencing data being obtained from amplified cell-free nucleic acid, the cell-free nucleic acid being generated by unbiased amplification of cell-free nucleic acid in the sample comprising cell-free nucleic acid; (b) performing a bioinformatics analysis to subtract a sequence of interest from the high-throughput sequencing data from one or more nucleic acids detected in the sample of cell-free nucleic acid; and (c) identifying the presence or prevalence of cell-free microbial sequences within components of the microbiome of the non-microbial host, at the viral strain or species level; The computer-readable medium on which the

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