Compositions and methods for enriching populations of nucleic acids

Abstract

To provide methods and compositions useful for enriching a particular nucleic acid population (population of interest) in a complex mixture of nucleic acids.SOLUTION: The population of interest may make up a minor portion of a complex mixture of nucleic acids. Methods described herein are methods to identify non-host nucleic acids in a sample where host nucleic acids are present and involve the selective enrichment of non-host nucleic acids relative to the host nucleic acids in a host sample, such as a cell-free plasma sample from the host. The enriched nucleic acids may then be analyzed in order to identify the presence of the non-host nucleic acids and the presence of a pathogen or infectious organism within the host. The methods are useful for detecting, predicting, diagnosing, or monitoring a disease or disorder, particularly a disease or disorder caused by a foreign microbe or pathogen.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 163,273, filed May 18, 2015 and U.S. Provisional Application No. 62 / 334,348, filed May 10, 2016, the disclosures of which are hereby incorporated by reference in their entirety.

Background Art

[0002] Infectious diseases and disorders pose challenges to both primary care providers and patients alike, and in particular often have low detection rates compared to other diseases. Inadequate detection of infectious diseases can result from several factors, including the lack of a meaningful test that can quickly generate an accurate response. Given the slow speed of some diagnostic tests, many physicians choose to treat patients based on suspicion of the cause before receiving test results, rather than risk the worsening of symptoms during the waiting period. Also, since tests for infectious diseases are generally pathogen-specific, physicians must have some idea of the causal factors of the patient's symptoms before ordering the test. Another confounding factor for some infectious diseases is that the infectious agent may mutate during the course of the infection, such that the initial diagnosis may not accurately reflect the nature of the patient's condition at a later time. Secondary and co-infections can also obscure other sources of infection or completely escape detection, thus confounding diagnosis and treatment.

Summary of the Invention

Problems to be Solved by the Invention

[0003] False and under-diagnosis of pathogen infections can have tragic consequences for patients as well as the community as a whole. ​​​​​​​​​​​​There is a risk of this happening. For example, the overuse or misuse of antibiotics can lead to an increase in antibiotic-resistant bacteria. This could exacerbate the condition, which is dangerous not only for the patient but also for other people who come into contact with them. Therefore, it is a reliable, comprehensive, and affordable way to identify pathogens in a sample. Inspection is required in this field of technology. [Means for solving the problem]

[0004] This disclosure generally refers to non-host nucleic acids in samples taken from a host and in which host nucleic acids are present. This provides a method for identifying a cell-free sample taken from a host. It has various applications, including the identification of infectious or pathogenic organisms within a host through analysis. Generally, the methods described herein involve host samples such as cell-free plasma samples from a host. This may include selective enrichment of non-host nucleic acids relative to host nucleic acids derived from within. Then, the enriched Nucleic acids are used to identify the presence of non-host nucleic acids and the presence of pathogens or infectious organisms within the host. It can be analyzed. Identification of the presence of non-host nucleic acids, pathogens, or infectious organisms indicates that the host This may enable the detection, diagnosis, prognosis, monitoring, or staging of infectious diseases or disorders experienced. .

[0005] For example, the present disclosure relates to a method for identifying a pathogen in a host, wherein the method involves a cell-free blood sample from the host. Alternatively, a method is provided to begin by preparing a plasma sample. Then, blood or The plasma sample is concentrated with respect to non-host nucleic acids relative to host nucleic acids, and then concentrated The sample was analyzed for non-host nucleic acids, and then the pathogens in the host were identified from the non-host nucleic acids. It can be determined.

[0006] In one aspect, the disclosure relates to priming or priming non-host sequences in nucleic acid samples from a host. A method for capturing, comprising the steps of (a) preparing a nucleic acid sample from a host, (b) Nucleic acid samples from the host include host nucleic acids and non-host nucleic acids; (b) from the host A nucleic acid sample is mixed with a collection of oligonucleotides, thereby obtaining a mixture. The step involves a collection of oligonucleotides having different nucleotide sequences. It contains at least 1000 oligonucleotides, and the different nucleotide sequences are few. Steps specifically selected to contain at least a 10-nucleotide-length non-host nucleic acid sequence. (c) In the mixture, contact the collection of oligonucleotides with the nucleic acid sample. The step involves bringing the non-host nucleic acids in the mixture into contact with at least 10 nucleotides. It binds to a long non-host nucleic acid sequence, thereby priming or capturing the non-host nucleic acid. Upon contact, up to 10% of the host nucleic acid is converted into a non-host nucleic acid sequence of at least 10 nucleotides in length. The present invention provides a method that includes the step of combining with

[0007] In some embodiments, this method involves priming or capturing non-inhabitants in the reaction. The method further includes a step of preferentially amplifying the principal nucleic acid. In some embodiments, the method is Next-generation sequencing assays, high-throughput sequencing assays, high-volume parallel sequencing Scaling assay, nanopore sequencing assay, or Sanger sequencing By performing sequencing assays such as assays, priming or capture can be achieved. The method further includes the step of sequencing the non-host nucleic acid. In some embodiments, this method However, the method further includes a step of preferentially isolating primed or captured non-host nucleic acids. . In some embodiments, the step of preferentially isolating comprises performing a pull-down assay. . In some embodiments, the method further comprises performing a primer extension reaction on the primed or captured non-host nucleic acid. . In some embodiments, at least 1000 oligonucleotides having different nucleotide sequences contain nucleic acid labels. . In some embodiments, the primed or captured non-host nucleic acid is an RNA non-host nucleic acid. . In some embodiments, the method further comprises performing a polymerization reaction on the primed or captured RNA non-host nucleic acid. . In some embodiments, the polymerization reaction is performed by reverse transcriptase.

[0008] In another aspect, the present disclosure provides a method for sequencing non-host sequences in a nucleic acid sample from a host, comprising: (a) preparing a nucleic acid sample from a host, wherein the nucleic acid sample from the host comprises host nucleic acid and non-host nucleic acid; (b) mixing the nucleic acid sample from the host with a collection of oligonucleotides to obtain a mixture, wherein the collection of oligonucleotides comprises at least 1000 oligonucleotides having different nucleotide sequences, and the different nucleotide sequences are specifically selected to contain non-host nucleic acid sequences of at least 10 nucleotide lengths; (c) contacting the collection of oligonucleotides with the nucleic acid sample in the mixture, whereby binding the non-host nucleic acid in the mixture to a non-host nucleic acid sequence of at least 10 nucleotide lengths and binding at most 10% of the host nucleic acid to a non-host nucleic acid sequence of at least 10 nucleotide lengths by the contact. ​​​​​​​​​​​(d) The steps of binding to a non-host nucleic acid sequence and performing a sequencing assay. Therefore, the non-host nucleic acid bound to a non-host nucleic acid sequence of at least 10 nucleotides in length is sequenced. The present invention provides a method that includes the step of determining

[0009] In some embodiments, the method preferentially amplifies non-host nucleic acids in the reaction. Further includes a .In some embodiments, the sequencing assay is a next-generation sequencer. Sequencing assays, high-throughput sequencing assays, high-volume parallel sequencing assays This is a nanopore sequencing assay or Sanger sequencing assay. In some embodiments, the method further includes the step of preferentially isolating non-host nucleic acids. Includes. In some embodiments, the isolation step is performed by performing a pull-down assay. Includes. In some embodiments, the method performs a primer extension reaction on non-host nucleic acids. The further step includes having a few different nucleotide sequences. In some embodiments, a few have different nucleotide sequences. At least 1000 oligonucleotides contain nucleic acid labels. In some embodiments, In some embodiments, the non-host nucleic acid is RNA non-host nucleic acid. The further step includes carrying out a polymerization reaction with non-host nucleic acids. In some embodiments, The reaction is carried out by reverse transcriptase.

[0010] In some embodiments of the methods provided herein, different nucleotide sequences are used. At least 1000 oligonucleotides have different nucleotide sequences. At most 10,000 oligonucleotides. Some of the methods provided herein In this embodiment, at least 1000 oligonucleotides having different nucleotide sequences Otide contains at least 100,000 oligonucleotides with different nucleotide sequences. It is a nucleotide. In some embodiments of the methods provided herein, different nucleotides At least 1000 oligonucleotides having a sequence, with different nucleotide sequences It is an oligonucleotide having at least 1,000,000 oligonucleotides. Provided herein In some embodiments of the method, at least 1000 nucleotides having different nucleotide sequences are used. The oligonucleotide is not conjugated to a solid support. Provided herein In some embodiments of the method, at least 1000 nucleotides having different nucleotide sequences are used. The oligonucleotides have a length of up to 200 nucleotides. Provided herein In some embodiments of the method, at least 10 having different nucleotide sequences Each of the 00 oligonucleotides is a nucleotide dormant with a length of 10-20 nucleotides. Each nucleotide domain, containing nucleotides 10-20 nucleotides long, is a different nucleotype. Includes a sequence of nucleotides. In some embodiments of the methods provided herein, 10 to 20 nucleotides are used. Each domain of the nucleotide in the octide length is 12 to 15 nucleotides long. In some embodiments of the method provided, each of the nucleotides is 10 to 20 nucleotides long. The domain is 13 to 15 nucleotides long. Some practical applications of the methods provided herein In the application form, each domain of a nucleotide 10-20 nucleotides in length is a mammalian nucleic acid sequence. No. In some embodiments of the methods provided herein, the host is a mammalian host. Furthermore, nucleic acid samples from mammalian hosts contain both mammalian host nucleic acids and non-mammalian nucleic acids. In some embodiments of the methods provided herein, the host is a human host, and the human host Nucleic acid samples from the source include human host nucleic acids and non-human nucleic acids. The method provided herein In some embodiments, non-human nucleic acids include microbial nucleic acids. The method provided herein In some embodiments, the non-human nucleic acid includes bacterial nucleic acid. In some embodiments, a nucleic acid sample from the host contains at least five non-host nucleic acid sequences. The present invention further includes the step of detecting at least five non-host nucleic acid sequences. In some embodiments of the methods provided in the document, nucleic acid samples from a host are taken from blood, plasma, etc. , serum, saliva, cerebrospinal fluid, synovial fluid, lavage fluid, urine and feces, for example, blood, plasma and serum Selected from the group. In some embodiments of the methods provided herein, sample The is selected from the group consisting of blood, plasma, and serum. Some of the methods provided herein In that embodiment, the nucleic acid sample from the host is a circulating nucleic acid sample. Provided herein In some embodiments of the method, nucleic acid samples from a host are circulated in a cell-free nucleic acid sample. In some embodiments of the methods provided herein, nucleic acid samples from a host are obtained. The library includes a nucleic acid sequencing library. Some practical applications of the methods provided herein In the application, the nucleic acid sample from the host contains single-stranded DNA or cDNA. In some embodiments of the methods provided, the nucleic acid is DNA. In some embodiments of the method, the nucleic acid is RNA. In several embodiments, the nucleic acid sample from the host does not contain artificially fragmented nucleic acids. In some embodiments of the methods provided herein, the collection of oligonucleotides The DNA includes DNA, RNA, PNA, LNA, BNA, or any combination thereof. In some embodiments of the methods provided herein, the collection of oligonucleotides The DNA oligonucleotide is included. Some embodiments of the method provided herein So, the collection of oligonucleotides includes RNA oligonucleotides.

[0011] In some embodiments of the methods provided herein, oligonucleotide collection The compound is a DNA oligonucleotide. Several implementations of the method provided herein In this state, the collection of oligonucleotides is RNA oligonucleotides. In some embodiments of the method provided in this book, the collection of oligonucleotides is nucleus Labeled with acid or chemical labels. In some embodiments of the methods provided herein The chemical label is biotin. In some embodiments of the methods provided herein, The collection of oligonucleotides does not contain artificially fragmented nucleic acids. In some embodiments of the provided method, non-host nucleic acids are pathogenic nucleic acids, microbial nucleic acids, and bacterial nucleic acids. Consists of nucleic acids, viral nucleic acids, fungal nucleic acids, parasitic nucleic acids, and any combination thereof. Selected from the group. In some embodiments of the methods provided herein, non-host nucleic acids are It is a microbial nucleic acid. In some embodiments of the methods provided herein, a non-host nucleic acid is It is a bacterial nucleic acid. In some embodiments of the methods provided herein, a non-host nucleic acid is used. It is a virus nucleic acid.

[0012] In yet another aspect, the disclosure relates to a method for enriching non-host sequences in nucleic acid samples from a host. A method comprising the step of preparing a nucleic acid sample from a host, wherein the nuclei from the host Acid samples are single-stranded nucleic acid samples from a host, and contain host nucleic acids and non-host nucleic acids. (b) Regenerate at least a portion of the single-stranded nucleic acid from the host, thereby, (c) A step of generating a population of double-stranded nucleic acids during the pull process; and a sample using a nuclease. By removing at least a portion of the double-stranded nucleic acid inside, thereby the nucleic acid sample from the host The present invention provides a method comprising the step of enriching a non-host sequence.

[0013] In some embodiments, the method further extends the step of performing a sequencing assay. Includes. In some embodiments, a nucleic acid sample from the host contains at least five non-host nucleic acids. The method further includes the step of detecting at least five non-host nucleic acid sequences, which include a sequence. In some embodiments, the host is human. In some embodiments, the nucleus from the host The acid sample is the circulating nucleic acid sample. In some embodiments, the nucleic acid sample from the host The sample is a circulating cell-free nucleic acid sample. In some embodiments, nucleic acid samples are obtained from a host. This includes blood, plasma, serum, saliva, cerebrospinal fluid, synovial fluid, lavage fluid, urine and feces, for example, blood, plasma It is selected from the group consisting of and serum. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the method is one The procedure further includes the step of adding a stranded host sequence to a single-stranded nucleic acid sample from a host. In this embodiment, the method uses heat to denature at least a portion of the nucleic acid in the nucleic acid sample. The method further includes the step of preparing a single-stranded nucleic acid sample. In some embodiments, The regeneration of at least a portion of the nucleic acid occurs within a set time frame. In some embodiments, Regeneration of at least a portion of the nucleic acid occurs within 96 hours. In some embodiments, The regeneration process includes regeneration in the presence of trimethylammonium chloride. In some embodiments, The nuclease is a double-strand specific nuclease, BAL-31, a double-strand specific DNase. These are combinations of these. In some embodiments, the nuclease is double-strand specific. It is a nuclease. In some embodiments, the nuclease is BAL-31. In some embodiments, the nuclease is active against double-stranded nucleic acids. Morphologically, the nuclease is inactive against single-stranded nucleic acids. In some embodiments, the nuclease The acid has not been artificially fragmented.

[0014] In yet another aspect, the disclosure relates to a method for enriching non-host sequences in nucleic acid samples from a host. A method comprising the step of preparing a nucleic acid sample from a host, wherein the nuclei from the host The acid sample includes a step in which host nucleic acids and non-host nucleic acids associated with nucleosomes are prepared; ( b) Remove at least a portion of the host nucleic acid associated with the nucleosome, thereby the host The present invention provides a method comprising the step of enriching non-host nucleic acids in a nucleic acid sample from a source.

[0015] In some embodiments, the method further extends the step of performing a sequencing assay. Includes. In some embodiments, a nucleic acid sample from the host contains at least five non-host nucleic acids. The method further includes the step of detecting at least five non-host nucleic acid sequences, which include a sequence. In some embodiments, the host is human. In some embodiments, the nucleus from the host The acid sample is the circulating nucleic acid sample. In some embodiments, the nucleic acid sample from the host The sample is a circulating cell-free nucleic acid sample. In some embodiments, nucleic acid samples are obtained from a host. This includes blood, plasma, serum, saliva, cerebrospinal fluid, synovial fluid, lavage fluid, urine and feces, for example, blood, plasma Selected from the group consisting of and serum. In some embodiments, step (b) Removal includes performing electrophoresis. In some embodiments, the removal in step (b) The procedure includes performing isokinetic electrophoresis. In some embodiments, step (b) The removal process involves using a porous filter. In some embodiments, step (b) In some embodiments, the removal in the process involves using an ion exchange column. The removal in step (b) involves one or more antibodies specific to one or more histones. Includes use. In some embodiments, one or more histones H2A N-terminus, histone H2A solvent-exposed epitope, molar on Lys9 in histone H3 Nomethylation, dimethylation of Lys9 on histone H3, Lys56 on histone H3 Trimethylation of histone H2B, phosphorylation of Ser14 in histone H2A.X Selected from the group consisting of phosphorylation on Ser139 in the middle. In some embodiments, 1 One or more antibodies are immobilized on the column. In some embodiments, this method is 1 The process further includes the step of removing one or more antibodies.

[0016] In another aspect, the disclosure relates to a method for enriching non-host sequences in a nucleic acid sample from a host. (a) The step of preparing nucleic acid samples from the host, The pull includes a step comprising host nucleic acids and non-host nucleic acids; (b) one or more length intervals DNA is removed or isolated, thereby concentrating non-host nucleic acids in nucleic acid samples from the host. The present invention provides a method that includes a step of shrinking.

[0017] In some embodiments, step (b) removes DNA at one or more length intervals. This includes doing so. In some embodiments, step (b) is performed at intervals of one or more lengths. This includes isolating the DNA. In some embodiments, one or more length intervals are Approximately 180 base pairs, approximately 360 base pairs, approximately 540 base pairs, approximately 720 base pairs, and approximately 900 bases Selected from a group consisting of pairs. In some embodiments, one or more length intervals are approximately 150 base pairs, approximately 300 base pairs, approximately 450 base pairs, approximately 600 base pairs, and approximately 750 base pairs Selected from the group consisting of. In some embodiments, one or more length intervals are about 1 60 base pairs, approximately 320 base pairs, approximately 480 base pairs, approximately 640 base pairs, and approximately 800 base pairs. Selected from the group. In some embodiments, one or more length intervals are about 17 Starting from 0 base pairs, approximately 340 base pairs, approximately 510 base pairs, approximately 680 base pairs, and approximately 850 base pairs Selected from the group. In some embodiments, one or more length intervals are about 190 Consists of base pairs, approximately 380 base pairs, approximately 570 base pairs, approximately 760 base pairs, and approximately 950 base pairs. Selected from the group. In some embodiments, one or more length intervals are 150 bases. A pair or multiple thereof, 160 base pairs or multiple thereof, 170 base pairs or multiple thereof, 190 It is selected from the group consisting of base pairs or multiples thereof, and any combination thereof. In some embodiments, step (b) is approximately 100, 120, 150, 175, 200, This includes removing DNA longer than 250, 300, 400, or 500 base pairs. In one embodiment, step (b) is approximately 100, 120, 150, 175, 200 This includes isolating DNA that is 250 or 300 base pairs long. In some embodiments, Step (b) is approximately 10 base pairs to approximately 100 base pairs, approximately 10 base pairs to approximately 120 base pairs. Approximately 10 base pairs to approximately 150 base pairs, approximately 10 base pairs to approximately 175 base pairs, approximately 10 base pairs to approximately 200 base pairs Base length, approximately 10 bases to approximately 250 bases, approximately 10 bases to approximately 300 bases, approximately 30 bases ~approximately 100 base pairs, approximately 30 base pairs to approximately 120 base pairs, approximately 30 base pairs to approximately 150 base pairs, approximately 30 base pairs to approximately 175 base pairs, approximately 30 base pairs to approximately 200 base pairs, approximately 30 base pairs to approximately 250 base pairs This involves isolating DNA with a base length of approximately 30 to 300 bases.

[0018] In some embodiments, the method further extends the step of performing a sequencing assay. Includes. In some embodiments, a nucleic acid sample from the host contains at least five non-host nucleic acids. The method further includes the step of detecting at least five non-host nucleic acid sequences, which include a sequence. In some embodiments, the host is human. In some embodiments, the nucleus from the host The acid sample is the circulating nucleic acid sample. In some embodiments, the nucleic acid sample from the host This is a circulating cell-free nucleic acid sample.

[0019] In yet another aspect, the disclosure relates to a method for enriching non-host sequences in nucleic acid samples from a host. A method comprising the step of preparing a nucleic acid sample from a host, wherein the nuclei from the host The acid sample includes a step containing host nucleic acids, non-host nucleic acids and exosomes; (b) exosomes Remove or isolate at least a portion of the somes, thereby in the nucleic acid sample from the host. The present invention provides a method comprising the step of enriching the non-host sequence.

[0020] In some embodiments, step (b) removes at least a portion of the exosomes. This includes, in some embodiments, step (b) is performed on at least a portion of the exosome This includes isolating the substance. In some embodiments, this method is used to perform a sequencing assay. The steps to be performed further include: In some embodiments, a small amount of nucleic acid sample is obtained from the host. Each contains five non-host nucleic acid sequences, and this method detects at least five non-host nucleic acid sequences. Further steps are included. In some embodiments, the host is human. In this state, nucleic acid samples from the host are blood, plasma, serum, saliva, cerebrospinal fluid, synovial fluid, lavage fluid, Selected from the group consisting of urine and feces, for example, blood, plasma and serum. Several implementations In this state, the nucleic acid sample from the host is the circulating nucleic acid sample. In some embodiments, The nucleic acid sample from the host is a circulating cell-free nucleic acid sample. In some embodiments, The method further includes the step of removing host nucleic acids from the exosome. Several implementations In some embodiments, the method further includes the step of isolating non-host nucleic acids. In step (b), the removal or isolation of leukocyte-derived exosomes is performed. This includes doing so. In some embodiments, the white blood cell is a macrophage. In the embodiment, the removal or isolation in step (b) removes leukocyte-derived exosomes. This may include using immunoprecipitation for isolation.

[0021] In some embodiments of the methods provided herein, the method involves one or more nuclear The further step involves adding an acid barcode to one or more samples. In some embodiments of the provided method, the method involves one or more pathogenic gene loci. Antimicrobial resistance markers; Antibiotic resistance markers; Antiviral resistance markers; Antiparasitic Bioagent resistance markers; useful genotyping regions; two or more microorganisms, pathogens, bacteria, or viruses Sequences common to fungi and / or parasites; non-host sequences incorporated into the host genome Column; masking non-host sequence; non-host mimic sequence; masking host sequence; host mimic sequence; if In addition, one or more microorganisms, pathogens, bacteria, viruses, fungi and / or parasites The method further includes the step of adding one or more nucleic acids specific to a specific sequence to the sample. nothing.

[0022] In yet another aspect, the disclosure relates to priming sequences in nucleic acid samples from a host. This is a method of capture, comprising the steps of (a) preparing a nucleic acid sample from a host; and (b) Nucleic acid samples from the host, one or more pathogenicity loci; antimicrobial agent resistance markers; Antibiotic resistance markers; antiviral drug resistance markers; antiparasitic drug resistance markers; beneficial Genotyping region; two or more microorganisms, pathogens, bacteria, viruses, fungi and / or parasites Sequences common to living organisms; non-host sequences integrated into the host genome; masking non-host sequences; Non-host mimicry sequences; masking host sequences; host mimicry sequences; and one or more microorganisms Targets specific to sequences specific to pathogens, bacteria, viruses, fungi, and / or parasites. The steps include: mixing one or more nucleic acid regions with the mixture to obtain a mixture; (c) In the mixture, the nucleic acid of one or more target regions is brought into contact with the nucleic acid sample. A step in which nucleic acids in a nucleic acid sample are brought into contact with one or more target regions. The process includes the step of binding to the nucleic acid in the region, thereby priming or capturing the nucleic acid. To provide a method.

[0023] In some embodiments, the method involves one or more pathogenicity loci; antimicrobial resistance Sex markers; antibiotic resistance markers; antiviral drug resistance markers; antiparasitic drug resistance markers Carr; useful genotyping region; two or more microorganisms, pathogens, bacteria, viruses, fungi and Sequences common to both host and / or parasites; non-host sequences integrated into the host genome; masking Non-host sequence; non-host mimic sequence; masking host sequence; host mimic sequence; and one or Sequences specific to multiple microorganisms, pathogens, bacteria, viruses, fungi, and / or parasites The method further includes the step of performing a nucleic acid amplification reaction using one or more specific nucleic acids. In some embodiments, the nucleic acid amplification reaction is polymerase chain reaction, reverse transcription, transcription-mediated amplification, and or includes ligase chain reaction. In some embodiments, the method involves one or more diseases Genetic locus; antimicrobial agent resistance marker; antibiotic resistance marker; antiviral agent resistance marker Carr; antiparasitic agent resistance marker; useful genotyping region; two or more microorganisms, pathogens sequences common to bacteria, viruses, fungi and / or parasites; incorporated into the host genome Non-host sequence; masking non-host sequence; non-host mimic sequence; masking host sequence; host mimic Imitation sequences; and one or more microorganisms, pathogens, bacteria, viruses, fungi and / or The method further includes the step of isolating nucleic acids specific to the parasite-specific sequence. In some embodiments, the isolation step includes performing a pull-down assay. In embodiments, the method further includes the step of performing a sequencing assay. In one embodiment, the host is human. In some embodiments, nucleic acid samples are taken from the host. The sample is a circulating nucleic acid sample. In some embodiments, the nucleic acid sample from the host is circulating. This is a cell-free nucleic acid sample.

[0024] In another aspect, the disclosure relates to at least 1000 sequences linked to a sequencing adapter sequence. A collection of oligonucleotides, including (a) Each of the 1000 oligonucleotides contains a nucleotide domain; (b) nucleus The domain of a rheotide has a length of 10-20 nucleotides; (c) 10-20 nucleos Each domain of a nucleotide having a length of 1 nucleotide has a different nucleotide sequence; (d) Each nucleotide domain, having a length of 10 to 20 nucleotides, contains one or more genes. At least 1000 oligonucleotides specifically selected so as not to be present in NOM This provides a collection of oligonucleotides including [specific oligonucleotides].

[0025] In some embodiments, at least 1000 oligonucleotides are present in 200 nucleotides. It is less than or equal to the ocide length. In some embodiments, one or more genomes are one or multiple A number of mammalian genomes. In some embodiments, one or more mammalian genomes This includes the human genome, dog genome, cat genome, rodent genome, pig genome, and bovine genome. Sheep genome, goat genome, rabbit genome, horse genome, and any combination thereof Selected from. In some embodiments, one or more mammalian genomes are human genomes. It is a single genome. In some embodiments, one or more genomes constitute a single genome. In some embodiments, each domain of nucleotides having a length of 10 to 20 nucleotides The nucleotide length is 12-15 nucleotides. In some embodiments, 10-20 nucleotides Each domain of a nucleotide with a given length is 13 to 15 nucleotides long. In that embodiment, the oligonucleotide is a DNA oligonucleotide. In some embodiments, the oligonucleotide is an RNA oligonucleotide. In terms of form, the oligonucleotide is a synthetic oligonucleotide. In some embodiments, The oligonucleotide does not contain nucleic acids that have been artificially fragmented. In some embodiments, In some cases, the oligonucleotide is labeled with nucleic acid, chemical, or optical labels. In one embodiment, at least 1000 oligonucleotides are present in at least 5000 oligonucleotides. It is a ligonucleotide. In some embodiments, at least 1000 oligonucleotides The rheotide is at least 10,000 oligonucleotides. In some embodiments, This is a combination of at least 1,000 oligonucleotides and at least 100,000 oligonucleotides. It is a creotide. In some embodiments, at least 1000 oligonucleotides D is at least 1,000,000 oligonucleotides. In some embodiments, At least 1000 oligonucleotides are found to be one or more microorganisms, pathogens, and 10-20 nuclei containing different sequences present in the genomes of bacteria, viruses, fungi, or parasites. Contains nucleotide domains of octide length.

[0026] In yet another aspect, the disclosure relates to a method for preparing a collection of oligonucleotides. (a) a step of preparing at least 1000 oligonucleotides At least 1000 oligonucleotides have different sequences, and 10 to 20 nucleos (b) A step involving a nucleotide domain of tide length; (b) Using a nucleic acid sample from a host The intended steps are: (c) at least 1000 oligonucleotides from the host nucleic acid (d) a step of mixing with; (d) at least 100 nucleic acids from the host that do not hybridize. Oligonucleo The present invention provides a method that includes the step of creating a collection of chids.

[0027] In some embodiments, at least 1000 oligonucleotides are present in up to 200 nucleotides. It has the length of a creotide. In some embodiments, the nucleotide domain is 12~ It is 15 nucleotides long. In some embodiments, the nucleotide domain is 13~ It is 15 nucleotides long. In some embodiments, the oligonucleotide is DNA or It is RNA. In some embodiments, the oligonucleotide is single-stranded. In that embodiment, the oligonucleotide is a synthetic oligonucleotide. In the application form, nucleic acids from the host are labeled with nucleic acid labels or chemical labels. Several implementation forms In this state, the chemical label is biotin. In some embodiments, the isolation in step (d) This includes performing electrophoresis. In some embodiments, the isolation in step (d) is performed by plutow This includes performing an assay. In some embodiments, this method uses heat to remove host cells from the host. The further step includes denaturing at least a portion of the nucleic acid.

[0028] In yet another aspect, the disclosure relates to a method for preparing a collection of oligonucleotides. (a) Selected from the group consisting of genome, exome and transcriptome Domains of nucleotides 10-20 nucleotides in length present in the background population (b) a step to determine the sequence that is not present in the background population at least 1000 nucleotide domains, each 10-20 nucleotides long The step includes preparing a collection of oligonucleotides containing ligonucleotides. Provide a method.

[0029] In some embodiments, at least 1000 oligonucleotides are present in up to 200 nucleotides. It has the length of a creotide. In some embodiments, the nucleotide domain is 12~ It is 15 nucleotides long. In some embodiments, the nucleotide domain is 13~ It is 15 nucleotides long. In some embodiments, the host is human. In the embodiment, the background population is the genome. In some embodiments, the background The round population is the exome. In some embodiments, the background population is It is a scriptome. In some embodiments, the decision is made computationally.

[0030] In another aspect, the present disclosure relates to a method for identifying a pathogen in a host, wherein a sample from the host The steps include preparing the sample and concentrating the non-host nucleic acid against the host-derived nucleic acid. This is a step in which concentration preferentially extracts nucleic acids from the sample that are longer than approximately 300 base pairs. A step including removing; a step of analyzing non-host nucleic acids; and a step including removing non-host nucleic acids The present invention provides a method comprising the step of identifying a pathogen in a host.

[0031] In some embodiments of the methods described herein, the concentration step is about 120 Nucleic acids longer than approximately 150, 200, or 250 base pairs are preferentially removed from the sample. This includes the step of concentrating. In some embodiments of the methods described herein, the step of concentrating is included. However, from the sample, approximately 10 base pairs to approximately 60 base pairs, approximately 10 base pairs to approximately 120 base pairs, approximately 1 0 bases to approximately 150 bases, approximately 10 bases to approximately 300 bases, approximately 30 bases to approximately 60 bases Length, approximately 30 base pairs to approximately 120 base pairs, approximately 30 base pairs to approximately 150 base pairs, approximately 30 base pairs to approximately This includes preferentially enriching nucleic acids that are 200 base pairs long and approximately 30 to 300 base pairs long. In some embodiments of the methods described herein, the concentration step is derived from the host. This includes preferentially digesting nucleic acids. In some embodiments of the methods described herein, The enrichment step includes preferentially replicating non-host nucleic acids. In some embodiments of the described method, non-host nucleic acids are present in the host-derived nucleic acids. One or more priming oligos complementary to one or more domains of the creotide They are preferentially replicated using nucleotides or captured oligonucleotides. In some embodiments of the described method, the nucleotide domain has 10 to 20 nucleotides. This is the nucleotide length. In some embodiments of the methods described herein, the nucleotide The domain is 12 to 15 nucleotides long. Some practical applications of the methods described herein In the application form, the nucleotide domain is 13mer or 14mer. In some embodiments of the methods described herein, the host is a eukaryotic host. In some embodiments of the method described herein, the host is a vertebrate host. In some embodiments of the method, the host is a mammalian host. In some embodiments of the law, non-host DNA includes DNA from a pathogenic organism. In some embodiments of the methods described, the sample includes a blood or plasma sample. In some embodiments of the methods described herein, the sample is a cell-free sample. In some embodiments of the methods described herein, the concentration step is performed by the host The ratio of non-host-derived nucleic acids to host nucleic acids is at least 2 times, at least 3 times, and at least 4 times. times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 double, at least 10 times, at least 11 times, at least 12 times, at least 13 times, less At least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 1 8 times, at least 19 times, at least 20 times, at least 30 times, at least 40 times, less At least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 1000 times, at least 5000 times or less At best, increase it by 10,000 times.

[0032] In another aspect, the present disclosure provides a single oligonucleotide containing 10 oligonucleotides. An ultramer oligonucleotide comprising a row, (a) 10 Each of the oligonucleotide sequences is either a uracil residue or an aprine / apyrimidine moiety. (b) Each of the 10 oligonucleotide sequences is a nucleotide (c) The nucleotide domain has a length of 10 to 20 nucleotides; (d) Each domain of a nucleotide having a length of 10 to 20 nucleotides is a different nucleotide. We provide ultramer oligonucleotides having an ocide sequence. In some embodiments, The 10 oligonucleotide sequences are 200, 150, 100, 50, 40, and 30. It is 20 nucleotides or less in length. In some embodiments, 10 oligonucleotides The sequences are of the same length. In some embodiments, they have a length of 10 to 20 nucleotides. Each domain of the nucleotide is absent in one or more genomes. In the application form, one or more genomes are one or more mammalian genomes. In that embodiment, one or more mammalian genomes are the human genome, dog genome, cat genome, etc. Nomu, rodent genome, pig genome, cattle genome, sheep genome, goat genome, rabbit Selected from genomes, horse genomes, and any combination thereof. Several implementations In this configuration, one or more mammalian genomes constitute the human genome. In some embodiments, One or more genomes constitute a single genome. In some embodiments, 10 to 20 Each domain of a nucleotide, which has the length of a nucleotide, is 12 to 15 nucleotides long. In some embodiments, each of the nucleotides having a length of 10 to 20 nucleotides The domain is 13-15 nucleotides long. In some embodiments, it is 10-20 nucleotides. Each domain of a nucleotide having the length of a rheotide contains mixed bases. Several implementations In this state, each nucleotide has a length of 10 to 20 nucleotides and there are one or more domains. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, Contains 10 or more mixed bases, 11 or more, 12 or more, or 13 or more. In the application form, the mixed bases are N(A, C, G, T), D(A, G, T), V(A, C, G), B(C, G, T), H(A, C, T), W(A, T), S(C, G), K(G, T), M A group consisting of (A, C), Y(C, T), R(A, G), and any combination thereof. Selected from. In some embodiments, the 10 oligonucleotide sequences are the same degenerate. It has a degree. In some embodiments, the sequence of 10 oligonucleotides is 2, 3, 4 6, 8, 9, 12, 16, 18, 24, 27, 32, 36, 48, 54, 64, 72, 8 Contractions of 1, 96, 108, 128, 144, 162, 192, 216, 243 or 256 It has a severe effect. In some embodiments, the 10 oligonucleotide sequences are 2 and 3 It has a degenerate prime factor selected from. In some embodiments, 10 oligonucleotides The rheotide sequence is DNA. In some embodiments, 10 oligonucleotides are arranged The column is RNA. In some embodiments, 10 oligonucleotide sequences are synthesized. In some embodiments, ultramer oligonucleotides are present in about or less Approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 30 It contains 0, 400, or 500 oligonucleotide sequences. In some embodiments, Ultramer oligonucleotides can reach up to approximately 10, 20, 30, 40, 50, 60, and 70 units. 80, 90, 100, 200, 300, 400, or 500 oligonucleotide sequences Includes. In some embodiments, the 10 oligonucleotide sequences are one or more Different sequences present in the genomes of microorganisms, pathogens, bacteria, viruses, fungi, or parasites It contains a nucleotide domain that is 10 to 20 nucleotides long.

[0033] In another aspect, the present disclosure is a method for preparing a collection of oligonucleotides. (a) the step of preparing an ultramer oligonucleotide disclosed herein; (b) Hydrolyze the ultramer oligonucleotide, thereby producing oligonucleotides The present invention provides a method that includes the step of creating a collection of [something]. In some embodiments, Step (b) hydrolyzes the aprine / apyrimidine moiety in the ultramer. Includes. In some embodiments, step (b) is endonuclease IV or endonuclease IV. This is carried out using nuclease VII. In some embodiments, this method is performed using ultra - Hydrolyze the uracil residue in the oligonucleotide to obtain the aprine / apyrimidine moiety. The process further includes the step of forming. In some embodiments, the uracil residue is hydrolyzed. The step is carried out using uracil DNA glycosylase. In some embodiments, This method biotinylates oligonucleotides in a collection of oligonucleotides. The further step includes the following: In some embodiments, biotinylation is performed enzymatically (e.g., T (using polynucleotide kinases) or chemically. Several embodiments So, this method probes oligonucleotides in an oligonucleotide collection. The further step includes attaching the probe to the digoxigenous substance. In some embodiments, the probe is attached to the digoxigenous substance. It is either a fluorescent probe or a fluorescent probe.

[0034] Built-in by reference All publications, patents, and patent applications referred to herein are treated as if each individual publication were a separate publication. It is specifically and individually indicated that an object, patent, or patent application is incorporated by reference. The entire text is incorporated herein by reference to the same extent as it is incorporated by reference.

[0035] Novel features of the present invention are specifically described in the appended claims. Features and advantages of the present invention A better understanding of this invention can be found in the following detailed description illustrating exemplary embodiments in which the principles of this invention are utilized. This can also be obtained by referring to the attached drawings. [Brief explanation of the drawing]

[0036] [Figure 1] This diagram shows a priming strategy that uses a collection of oligonucleotides to enrich a population of nucleic acids. [Figure 2] This diagram illustrates a pull-down strategy that uses a collection of oligonucleotides to enrich a population of nucleic acids. [Figure 3] This figure shows a method for preparing an oligonucleotide collection using a hybridization-based approach. [Figure 4] This figure shows the length and sequence coverage of nucleotide domains. Approximately 96.5% of nucleotide domains with a length of 13 nucleotides are human. [Figure 5] This figure shows the sequence coverage of the target population estimated using a collection of oligonucleotides. The coverage of the E. coli genome is shown using a collection of oligonucleotides, which include non-human nucleotide domains of 13 nucleotides in length. [Figure 6] This figure shows the periodicity of human cell-free DNA length. [Figure 7]This figure shows nucleosome depletion by nucleosome-specific antibodies. [Figure 8A] This figure shows a plot of cell-free DNA quantity versus DNA fragment length for both host and non-host cell-free DNA. [Figure 8B] This figure shows the results of a selective enrichment process of cell-free DNA short fragments. [Figure 9A] This figure shows a typical ultramer oligonucleotide design. [Figure 9B] This figure shows an ultramer oligonucleotide containing an oligonucleotide sequence having N mixed bases. [Figure 9C] This figure shows an ultramer oligonucleotide containing an oligonucleotide sequence having two N mixed bases. [Figure 9D] This figure shows an ultramer oligonucleotide containing an oligonucleotide sequence having two mixed bases. [Figure 10A] This figure shows the reaction scheme for preparing an oligonucleotide collection from ultramer oligonucleotides. [Figure 10B] This figure shows examples of digestion reaction products after digestion of ultramer oligonucleotides. [Figure 11A] This figure shows representative oligonucleotides that have a mixed base moiety. [Figure 11B] This figure shows the analysis of a pool of non-human 13mer probes grouped according to their degeneracy. [Figure 12A] This is a schematic diagram of the process for enzymatic biotinylation of oligonucleotides. [Figure 12B] This is a schematic diagram of the process for chemical biotinylation of oligonucleotides. [Modes for carrying out the invention]

[0037] overview This disclosure concerns patient samples in which an overwhelmingly high proportion of the sample consists of the patient's own nucleic acids. This disclosure provides a novel and rapid method for enriching pathogen nucleic acids in samples. This allows for the detection of multiple pathogen nucleic acids, enabling caregivers to determine which pathogens are infecting the patient. A situation where there is no clear idea or doubt as to whether it is possible, or a situation where there is no hypothesis. Pathogens can be detected even in such conditions. Generally, the composition provided herein The substance and method increase the expression of pathogen nucleic acids compared to nucleic acids from the host. Designed to concentrate biological samples containing [specific substances]. Concentration of pathogen nucleic acids in samples. This is particularly true when the analysis involves sequencing reactions, as it involves the time and cost associated with analyzing the sample. It can be reduced.

[0038] This disclosure provides several methods for performing enrichment. In some examples, this disclosure provides methods for performing enrichment. A novel collection of alkyl groups that preferentially bind to sets of non-host nucleic acids in the nucleotide. This provides a method for producing oligonucleotide collections of multiple different types. It can be used to preferentially detect non-host nucleic acids in molecular biology assays. For example, Then, the oligonucleotide collection is subjected to a primer extension reaction, a PCR reaction, or a reverse reaction. It can be used as a primer in photochemical reactions. Furthermore, oligonucleotides The collection can be used in hybridization assays and / or pull-down assays. It can be used to preferentially bind and isolate pathogen nucleic acids. In some cases, The collection of nucleotides is not only for enriching pathogen nucleic acids, but also for such nuclei It can also be used to tag acids.

[0039] Additional enrichment techniques are also provided herein. Such techniques can be used alone or with oligonucleotides. This can be used in combination with a collection of leotide or another concentration method. Examples of additional enrichment techniques include (a) the major population in the nucleic acid sample is in the sample Self-hybridization (sel) which self-hybridizes more rapidly than a small group of organisms. f-hybridization) technology, (b) nucleosome association from free DNA D (c) Depletion of NA; (d) Removal and / or isolation of DNA at specific length intervals; (c) Exosol (e) Depletion or concentration of the sphere; and strategic capture of the target area.

[0040] The concentration method provided herein is for the circulation present in blood samples from infected patients. Alternatively, it is particularly suitable for detecting microbial nucleic acids, such as circulating cell-free microbial nucleic acids. The method also includes any other methods, such as detecting a small population of nucleic acids in a mixture dominated by a large population. It is also useful in this situation.

[0041] Figure 1 shows the details for detecting pathogens or other non-human nucleic acids in human patient samples. The book provides general methods disclosed therein. In some examples, a blood sample (120) or The plasma sample (130) contains pathogens (e.g., microorganisms, bacteria, viruses, fungi or parasites). Obtained from human patients (110) who are infected (or suspected of being infected) with a living organism. The liquid sample is brought into contact with the collection of oligonucleotides (150) provided herein. It may contain nucleic acids such as circulating cell-free nucleic acid (140) that can be brought into contact with other substances. The collection of otide can preferentially bind to pathogen or non-human nucleic acid sequences (170). The oligonucleotide collection includes nucleic acid labeling, barcoding, and sample-specific barcoding. Code, universal primer sequence, sequencing primer binding site, barcode amplification Primer binding sites, sequencer-compatible sequences and / or adapters that enable this (e.g.) For example, it can be linked to a label (160) which may include a sequencing adapter. If the nucleic acid sample contains RNA, the collection of oligonucleotides will be from non-host RNA. To preferentially prime A, cDNA synthesis from RNA template (170) is performed. It can be used for timing. In some cases, for oligonucleotide collection. This is used in the primer extension reaction (170) to preferentially select the pathogen DNA sequence in the sample. It can be primed. The primer extension reaction also allows the overhang sequence to become pathogenic. It can also be added to somatic nucleic acids. For example, labeling (160) via a primer extension reaction. The sequence (e.g., adapter, barcode, etc.) can be added to the pathogen nucleic acid. The final library (180) can be prepared by performing a CR reaction, and this can be used in the sample. Sequencing assays to aid in the detection and identification of pathogen species (195) (19 It can be used for 0).

[0042] Figure 2 is provided herein, including the hybridization and pull-down steps. The steps in another method are shown. The sample can be prepared as shown in Figure 1 (210, (220, 230). A sequencing-ready library can be prepared using the samples. Nucleic acids (240) in the sample can be tagged with a label (250). Each of these can be conjugated with a label such as a biotin tag (270). It can be brought into contact with a collection of oligonucleotides (260). Otid hybridizes to non-human sequences in the sample (280, 285), and then, for example, For example, a pull-down using avidin or streptavidin bound to a solid support. It can be preferentially pulled down by (285). Next, the infected host (e.g., Hi To identify non-host species (e.g., pathogens) within (295), the library is sequenced. It can be decided (290).

[0043] The methods and compositions provided herein are for a small group of targets in a complex mixture. It offers many advantages over current methods for detecting nucleic acids. One advantage is that the library... Reducing the number of sequence reads analyzed or the number of sequences used to analyze reduces the sequencing cost. This is the case. Also, targets such as the collection of oligonucleotides provided herein. By using nucleotide polymorphisms, in particular, background population nucleic acids (for example) This method is compared to other methods, such as those that rely on depleting human nucleic acids from a sample. This allows for speeding up the process of priming, capturing, or amplifying small groups. These can be used to detect a specific population (e.g., pathogens or microorganisms) in a sample. It can dramatically increase the sensitivity and specificity of the oligonucleotides. Using this collection, you can create a DNA or RNA sequencing library. Yes; in some cases, these can be used to create DNA sequencing libraries from the same sample. Both RNA sequencing libraries can be prepared. As a result, in this specification The methods and compositions provided are for treating pneumonia, tuberculosis, HIV infection, hepatitis (e.g., A, Hepatitis B or C), sepsis, human papillomavirus (HPV) infection, chlamydia infection Infectious diseases, syphilis infection, Ebola infection, multidrug-resistant infections, Staphylococcus aureus infection, enterococcal infection This provides a new and efficient method for detecting infectious diseases, including influenza. In this example, the present method and composition were used even by those who had not experienced symptoms related to an infection. In a good host, for example, the presence of one or more microorganisms in the microbiome. It can be monitored.

[0044] Collection of oligonucleotides for sequence enrichment This disclosure describes how to concentrate and capture a specific nucleic acid population ("Target Population") within a complex mixture of nucleic acids. Provides a collection of oligonucleotides useful for capturing or priming. The population in question is a population that contains both host (e.g., human) nucleic acids and non-host nucleic acids, including non-host nucleic acids. The primary nucleic acid may be a non-human, microbial, or pathogenic nucleic acid. Typically, the target population is... This is another group of nucleic acids that make up a larger portion of the complex mixture of nucleic acids ("background"). It constitutes a small portion of a complex mixture of nucleic acids, which may include a group, for example, host nucleic acids. In that case, the methods and compositions provided herein are for the entire population in the sample. It is particularly useful when it constitutes up to 1% or 0.1% of nucleic acids.

[0045] Generally, the methods provided herein involve priming, capturing, or targeting a population. This includes using oligonucleotides for concentration. Typically, the target population is particularly Having specific attributes or characteristics, oligonucleotides prime and capture the target population. To capture or concentrate another population that can constitute the majority of the entire population of nucleic acids (for example, "background data") It is designed not to prime, capture, or concentrate the "Wound" group. For example, against The population that becomes the elephant contains both host and non-host nucleic acids in the population (e.g., non-mammalian Subsets of nucleic acids (of substances, non-humans, pathogens, microorganisms, viruses, bacteria, fungi, or parasites) It is possible. In some cases, oligonucleotides are part of the background population (e.g., To remove, isolate, or deplete human nucleic acids, thereby affecting the target population (e.g., Oligonucleotides can be designed to concentrate non-human nucleic acids. In some cases, oligonucleotides are The sequence of a certain target population (e.g., a non-human nucleic acid sequence) is (completely or substantially) identical. Nucleotides having sequences that can be or (completely or substantially) complementary It may include a domain name.

[0046] The collection of oligonucleotides provided herein binds to the target population. It may contain nucleic acid sequences that can recognize or target populations. Using a collection of samples, for example, a background population (for example, a host population) Sun cells containing nucleic acids from the target population (e.g., non-host or non-human) and humans. It is possible to specifically target and detect the target population during the pull. For example, oligo A collection of nucleotides, including non-host nucleic acids in a sample containing both host and non-host nucleic acids. Used as a primer to specifically prime, capture, amplify, replicate, or detect It is possible. More specifically, in some cases, these can be used during the sample. cDNA synthesis can be primed from existing RNA templates in some cases. These are used in primer extension reactions to label target sequences with nucleic acids or to add sequences to them. It is possible to use these in a DNA, cDNA, or RNA library. It can be used as bait to capture non-host sequences from Lee. The amplified or captured non-host nucleic acids are sequenced in sequencing assays, especially high-throughput assays. Sequencing assays, next-generation sequencing platforms, high-volume parallel sequencing A goplatform, nanopore sequencing assay, or another sequencing assay known in the art. Identification is performed by methods known in the art, such as by conducting a quenching assay. It is possible.

[0047] A collection of oligonucleotides may contain one or more oligonucleotides. In some cases, the collection of oligonucleotides is approximately 100; 200; 300; 400;500;600;700;800;900;1000;2000;3000;4 000;5000;6000;7000;8000;9000;10000;20000 ;30000;40000;50000;60000;70000;80000;900 00;100000;200000;300000;400000;500000;60 0000;700000;800000;900000;106;1.1×106;1. 2×10⁶; 1.3×10⁶; 1.4×10⁶; 1.5×10⁶; 1.6×10⁶; 1. 7×10⁶; 1.8×10⁶; 1.9×10⁶; 2×10⁶; 2.1×10⁶; 2.2× 10⁶;2.3×10⁶;2.4×10⁶;2.5×10⁶;2.6×10⁶;2.7× 106;2.8×106;2.9×106;3×106;4×106;5×106;6× 106;7×106;8×106;9×106;107;5×107;108;5×10 It may contain 8; or 109 oligonucleotides. In some cases, oligonucleotides Chido's collection can be up to 100; 200; 300; 400; 500; 600; 700; 800;900;1000;2000;3000;4000;5000;6000;70 00;8000;9000;10000;20000;30000;40000;500 00;60000;70000;80000;90000;100000;200000 ;300000;400000;500000;600000;700000;8000 00;900000;106;1.1×106;1.2×106;1.3×106;1. 4×10⁶; 1.5×10⁶; 1.6×10⁶; 1.7×10⁶; 1.8×10⁶; 1. 9×10⁶; 2×10⁶; 2.1×10⁶; 2.2×10⁶; 2.3×10⁶; 2.4× 10⁶;2.5×10⁶;2.6×10⁶;2.7×10⁶;2.8×10⁶;2.9× 106; 3×106; 4×106; 5×106; 6×106; 7×106; 8×106; 9 × 10⁶; 10⁷; 5 × 10⁷; 10⁸; 5 × 10⁸; or 10⁹ oligonucleotides It may contain oligonucleotides. In some cases, the collection of oligonucleotides contains at least one 00;200;300;400;500;600;700;800;900;1000; 2000;3000;4000;5000;6000;7000;8000;9000; 10000;20000;30000;40000;50000;60000;7000 0;80000;90000;100000;200000;300000;40000 0;500000;600000;700000;800000;900000;106 ;1.1×10⁶;1.2×10⁶;1.3×10⁶;1.4×10⁶;1.5×10⁶ ;1.6×10⁶;1.7×10⁶;1.8×10⁶;1.9×10⁶;2×10⁶;2 .1×10⁶; 2.2×10⁶; 2.3×10⁶; 2.4×10⁶; 2.5×10⁶; 2 0.6×10⁶; 2.7×10⁶; 2.8×10⁶; 2.9×10⁶; 3×10⁶; 3.5 ×106;4×106;4.5×106;5×106;5.5×106;6×106;6 0.5×10⁶; 7×10⁶; 7.5×10⁶; 8×10⁶; 8.5×10⁶; 9×10⁶ ;9.5×10⁶;10⁷;5×10⁷;10⁸;5×10⁸;or 10⁹ oligos It may contain nucleotides.

[0048] Oligonucleotides in a collection of oligonucleotides may be different even if they have the same length. They may have a length. In some cases, two or more oligonucleotides in a collection. The oligonucleotides above have the same length. In some cases, the oligonucleotides All oligonucleotides in the collection have the same length. In some cases, the oligonucleotides The oligonucleotides in the collection of nucleotides have different lengths. In this case, the oligonucleotides in the oligonucleotide collection are approximately 1, 2, 3, 4. It has different lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20. In some cases, the oligonucleotide in the oligonucleotide collection is the most Roughly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or It has a length of 20. In some cases, oligonucleotides in a collection of oligonucleotides Cleotide contains at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 It has lengths of 3, 14, 15, or 20.

[0049] In some cases, oligonucleotides number approximately 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 , 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 , 280, 290, 300, 400, 500, 600, 700, 800, 900 or 1 It can be 000 nucleotides long. In some cases, oligonucleotides can be up to 10,1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 , 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 , 240, 250, 260, 270, 280, 290, 300, 400, 500, 600 The length can be 700, 800, 900, or 1000 nucleotides. In some cases, Oligonucleotides are at least 10, 11, 12, 13, 14, 15, 16, 17, 1 8, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80 , 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 400, 500, 600, 700, 800, 900 or 1000 Nuk The rheoside length can be 10 to 1000, 10 to 500, 10-400, 10-300, 10-200, 10-100, 10-90, 10 ~80, 10~70, 10~60, 10~50, 10~40, 10~30, 10~20, 10-15, 11-1000, 11-500, 11-400, 11-300, 11-20 0, 11-100, 11-90, 11-80, 11-70, 11-60, 11-50, 1 1-40, 11-30, 11-20, 11-15, 12-1000, 12-500, 12 ~400, 12~300, 12~200, 12~100, 12~90, 12~80, 12 ~70, 12~60, 12~50, 12~40, 12~30, 12~20, 12~15, 13-1000, 13-500, 13-400, 13-300, 13-200, 13-1 00, 13-90, 13-80, 13-70, 13-60, 13-50, 13-40, 1 3-30, 13-20, 13-15, 13-14, 14-1000, 14-500, 14 ~400, 14~300, 14~200, 14~100, 14~90, 14~80, 14 ~70, 14~60, 14~50, 14~40, 14~30, 14~20 or 14~1 It can be 5 nucleotides long.

[0050] The oligonucleotides in the collection of oligonucleotides have different nucleotide sequences. It may have. In some cases, the collection of oligonucleotides is different nucleotides. Approximately 100;200;300;400;500;600;700;800;9 have the sequence 00;1000;2000;3000;4000;5000;6000;7000;80 00;9000;10000;20000;30000;40000;50000;60 000;70000;80000;90000;100000;200000;3000 00;400000;500000;600000;700000;800000;90 0000;106;1.1×106;1.2×106;1.3×106;1.4×106 ;1.5×10⁶;1.6×10⁶;1.7×10⁶;1.8×10⁶;1.9×10⁶ ;2×10⁶;2.1×10⁶;2.2×10⁶;2.3×10⁶;2.4×10⁶;2 0.5×10⁶; 2.6×10⁶; 2.7×10⁶; 2.8×10⁶; 2.9×10⁶; 3 ×106; 4×106; 5×106; 6×106; 7×106; 8×106; 9×106 ; or may contain 107 oligonucleotides. In some cases, oligonucleotides The collection of nucleotides has a maximum of 100;200;300;40 different nucleotide sequences. 0;500;600;700;800;900;1000;2000;3000;400 0;5000;6000;7000;8000;9000;10000;20000;3 0000;40000;50000;60000;70000;80000;90000 ;100000;200000;300000;400000;500000;6000 00;700000;800000;900000;106;1.1×106;1.2× 10⁶; 1.3 × 10⁶; 1.4 × 10⁶; 1.5 × 10⁶; 1.6 × 10⁶; 1.7 × 10⁶; 1.8 × 10⁶; 1.9 × 10⁶; 2 × 10⁶; 2.1 × 10⁶; 2.2 × 10 6; 2.3 × 10⁶; 2.4 × 10⁶; 2.5 × 10⁶; 2.6 × 10⁶; 2.7 × 10 6; 2.8 × 10⁶; 2.9 × 10⁶; 3 × 10⁶; 4 × 10⁶; 5 × 10⁶; 6 × 10 Contains 6, 7 × 10⁶, 8 × 10⁶, 9 × 10⁶, or 10⁷ oligonucleotides Obtain. In some cases, a collection of oligonucleotides contains different nucleotide sequences. Having at least 100;200;300;400;500;600;700;800; 900;1000;2000;3000;4000;5000;6000;7000;8 000;9000;10000;20000;30000;40000;50000;6 0000;70000;80000;90000;100000;200000;300 000;400000;500000;600000;700000;800000;9 00000;106;1.1×106;1.2×106;1.3×106;1.4×10 6; 1.5 × 10⁶; 1.6 × 10⁶; 1.7 × 10⁶; 1.8 × 10⁶; 1.9 × 10 6; 2 × 10⁶; 2.1 × 10⁶; 2.2 × 10⁶; 2.3 × 10⁶; 2.4 × 10⁶; 2.5×10⁶; 2.6×10⁶; 2.7×10⁶; 2.8×10⁶; 2.9×10⁶; 3×10⁶; 4×10⁶; 5×10⁶; 6×10⁶; 7×10⁶; 8×10⁶; 9×10 It may contain 6 or 107 oligonucleotides.

[0051] Oligonucleotides with different sequences are multiple within an oligonucleotide collection. It can exist in multiple copies. A collection of oligonucleotides is an identical nucleotide sequence. It may contain oligonucleotides having columns. In some cases, oligonucleotides The selection is approximately 1;2;3;4;5;6;7;8;9;10;11;12;13;14; 15;16;17;18;19;20;25;30;35;40;45;50;55;6 0;65;70;75;80;85;90;95;100;150;200;250;3 00;350;400;450;500;600;700;800;900;or 10 It may contain 0 copies of the same nucleotide sequence. In some cases, oligonucleotides Collections can be up to 1;2;3;4;5;6;7;8;9;10;11;12;13;1 4;15;16;17;18;19;20;25;30;35;40;45;50;55 ;60;65;70;75;80;85;90;95;100;150;200;250 ;300;350;400;450;500;600;700;800;900;or It may contain 1000 copies of the same nucleotide sequence. In some cases, oligonucleotides The collection of D includes at least 1;2;3;4;5;6;7;8;9;10;11;12 ;13;14;15;16;17;18;19;20;25;30;35;40;45; 50;55;60;65;70;75;80;85;90;95;100;150;20 0;250;300;350;400;450;500;600;700;800;90 It may contain 0 or 1000 copies of the same nucleotide sequence.

[0052] In some cases, one or more oligonucleotides in a collection of oligonucleotides Rheotide is not labeled; in some cases, one of the oligonucleotides in the collection Alternatively, multiple oligonucleotides are labeled. In some cases, one or more oligonucleotides are labeled. Gonucleotides are labeled, for example, with nucleic acid labels, chemical labels, or optical labels. In this case, one or more oligonucleotides are conjugated onto a solid support. In some cases, one or more oligonucleotides are conjugated onto a solid support. No. In some cases, the label is placed at the 5' or 3' end of the oligonucleotide, or It can be attached inside the oligonucleotide. In some cases, one or more oligonucleotides in a collection of oligonucleotides are labeled with two or more labels.

[0053] The oligonucleotide can contain a nucleic acid label. In some cases, the nucleic acid label can include one or more of the following: barcode (e.g., sample barcode), universal primer array, primer binding site (e.g., but not limited to, DNA sequencing primer binding site, sample barcode sequencing primer binding site, and amplification primer binding site that conforms to various sequencing platform requirements, for sequencing or barcode reading), sequencer compatibility sequence, sequence for attaching to a sequencing platform, sequencing adapter sequence or adapter. The nucleic acid label can be attached to the oligonucleotide (e.g., by ligation or synthetic design). In some cases, the length of the nucleic acid label is included in the length of the oligonucleotide. In some cases, the length of the nucleic acid label is not included in the length of the oligonucleotide.

[0054] The oligonucleotide can contain a chemical label. Some non-limiting examples of chemical labels include biotin, avidin, streptavidin, radioactive label, polypeptide, and polymer. The oligonucleotide can contain an optical label. Some non-limiting examples of optical labels include fluorophore, fluorescent protein, dye, and quantum dot. The oligonucleotide can be conjugated to a solid support. In some cases, the oligonucleotide is not conjugated to a solid support. Some non- ​​​​​​​​​​​​​​​​Limited examples include beads, magnetic beads, polymers, slides, chips, surfaces, and plates. Examples include tors, channels, cartridges, microfluidic devices, and microarrays. . One or more oligonucleotides for affinity chromatography It can be conjugated to a body support. In some cases, each oligonucleotide They have different labels. In some cases, each oligonucleotide has the same label. In some cases, each copy of the oligonucleotide has the same label. In some cases, different labels are present. Each oligonucleotide having a specific sequence has a different label.

[0055] The oligonucleotides provided herein generally consist of one or more nucleotides. It contains nucleotides. In some cases, nucleotides are deoxyribonucleotides (e.g., A, C, G or T), ribonucleotides (e.g., A, C, G or U), modified nucleotides These can be synthetic nucleotides. In some cases, the oligonucleotides are DNA, RNA, etc. , may include cDNA, dsDNA, ssDNA, mRNA or cRNA. In some cases, oligonucleotides may contain DNA or RNA. In some cases, oligonucleotides Cleotides can include DNA and RNA. In some cases, oligonucleotides are D It may contain NA. In some cases, oligonucleotides may contain RNA. In some cases, oligonucleotides may include modified or synthetic nucleotides. In this case, oligonucleotides are peptide nucleic acids (PNA) and locked nucleic acids (LNA). It may contain one or more modified or synthetic nucleic acids, such as cross-linked nucleic acids (BNA). In that case, the oligonucleotide is DNA, RNA, PNA, LNA, BNA or these It may include any combination of these. In some cases, oligonucleotides are artificially fragmented. It does not contain synthesized nucleic acids. In some cases, the collection of oligonucleotides is artificial. It does not contain nucleic acids that are fragmented. In some cases, oligonucleotides are (for example, Includes synthetic nucleic acids (by DNA synthesis). In some cases, a collection of oligonucleotides. The term includes synthetic nucleic acids (e.g., those produced by DNA synthesis). In some cases, oligonucleotides are also included. The collection of cidoes can be freeze-dried or dried. In some cases, oligosaccharides A collection of nucleotides may contain water or a buffer solution (e.g., a buffered aqueous solution).

[0056] In some cases, oligonucleotides are approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 1 30, 140, 150, 160, 170, 180, 190, 200, 210, 220, 2 30, 240, 250, 260, 270, 280, 290, 300, 400, 500, 6 00, 700, 800, 900, 1000 or more PNAs, LNAs and / or It may contain BNA bonds. In some cases, the oligonucleotide is approximately 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 5 5, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96 can contain 97, 98, 99 or 100% PNA, LNA and / or BNA binding be possible.

[0057] Domain of nucleotides An oligonucleotide can include one or more domains of nucleotides. In some cases, the oligonucleotide includes one domain of nucleotides. In some cases, the domain of nucleotides is an oligonucleotide. In some cases, portions of oligonucleotides having different nucleotide sequences appear within the domain of nucleotides. In some cases, portions of oligonucleotides having different nucleotide sequences constitute the entire oligonucleotide. In some cases, portions of oligonucleotides having different nucleotide sequences constitute a subset of oligonucleotides such as a domain of nucleotides within the oligonucleotide. In some cases, a collection of oligonucleotides may include oligonucleotides, the oligonucleotides include domains of nucleotides having different sequences, and the oligonucleotides may be present in multiple copies. In some cases, the domain of nucleotides does

[0058] not include artificially fragmented nucleic acids. In some cases, the domain of nucleotides is synthesized (e.g., by DNA synthesis). In some cases, a collection of oligonucleotides can include oligonucleotides, each oligonucleotide including a domain of nucleotides having a different 0;4000;5000;6000;7000;8000;9000;10000;20 000;30000;40000;50000;60000;70000;80000; 90000;100000;200000;300000;400000;500000 ;600000;700000;800000;900000;106;1.1×106 ;1.2×10⁶;1.3×10⁶;1.4×10⁶;1.5×10⁶;1.6×10⁶ ;1.7×10⁶;1.8×10⁶;1.9×10⁶;2×10⁶;2.1×10⁶;2 0.2×10⁶; 2.3×10⁶; 2.4×10⁶; 2.5×10⁶; 2.6×10⁶; 2 0.7×10⁶; 2.8×10⁶; 2.9×10⁶; 3×10⁶; 4×10⁶; 5×10⁶ ;6×10⁶;7×10⁶;8×10⁶;9×10⁶;or 10⁷ oligonucleos They may contain nucleotides, and each oligonucleotide has a different nucleotide sequence. Contains the rheoside domain. In some cases, the oligonucleotide collection is the largest 100;200;300;400;500;600;700;800;900;1000 ;2000;3000;4000;5000;6000;7000;8000;9000 ;10000;20000;30000;40000;50000;60000;700 00;80000;90000;100000;200000;300000;4000 00;500000;600000;700000;800000;900000;10 6; 1.1 × 10⁶; 1.2 × 10⁶; 1.3 × 10⁶; 1.4 × 10⁶; 1.5 × 10 6; 1.6 × 10⁶; 1.7 × 10⁶; 1.8 × 10⁶; 1.9 × 10⁶; 2 × 10⁶; 2.1×10⁶; 2.2×10⁶; 2.3×10⁶; 2.4×10⁶; 2.5×10⁶; 2.6×10⁶; 2.7×10⁶; 2.8×10⁶; 2.9×10⁶; 3×10⁶; 4× 106; 5×106; 6×106; 7×106; 8×106; 9×106; or 107 It may contain several oligonucleotides, and each oligonucleotide may have a different nucleotype. Contains a nucleotide domain having a sequence. In some cases, oligonucleotides The collection contains at least 100;200;300;400;500;600;700; 800;900;1000;2000;3000;4000;5000;6000;70 00;8000;9000;10000;20000;30000;40000;500 00;60000;70000;80000;90000;100000;200000 ;300000;400000;500000;600000;700000;8000 00;900000;106;1.1×106;1.2×106;1.3×106;1. 4×10⁶; 1.5×10⁶; 1.6×10⁶; 1.7×10⁶; 1.8×10⁶; 1. 9×10⁶; 2×10⁶; 2.1×10⁶; 2.2×10⁶; 2.3×10⁶; 2.4× 10⁶;2.5×10⁶;2.6×10⁶;2.7×10⁶;2.8×10⁶;2.9× 106; 3×106; 4×106; 5×106; 6×106; 7×106; 8×106; It may contain 9 × 10⁶; or 10⁷ oligonucleotides, each oligonucleotide Otide contains nucleotide domains with different nucleotide sequences.

[0059] Oligonucleotides in an oligonucleotide collection are of the same nucleotide type. It may contain a domain of nucleotides having a column. In some cases, oligonucleotides The collection contains approximately 1;2;3;4;5;6;7;8;9;10;11;12;13;14;15;16;1 7;18;19;20;25;30;35;40;45;50;55;60;65;70 ;75;80;85;90;95;100;150;200;250;300;350; 400; 450; 500; 600; 700; 800; 900; or 1000 oligos It may contain nucleotides. In some cases, a collection of oligonucleotides is identical. Contains up to 1;2;3;4;5 nucleotide domains having the nucleotide sequence ;6;7;8;9;10;11;12;13;14;15;16;17;18;19;2 0;25;30;35;40;45;50;55;60;65;70;75;80;85 ;90;95;100;150;200;250;300;350;400;450;5 Contains 00;600;700;800;900; or 1000 oligonucleotides In some cases, a collection of oligonucleotides may have identical nucleotide sequences. At least 1;2;3;4;5;6;7;8 nucleotide domains containing the domains of nucleotides having ;9;10;11;12;13;14;15;16;17;18;19;20;25;3 0;35;40;45;50;55;60;65;70;75;80;85;90;95 ;100;150;200;250;300;350;400;450;500;600 It may contain 700, 800, 900, or 1000 oligonucleotides.

[0060] In some cases, each oligonucleotide in the collection of oligonucleotides is 1 It may contain domains of nucleotides that are not present in one or more background populations. In some cases, each oligonucleotide in the oligonucleotide collection is Domains of nucleotides that are not present in the main genome, exome, or transcriptome It may contain. In some cases, each oligonucleotide in the collection of oligonucleotides Otide is present in the genome, exome, or transcriptome of one or more vertebrate animals. It may contain nucleotide domains that are not present in the original. In some cases, oligonucleotides Each oligonucleotide in the collection is from one or more mammalian genomes, exo. It may contain nucleotide domains that are not present in the chromosome or transcriptome. In some cases, each oligonucleotide in the collection of oligonucleotides is one or nucleos that are not present in multiple human genomes, exomes, or transcriptomes It may contain the domain of the oligonucleotide. In some cases, each of the oligonucleotides in the collection Oligonucleotides are derived from the genomes of one or more humans and one or more bacteria. It may contain nucleotide domains that are not present in the nucleotide or transcriptome. In some cases, each oligonucleotide in the collection of oligonucleotides is 1 The genomes, exomes, or transcripts of one or more humans and one or more viruses. It may contain domains of nucleotides that are not present in the scriptome.

[0061] In some cases, the nucleotide domain is identical to that of one or more populations in question. They may contain nearly identical, complementary, or nearly complementary nucleotide sequences. In some cases, The nucleotide domains are identical, nearly identical, complementary, or It may contain nearly complementary nucleotide sequences. In some cases, the nucleotide domain This is approximately 0.0001, 0.0005, 0.001, and 0.005 of the nucleic acids in the target population. , 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 , 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 also It may contain nucleotide sequences that are identical, nearly identical, complementary, or nearly complementary to 100%. In some cases, the domain of a nucleotide is up to 0.000 of the nucleic acids in the population in question. 1, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 4 0, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93 , identical, nearly identical, complementary or 94, 95, 96, 97, 98, 99 or 100% It may contain nearly complementary nucleotide sequences. In some cases, the nucleotide domains are , at least 0.0001, 0.0005, 0.001, 0.001 of nucleic acids in the target population. 005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0. 7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 9 Contains nucleotide sequences identical, nearly identical, complementary, or nearly complementary to 9 or 100% In some cases, the nucleotide domain is about 0.0001 of the nucleotide sequence. , 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0. 3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 , 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% are identical to the nucleic acids in the target population. It may contain nearly identical, complementary, or nearly complementary nucleotide sequences. In some cases... The nucleotide domains are the maximum 0.0001, 0.0005, 0 of the nucleotide sequence. 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 , 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 , 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97%, 98%, 99%, or 100% are identical, nearly identical, or complementary to the nucleic acids in the target population. It may contain nucleotide sequences that are nearly complementary. In some cases, the nucleotides The main part is at least 0.0001, 0.0005, 0.001, 0 of the nucleotide sequence. 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 , 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or 100% are identical, nearly identical, complementary, or nearly complementary to the nucleic acids in the target population. It may contain a nucleotide sequence that is a pair. In some cases, the nucleotide domain is a pair The group that forms the elephant may be identical, nearly identical, complementary, or nearly complementary. It may contain nucleotide sequences that are not present. In some cases, the nucleotide domain is shown in Figure 4. As shown in Figure 5, the degree of inclusion of the target population can be maintained.

[0062] In some cases, the nucleotide domain is one or more background populations It may contain nucleotide sequences that are identical, nearly identical, complementary, or nearly complementary to the given sequence. In some cases, the nucleotide domain is a subset of the background population and It may contain nucleotide sequences that are identical, nearly identical, complementary, or not nearly complementary. In some cases, the nucleotide domain is approximately 0.0% of the nucleic acid in the background population. 0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0. 2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2. 5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 3 5, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92 Even if it is the same as 93, 94, 95, 96, 97, 98, 99 or 100%, or nearly the same. , may contain nucleotide sequences that are either complementary or nearly complementary. In some cases, nuclei The leotide domain is up to 0.0001, 0.000 in nucleic acids in the background population. 5, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 , 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, Identical to 96, 97, 98, 99, or 100%, nearly identical, nearly identical, complementary, or nearly equivalent. It may contain non-complementary nucleotide sequences. In some cases, the nucleotide domain is , at least 0.0001, 0.0005, 0.001 of nucleic acids in the background population , 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 , 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6 , 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 9 8, 99, or 100% are either identical, nearly identical, complementary, or not nearly complementary. It may contain a creotide sequence. In some cases, the nucleotide domain is a nucleotide Approximately 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05 of the array 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1. 5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 2 5, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% are backgrounds Nucleic acids in the und population that are identical, nearly identical, complementary, or nearly complementary It may contain an ocidal sequence. In some cases, the nucleotide domain is a nucleotide sequence Maximum values ​​of 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25 , 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% are backgrain Nucleotides that are identical, nearly identical, complementary, or nearly complementary to nucleic acids in the population It may contain a nucleotide sequence. In some cases, the nucleotide domain is a nucleotide sequence At least 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05 , 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1 0.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 9 0, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% are backing Nucleic acids in the round population that are identical, nearly identical, complementary, or nearly complementary It may contain a rheotide sequence. In some cases, the nucleotide domain may be one or more. Nucleotide sequences identical, nearly identical, complementary, or nearly complementary to the background population. It may include.

[0063] In some cases, the nucleotide domain is one or more background populations It may contain nucleotide sequences that bind through mismatch with nucleic acids. In some cases, nucleo The domain of the cydo is 1, 2, 3, 4, 5 with one or more background population nucleic acids. , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 Contains nucleotide sequences that are joined by 0, 21, 22, 23, 24, or 25 mismatches. It may appear that in some cases, the nucleotide domain is one or more backgrounds Maximum 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mistakes It may contain nucleotide sequences that are joined by matching. In some cases, the domain of nucleotides n is at least 1, 2, 3, 4, 5, with one or more background population nucleic acids. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , containing nucleotide sequences joined by 21, 22, 23, 24 or 25 mismatches obtain.

[0064] A nucleotide domain can be of one or more lengths. In some cases, the nucleotide The domain of an nucleotide is of a single length. In some cases, the domains of nucleotides are different. It is of a certain length. In some cases, the nucleotide domain is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 Nucle The nucleotide length can be 13 or 14 nucleotides. It is the nucleotide length. In some cases, the nucleotide domain is 13 nucleotides long. In some cases, the nucleotide domain is 14 nucleotides long. In total, the nucleotide domains can number up to 10, 11, 12, 13, 14, 15, 16, 17 It can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In some cases, the nucleotide domain is up to 13 or 14 nucleotides long. In some cases, the nucleotide domain is up to 13 nucleotides long. In total, a nucleotide domain is a maximum of 14 nucleotides long. In some cases, nucleotides Ochido's domains are at least 10, 11, 12, 13, 14, 15, 16, 17, 1 The length can be 8, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In that case, the nucleotide domain is at least 13 or 14 nucleotides long. In some cases, the nucleotide domain is at least 13 nucleotides long. In some cases, the nucleotide domain is at least 14 nucleotides long. In this case, the nucleotide domains are 10-20, 11-20, 12-20, 13-20, 14-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-1 4, 10-13, 11-19, 12-19, 13-19, 14-19, 11-18, 11 ~17, 11~16, 11~15, 11~14, 11~13, 12~18, 12~17, 12-16, 12-15, 12-14, 12-13, 13-18, 13-17, 13-1 6, 13-15, 13-14, 14-18, 14-17, 14-16 or 14-15 It can be the length of the nucleotide. In some cases, the nucleotide domain has 12 to 15 nucleotides. This is the nucleotide length. In some cases, the nucleotide domain is 13-15 nucleotides long. In some cases, the nucleotide domain is 13 to 14 nucleotides long. In some cases, the nucleotide domain is 10mer, 11mer, 12mer, 13 mer, 14mer, 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer, 23mer, 24mer or 25mer, etc. It is a k-mer. In some cases, the nucleotide domain is 13-mer or 14-mer. It is er. In some cases, the nucleotide domain is 13mer. In this case, the nucleotide domain is 14mer.

[0065] background groups As used herein, background population generally refers to the population being studied. It is a non-existent population. Using a background population of nucleic acids, the collection of oligonucleotides A sample can be created. For example, using a background population of nucleic acids, a target sample can be created. Create a collection of oligonucleotides that can hybridize into a population. (For example, background population nucleic acids, starting oligonucleotides) To use as a "bait" for extracting oligonucleotides from a seed collection. (This is possible). In some cases, the background population is the population of nucleic acids in the sample. The methods and compositions provided herein can be used to remove the nucleic acid background from a sample. The group can be preferentially removed or isolated; in some cases, this method and composition A substance is used to isolate, remove, or detect the target population, but the background population... To prevent this from happening, it is important to specifically target the target group in a preferred manner. can.

[0066] In some cases, the background population is the host organism or the host genome, exome. Or it may originate from the transcriptome sequence (for example, present in this sequence, (This sequence contains sequences that are identical, nearly identical, complementary, or nearly complementary to this sequence.) In this case, the hosts are mammals, humans, non-human mammals, and domesticated animals (for example, laboratory animals). This could be a domestic pet or livestock, or a non-domesticated animal (e.g., a wild animal). In some cases, the hosts are dogs, cats, rodents, mice, hamsters, cows, birds, and chickens. Birds, pigs, horses, goats, sheep, rabbits, microorganisms, pathogens, bacteria, viruses, fungi, It can be a parasite. In some cases the host is a mammal. In some cases the host The host is human. The host may also be a patient. In some cases, the host is an antimicrobial agent, antibacterial agent. They can be treated with antiviral or antiparasitic agents. In some cases, the host is treated with antimicrobial agents. They may be treated with antibacterial agents, antiviral agents or antiparasitic agents, or they may be treated with antibacterial agents, antiviral agents or antiparasitic agents. It may be. In some cases, the host is (for example, one or more microorganisms, pathogens, bacteria, Infected with a virus, fungus, or parasite. In some cases, the host is infected (for example, 1 Not infected with any particular microorganism, pathogen, bacterium, virus, fungus or parasite In some cases, the host is healthy. In some cases, the host is susceptible or susceptible. There is a risk of staining.

[0067] In some cases, the background population consists of dogs, cats, rodents, mice, and hamsters. - Cattle, birds, chickens, pigs, horses, goats, sheep, rabbits, microorganisms, pathogens, bacteria It may originate from viruses, fungi, or parasites. In some cases, it may be a background population. It is a mammal. In some cases the background population is human. In some cases In some cases, the background population may originate from the host. This refers to nucleic acids in multiple populations, such as humans and microbial populations (for example, humans only, humans and viruses, etc.). It contains (two- and bacteria, humans and fungi, humans and parasites, etc.).

[0068] In some cases, the background population consists of one or more genomes, exomes and It may be a bi / or transcriptome. In some cases, the background population is Approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 genomes, exomes and / or This can be a transcriptome. In some cases, the background population is up to 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 genomes, exomes and / or tranexes It could be a scriptome. In some cases, the background population is at least 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 genomes, exomes and / or tranexes It could be a scriptome.

[0069] In some cases, background populations feed on milk from multiple individual mammals of the same species. It can be an animal genome, exome, or transcriptome. In some cases, The Kuground population consists of mammalian genomes from multiple individual mammals of one or more species. It can be an exome or a transcriptome. In some cases, background A group consists of one or more males and one or more female mammals of the same species. This could be a mammalian genome, exome, or transcriptome. (Background) The population can be mammalian genomic DNA and mammalian RNA. In some cases, back Ground populations include mammalian genomes, exomes or transcriptomes and one Or it could be multiple microbial genomes, exomes, or transcriptomes. How many In that case, the background population is the mammalian genome, exome, or transcript. Tome and one or more pathogen genomes, exomes, or transcriptomes It is possible. In some cases, the background population is mammalian genome, exome or This refers to the transcriptome and one or more bacterial genomes, exomes, or trans. It could be a cryptome. In some cases, the background population is a mammalian genome. Exome or transcriptome and one or more viral genomes, exo It can be a background or transcriptome. In some cases, the background population is , mammalian genome, exome or transcriptome and one or more reticuli It can be the lovirus genome, exome, or transcriptome. In some cases... The background population consists of mammalian genomes, exomes, or transcriptomes. and one or more viral and one or more bacterial genomes, exomes or It can be a randicome. In some cases, the background population is a mammalian g Nom, exome or transcriptome and one or more parasitic genomes, It can be an exome or a transcriptome. In some cases, one or more are Approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, It could be 45, 50, 60, 70, 80, 90 or 100. In some cases, one The maximum number of options is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30. It could be 35, 40, 45, 50, 60, 70, 80, 90 or 100. In this case, one or more must be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 It is possible. In some cases, the mammalian genome, exome, or transcriptome is non This refers to the human genome, exome, or transcriptome.

[0070] In some cases, the mammalian genome, exome, or transcriptome is the human genome. It is an exome or transcriptome. In some cases, background The population is the human genome, exome, or transcriptome. In some cases, A mixed ground population consists of human genomes, exomes, or trans genomes from multiple individual humans. It could be a cryptome. In some cases, the background group is one or more people. Human genome, exome, or transcript from males and one or more females It could be a ptome. The background population consists of human genomic DNA and human RNA. In some cases, the background population is the human genome, exome, or transgender. Scriptome and one or more microbial genomes, exomes or transcripts It can be a genome. In some cases, the background population is the human genome, exome. or transcriptome and one or more pathogen genomes, exomes or It could be a randicome. In some cases, the background population is the human genome. , exome or transcriptome and one or more bacterial genomes, exo It could be a transcriptome or a background group. In some cases, the background group is The human genome, exome or transcriptome and one or more viral genes It can be a nom, exome, or transcriptome. In some cases, background The und population consists of the human genome, exome or transcriptome and one or more The number of retroviral genomes, which may be exomes or transcriptomes. In that case, the background population is the human genome, exome, or transcript. exome and one or more viral and one or more bacterial genomes, exome or This can be a transcriptome. In some cases, the background population is human hair Nom, exome or transcriptome and one or more parasitic genomes, It can be an exome or a transcriptome. In some cases, one or more are Approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, It could be 45, 50, 60, 70, 80, 90 or 100. In some cases, one The maximum number of options is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30. It could be 35, 40, 45, 50, 60, 70, 80, 90 or 100. In this case, one or more must be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 It is possible.

[0071] In some cases, the background population is the genome, exome, or transcript. Approximately 0.000001, 0.000005, 0.00001, 0.00005, 0. 0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0. 2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, It can make up 98, 99, or 100%. In some cases, the background population is geno. Maximum values ​​of 0.000001, 0.000005 for the exome or transcriptome. , 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.0 0.5, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 , 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 3 0, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 It can make up 92, 93, 94, 95, 96, 97, 98, 99 or 100%. In some cases, the background population is genome, exome, or transcriptome. At least 0.000001, 0.000005, 0.00001, 0.00005, 0 .0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 6 0, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 It can constitute 98, 99, or 100%.

[0072] In some cases, the background population is about 0.01 of the total population of nucleic acids in the sample. 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 , 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4 It can constitute 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In this case, the background population is up to 0.01, 0.05 of the total population of nucleic acids in the sample. 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 4 5, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94 , 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5 It can consist of 99.6%, 99.7%, 99.8%, or 99.9%. In some cases, The ground population is at least 0.01, 0.05, and 0.0 of the total nucleic acid population in the sample. 1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 9 5, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 9 It can consist of 9.6%, 99.7%, 99.8%, or 99.9%.

[0073] In some cases, the background population is DNA, RNA, cDNA, mRNA, cR NA, dsDNA, ssDNA, miRNA, circular nucleic acid, circular DNA, circular RNA, microorganism cell nucleic acid, cell-free DNA, cell-free RNA, circulating cell-free DNA, circulating cell-free RNA or genome It may contain DNA. The background population may be a mixture of DNA and RNA. The background population can be genomic DNA. In some cases, the background The population contains artificially fragmented nucleic acids. In some cases, the background population is It does not contain artificially fragmented nucleic acids. In some cases, the background population is (e.g.) For example, it includes synthetic nucleic acids (by DNA synthesis). In some cases, background population nucleic acids. It is synthesized (for example, by DNA synthesis). In some cases, the background population This could be the genome, exome, or transcriptome.

[0074] In some cases, one or more nucleic acids in the background population are not labeled; In some cases, one or more nucleic acids in the background population are labeled. In this case, one or more nucleic acids in the background population may be, for example, nucleic acid labeled, chemically labeled. Labeled with an identifier or optical label. In some cases, one or in the background population Multiple nucleic acids are conjugated onto a solid support. In some cases, the label is placed in the background. The 5' or 3' end of nucleic acids in the background population or within nucleic acids in the background population It can be attached. In some cases, one or more in the background population Nucleic acids are labeled with two or more labels.

[0075] Nucleic acids in the background population may contain nucleic acid labels. In some cases, the nucleus Acid labels may include one or more of the following: barcodes (e.g., sample barcodes) ), universal primer sequence, primer binding site (for example, not limited to just that) DNA sequencing primer binding site, sample barcode sequencing primer binding site and includes amplification primer binding sites that meet the requirements of various sequencing platforms. (For column determination or barcode reading), sequencer-compatible sequences, sequence determination programs Rat homing attachment sequence, sequencing adapter sequence or adapter. Nucleic acid labeling, ( For example, by ligation or synthetic design, nucleic acids in a background population attach to it. It can be made possible. In some cases, the length of the nucleic acid label is such that the nucleic acid in the background population It is included in the length of the nucleic acid label. In some cases, the length of the nucleic acid label is included in the length of the nucleic acid in the background population. It is not included in the length.

[0076] Nucleic acids in the background population may contain chemical labels. Some non-limiting chemical labels Examples include biotin, avidin, streptavidin, radiolabeled substances, polypeptides. Examples include polymers. Nucleic acids in the background population may contain optical labels. Some non-limiting examples of labeling include fluorophores, fluorescent proteins, dyes, and quantum particles. One example is conjugating nucleic acids from a background population onto a solid support. It is possible. Some non-limiting examples of solid supports include beads, magnetic beads, and Remar, slide, tip, surface, plate, channel, cartridge, microfluidic Examples include Vice and microarrays. Nucleic acids in a background population are affinity-based. It can be conjugated onto a solid support for chromatography. In some cases, each nucleic acid in the background population has a different label. Each nucleic acid in the ground population has the same label. In some cases, the background population Each copy of nucleic acid in a group has the same label. In some cases, the back has different sequences. Each nucleic acid in the ground sample has a different label.

[0077] Target group Generally, a group of subjects is distinguished from other components of a larger group by certain characteristics. This is a population that exhibits certain characteristics. The target population is present in a complex mixture of host nucleic acids and non-host nucleic acids. It may be a non-host nucleic acid (e.g., bacterial nucleic acid).

[0078] In some cases, the target population is DNA, RNA, cDNA, mRNA, cRNA, dsDNA, ssDNA, miRNA, circulating nucleic acid, circulating DNA, circulating RNA, cell-free nucleic acid Cell-free DNA, cell-free RNA, circulating cell-free DNA, circulating cell-free RNA, or genomic DNA It may include A. The target population may be a mixture of DNA and RNA. The population can be genomic DNA. In some cases, the population in question is artificially fragmented. It contains nucleic acids. In some cases, the target population contains artificially fragmented nucleic acids. No. In some cases, the population in question does not include synthetic nucleic acids (e.g., those produced by DNA synthesis). In some cases, the target population is synthesized (for example, by DNA synthesis). In some cases, the target population is the genome, exome, or transcriptome. obtain.

[0079] In some cases, the target population is non-host. In some cases, the non-host is something other than the host. It refers to the organism that is the host. In some cases, the non-host refers to a species other than the host. The term "non-host" can refer to other organisms of the same species as the host. In some cases, the non-host is a non-mammalian. Non-human, non-dog, non-cat, non-rodent, non-mouse, non-hamster, non-cow, non-bird, non It could be a chicken, non-pig, non-horse, non-goat, non-sheep, or non-rabbit. In combination with a non-host, the host is a microorganism, pathogen, bacteria, virus, fungus, parasite, or combination thereof. It can be a case of non-host. In some cases the non-host is a non-mammalian. It is non-human. In some cases, the non-host is non-patient.

[0080] In some cases, the target population may be non-mammalian or non-human. In addition, the target population includes microorganisms, bacteria, viruses, fungi, retroviruses, pathogens or It can be a parasite. In some cases, the target population is non-microorganism, non-bacterial, and non-viral. It may be a nonfungus, nonretrovirus, nonpathogen, or nonparasite. The target population may originate from microorganisms, pathogens, bacteria, viruses, fungi, or parasites. In some cases, the target population is non-mammalian. The population is non-human. In some cases, the population in question is a microorganism that infects a host or It may originate from a pathogen. In some cases, the target population is non-mammalian DNA or RNA. It is possible. In some cases, the target population may be non-human DNA or RNA. In some cases, the target population includes microorganisms, bacteria, viruses, fungi, retroviruses, It may be the DNA or RNA of a pathogen or parasite. In some cases, the target population The group is non-microorganism, non-bacterial, non-viral, non-fungal, non-retrovirus, non-pathogen, or non-parasitic. It could be the DNA or RNA of a living organism.

[0081] In some cases, the target population is one or more non-host genomes, exomes or This may include transcriptomes. In some cases, the target population is approximately 1;2;3 ;4;5;6;7;8;9;10;15;20;25;30;35;40;45;50; 60;70;80;90;100;500;1000;5000;10000;5000 0; or 100,000 non-host genomes, exomes, or transcriptomes It is possible. In some cases, the target population is up to 1;2;3;4;5;6;7;8;9 ;10;15;20;25;30;35;40;45;50;60;70;80;90; 100; 500; 1000; 5000; 10000; 50000; or 100000 pieces This may include the non-host genome, exome, or transcriptome. In some cases, The target populations are at least 1;2;3;4;5;6;7;8;9;10;15;20 ;25;30;35;40;45;50;60;70;80;90;100;500;1 000;5000;10000;50000; or 100000 non-host genomes, e It may include a genome or transcriptome. In some cases, the population being studied is Non-mammalian genomes, exomes, or transcripts from multiple individuals of the same non-mammalian species. It may include putomes. In some cases, the population in question is one or more non-mammals. Includes non-mammalian genomes, exomes, or transcriptomes from multiple individuals of a species. Obtain. In some cases, the target population is non-mammalian genomic DNA and non-mammalian R May include NA. In some cases, the target population is one or more microbial genomes. It may include the exome or transcriptome. In some cases, the population being studied is This may include one or more pathogen genomes, exomes, or transcriptomes. In some cases, the target population is one or more bacterial genomes, exomes, or It may include a randicome. In some cases, the population being studied is one or more This may include the viral genome, exome, or transcriptome. In some cases, The target population is one or more retroviral genomes, exomes, or trans genomes. It may include cryptomes. In some cases, the population in question may contain one or more viruses. Includes the genome, exome, or transcriptome of one or more bacteria. In some cases, the target population is obtained from the genome of one or more parasites, exo. It may include a 'm' or 'transcriptome'. In some cases, one or more are approximately 1, 2. , 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 5 It could be 0, 60, 70, 80, 90, or 100. In some cases, one or more. The maximum values ​​are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 4. It could be 0, 45, 50, 60, 70, 80, 90 or 100. In some cases, 1 One or more is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 2 It could be 5, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100. In some cases, non-mammalian genomes, exomes, or transcriptomes are non-human genomes. It is a nome, exome, or transcriptome. In some cases, it is a non-mammalian genotype. The exome, or transcriptome, is a group of microorganisms, bacteria, viruses, fungi, and retrograde organisms. The genome, exome, or transcriptome of a virus, pathogen, or parasite. ru.

[0082] The target population may include a portion of the genome, exome, or transcriptome. In some cases, the target population is approximately the genome, exome, or transcriptome of 0.000001, 0.000005, 0.00001, 0.00005, 0.0001 , 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0. 3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 9 It can constitute 9 or 100%. In some cases, the population being studied is the genome, exome Or the maximum of the transcriptome 0.000001, 0.000005, 0.0000 1, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01 , 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0. 9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 , 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, It can consist of 94, 95, 96, 97, 98, 99, or 100%. In some cases, The population that will become the elephant will have at least 0.000 of the genome, exome, or transcriptome. 001, 0.000005, 0.00001, 0.00005, 0.0001, 0.00 0.5, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4 , 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 1 It can constitute 00%.

[0083] The target population may represent a portion of the total population of nucleic acids in the sample. In some cases, The population that represents the elephant is approximately 0.000001, 0.000005 of the total nucleic acid population in the sample. 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.00 5, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, It can consist of 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50%. In this case, the target population is the maximum of 0.000001, 0 of the total population of nucleic acids in the sample. .000005, 0.00001, 0.00005, 0.0001, 0.0005, 0. 001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50% It can be composed of at least one part of the total population of nucleic acids in the sample. 0.000001, 0.000005, 0.00001, 0.00005, 0.000 1, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 4 It can consist of 0, 45, or 50%.

[0084] Background population nucleic acids are present in excess of the nucleic acids of the target population in the sample. It is possible. The ratio of the background population to the nucleic acids of the target population is approximately 1:2:3:4. ;5;6;7;8;9;10;11;12;13;14;15;16;17;18;19 ;20;25;30;35;40;45;50;55;60;65;70;75;80; 85;90;95;100;200;300;400;500;600;700;800 ;900;1000;2000;3000;4000;5000;6000;7000; 8000;9000;10000;20000;30000;40000;50000; 60000;70000;80000;90000;100000;200000;30 0000;400000;500000;600000;700000;800000; 900000;1000000;2000000;3000000;4000000;5 000000;6000000;7000000;8000000;9000000;1 It could be 0000000. The ratio of the background population to the nucleic acids of the target population is Max 1;2;3;4;5;6;7;8;9;10;11;12;13;14;15;16 ;17;18;19;20;25;30;35;40;45;50;55;60;65; 70;75;80;85;90;95;100;200;300;400;500;60 0;700;800;900;1000;2000;3000;4000;5000;6 000;7000;8000;9000;10000;20000;30000;400 00;50000;60000;70000;80000;90000;100000; 200000;300000;400000;500000;600000;70000 0;800000;900000;1000000;2000000;3000000; 4000000;5000000;6000000;7000000;8000000; It could be 9,000,000;1,000,000. The background for nucleic acids of the target population. The ratio of round groups is at least 1;2;3;4;5;6;7;8;9;10;11;12 ;13;14;15;16;17;18;19;20;25;30;35;40;45; 50;55;60;65;70;75;80;85;90;95;100;200;30 0;400;500;600;700;800;900;1000;2000;3000 ;4000;5000;6000;7000;8000;9000;10000;200 00;30000;40000;50000;60000;70000;80000;9 0000;100000;200000;300000;400000;500000; 600000;700000;800000;900000;1000000;2000 000;3000000;4000000;5000000;6000000;7000 It can be 000;8000000;9000000;10000000. The ratio is the concentration. It can be calculated in terms of moles or mass.

[0085] The target population may include nucleic acids derived from one or more species. In some cases, The target population is approximately 1;2;3;4;5;6;7;8;9;10;15;20;25; 30;35;40;45;50;60;70;80;90;100;200;300;4 00;500;1000;5000;10000;50000; or 100000 seeds It may contain nucleic acids derived from [the source]. In some cases, the target population may be up to 1;2;3;4; 5;6;7;8;9;10;15;20;25;30;35;40;45;50;60; 70;80;90;100;200;300;400;500;1000;5000;1 It may contain nucleic acids derived from 0000;50000; or 100000 species. In this case, the target population is at least 1;2;3;4;5;6;7;8;9;10;15 ;20;25;30;35;40;45;50;60;70;80;90;100;20 0;300;400;500;1000;5000;10000;50000;or 1 It may contain nucleic acids derived from 00000 species. In some cases, the species are non-mammalian species. In some cases, the species is a species other than human. In some cases, the species is a microorganism, bacteria, virus. It is a fungus, retrovirus, pathogen, or parasite. In some cases, it is the target. The groups were approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, and 35. , 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 50 It may contain nucleic acids derived from 0 or 1000 bacterial or viral species. In some cases, The target groups are up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, and 25. , 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, It may contain nucleic acids derived from 400, 500, or 1000 bacterial or viral species. In Tsuka's case, the target groups are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 , nuclei derived from 200, 300, 400, 500 or 1000 bacterial or viral species It may contain acid. In some cases, the target population is approximately 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 9 To 0, 100, 200, 300, 400, 500 or 1000 bacterial and viral species It may contain at least one nucleic acid of origin. In some cases, the population in question may be up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 , 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 10 It may contain at least one nucleic acid derived from 00 bacterial and viral species. In total, the target groups are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200 at least 300, 400, 500, or 1000 bacterial and viral species It may contain one nucleic acid. In some cases, the population in question contains approximately one bacterial species and one virus. S species, 2 bacterial species and 2 viral species, 3 bacterial species and 3 viral species, 4 bacterial species and 4 U Virus species, 5 bacterial species and 5 virus species, 6 bacterial species and 6 virus species, 7 bacterial species and 7 virus species, 8 bacterial species and 8 virus species, 9 bacterial species and 9 virus species, 10 bacterial species and 10 virus species, 15 bacterial species and 15 virus species, 20 bacterial species and 20 viruses S species, 25 bacterial species and 25 virus species, 30 bacterial species and 30 virus species, 35 bacterial species and 35 virus species, 40 bacterial species and 40 virus species, 45 bacterial species and 45 viruses S species, 50 bacterial species and 50 virus species, 60 bacterial species and 60 virus species, 70 bacterial species and 70 virus species, 80 bacterial species and 80 virus species, 90 bacterial species and 90 viruses S species, 100 bacterial species and 100 virus species, 200 bacterial species and 200 virus species, 3 00 bacterial species and 300 virus species, 400 bacterial species and 400 virus species, 500 bacteria Derived from a species and 500 virus species, or 1000 bacterial species and 1000 virus species. It may contain at least one nucleic acid. In some cases, the population in question may contain up to one bacterial species. and 1 virus species, 2 bacterial species and 2 virus species, 3 bacterial species and 3 virus species, 4 bacteria species and 4 virus species, 5 bacterial species and 5 virus species, 6 bacterial species and 6 virus species, 7 Bacterial species and 7 virus species, 8 bacterial species and 8 virus species, 9 bacterial species and 9 virus species , 10 bacterial species and 10 virus species, 15 bacterial species and 15 virus species, 20 bacterial species and 20 virus species, 25 bacterial species and 25 virus species, 30 bacterial species and 30 virus species , 35 bacterial species and 35 virus species, 40 bacterial species and 40 virus species, 45 bacterial species and 45 virus species, 50 bacterial species and 50 virus species, 60 bacterial species and 60 virus species , 70 bacterial species and 70 virus species, 80 bacterial species and 80 virus species, 90 bacterial species and 90 virus species, 100 bacterial species and 100 virus species, 200 bacterial species and 200 virus Virus species, 300 bacterial species and 300 virus species, 400 bacterial species and 400 virus species 500 bacterial species and 500 virus species, or 1000 bacterial species and 1000 viruses It may contain at least one nucleic acid derived from a species. In some cases, the population being studied is small At least 1 bacterial species and 1 virus species, 2 bacterial species and 2 virus species, 3 bacterial species and 3 Virus species, 4 bacterial species and 4 virus species, 5 bacterial species and 5 virus species, 6 bacterial species and 6 virus species, 7 bacterial species and 7 virus species, 8 bacterial species and 8 virus species, 9 bacterial species and 9 virus species, 10 bacterial species and 10 virus species, 15 bacterial species and 15 viruses Species, 20 bacterial species and 20 virus species, 25 bacterial species and 25 virus species, 30 bacterial species and 30 virus species, 35 bacterial species and 35 virus species, 40 bacterial species and 40 viruses 45 bacterial species and 45 virus species, 50 bacterial species and 50 virus species, 60 bacterial species and 60 virus species, 70 bacterial species and 70 virus species, 80 bacterial species and 80 viruses Species, 90 bacterial species and 90 virus species, 100 bacterial species and 100 virus species, 200 species 200 bacterial species and 200 virus species, 300 bacterial species and 300 virus species, 400 bacterial species and 400 virus species, 500 bacterial species and 500 virus species, or 1000 bacterial species and It may contain at least one nucleic acid derived from 1,000 virus species.

[0086] In some cases, one or more nucleic acids in the target population are not labeled; in some cases In some cases, one or more nucleic acids in the target population are labeled. One or more nucleic acids in the resulting population are, for example, labeled with nucleic acid labels, chemical labels, or optical labels. Labeled. In some cases, one or more nucleic acids from the target population are labeled onto a solid support. To conjugate. In some cases, the label is applied to the 5' or 5' of the nucleic acid in the target population. It can be attached to the 3' end or inside the nucleic acids in the target population. In addition, one or more nucleic acids in the target population are labeled with two or more labels.

[0087] Nucleic acids in the target population may contain nucleic acid labels. In some cases, nucleic acid labels are This may include one or more of the following: barcodes (e.g., sample barcodes), Uni Versal primer sequence, primer binding site (for example, not limited to that, DNA) Sequencing primer binding site, sample barcode sequencing primer binding site and type Includes amplification primer binding sites that meet the requirements of various sequencing platforms, (or for barcode reading), sequencer-compatible sequences, sequence determination platform A sequence to which a layer is attached, a sequencing adapter sequence, or an adapter. A nucleic acid label (for example, It can be attached to nucleic acids in the target population (by ligation or synthetic design). In some cases, the length of the nucleic acid label is included in the length of the nucleic acids in the population being studied. In some cases, the length of the nucleic acid label is not included in the length of the nucleic acids in the target population.

[0088] Nucleic acids in the target population may contain chemical labels. Some limitations on chemical labels Typical examples include biotin, avidin, streptavidin, radiolabeling, and polypeptides. Examples include polymers. Nucleic acids in the target population may include optical labels. Some non-limiting examples of optical labeling include fluorophores, fluorescent proteins, dyes, and quantities. One example is the use of child dots. This involves conjugating nucleic acids from the target population onto a solid support. This is possible. Some non-limiting examples of solid supports include beads, magnetic beads, and poly Marker, slide, tip, surface, plate, channel, cartridge, microfluidic device Examples include situ cells and microarrays. Nucleic acids in the target population are analyzed using affinity cross-linking. It can be conjugated to a solid support for matrixing. In some cases, Each nucleic acid in the target population has a different label. In some cases, in the target population Each nucleic acid has the same label. In some cases, each copy of nucleic acid in the population in question is the same. It has a label. In some cases, each nucleic acid in the target population having a different sequence is different Close the sign.

[0089] Microorganisms that can be detected by the methods provided herein (microorganisms) Some non-exclusive examples of nisms (microbes) include bacteria, archaea, and protozoa. Examples include insects, protists, fungi, algae, viruses, retroviruses, pathogens, or parasites. In some cases, microorganisms are prokaryotes. In other cases, microorganisms are eukaryotes. Yes. Some non-specific examples of bacteria include the genus Bacillus, Boll. The genera Bordetella, Borrelia, and Bursella are all related to the B rucella), Campylobacter genus, Chlamydia genus (Chlamydia), Chlamydophila genus, Clost Clostridium, Corynebacterium E. erium), Enterococcus, Escherichia scherichia), Francisella genus, Haemophilus genus (Haemophilus), Helicobacter genus, Regio Legionella, Leptospira, Lister Listeria, Mycobacterium, Icoplasma, Neisseria, and other genera Pseudomonas, Rickettsia, Sa Salmonella, Shigella, Staphylococcus Staphylococcus genus, Staphylococcus aureus Aures, Streptococcus genus, Treponema The genera Treponema, Vibrio, and Yersinia Examples of fungi include the Candida genus. dida), Aspergillus genus, Cryptococcus genus (Cr Histoplasma (Histoplasma), Pneumosis The genera Pneumocystis and Stachybotry Examples include s). In some cases, the microscopic particles detected by the methods provided herein are The organism is a drug-resistant microorganism or a multidrug-resistant pathogen. Non-limiting examples include: in some cases, Clostridium diff Drug resistance of Clostridium difficile (C. difficile) Sex strains, carbapenem-resistant Enterobacteriaceae (CRE), drug-resistant Neisseria gonorrhoeae (GO). Acinetobacter norrhoeae (cephalosporin resistant), multidrug resistant (Acin etobacter, drug-resistant Campylobacter, fluconazole-resistant Candida (fungus) ), broad-spectrum β-lactamase-producing Enterobacteriaceae (ESBL), vancomycin-resistant enterobacteria Cocci (Enterococcus) (VRE), multidrug-resistant Pseudomonas aeruginosa Salmonella aeruginosa, drug-resistant non-typhoid Salmonella , drug-resistant Salmonella typhi, drug-resistant Shigella ella), methicillin-resistant Staphylococcus aureus (MRSA), drug-resistant Streptococcus pneumoniae (Stre) ptococcus pneumonia), drug-resistant tuberculosis (MDR and XDR), multiple Drug-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus (VRSA), erythromycin Clindamycin-resistant Streptococcus group A or clindamycin-resistant Streptococcus group B.

[0090] In some cases, the methods and compositions provided herein can be used to retrovirus Alternatively, viruses such as lentiviruses can be detected. In some cases, viruses The Baltimore virus classification system includes groups I, II, III, IV, and It is a member of group V, group VI, or group VII. In some cases, the virus is adenovirus Adenoviridae, Anelloviridae dae), Arenaviridae, Astroviridae (As troviridae, Bunyaviridae, caliciwi Family Caliciviridae, Family Coronaviridae e) Filoviridae, Flaviviridae Hepadnaviridae, Hepevirus Family (Hepeviridae), Family (Herpesviridae), Orthomyxoviridae, Papillomaviridae (Papillomaviridae), Papovaviridae e) Paramyxoviridae, Parvoviridae Parvoviridae, Picornaviridae Polyomaviridae, Poxviridae viridae, Reoviridae, Retroviridae Troviridae, Rhabdoviridae, or Toga It is a member of the family Togaviridae. In some cases, viruses Adenovirus, Amur virus, Andean virus, animal virus, astrovirus S, Tri-nephritis virus, Tri-orthreovirus, Tri-reovirus, Bannavirus, Bass-Congo virus, bat-borne virus, BK virus, blueberry shock virus Viruses, avian anemia virus, bovine serovirus, bovine coronavirus, bovine herpesvirus Russ 4, bovine parvovirus, bulbul coronavirus HKU11, carisalvirus, Catfish virus, Chandipla virus, American catfish virus, Chokuroi virus Rus, Cortivirus, Coxsackievirus, Cricket paralysis virus, Crimeancon Goh hemorrhagic fever virus, cytomegalovirus, dengue virus, Dobrava bergredoui Rus, Ebola virus, Ebola virus, El Moro Canyon virus, endotheliotropic elephant Rupes virus, Epstein-Barr virus, feline leukemia virus, foot-and-mouth disease virus, Gou virus, Guanalitovirus, Hantan River virus, Ha HCoV-EMC / 2012, Hendravirus, Henipavirus, Type A Hepatitis viruses, hepatitis B virus, hepatitis C virus, hepatitis D, hepatitis E virus, single Pure herpes type 1, herpes simplex virus type 2, herpes simplex virus type 1, herpes simplex virus Type 2, HIV human astrovirus, human bocavirus, human cytomegalovirus, Human herpesvirus type 8, human immunodeficiency virus (HIV) Human metapneumovirus, human papillomavirus, imdin virus, influenza Nzavirus, Ira Vistavirus, JC virus, Junin virus, Khabarovsk Viruses, koi herpesvirus, kunjin virus, lassa virus, limestone virus Yanion virus, Lloviu cueva virus, Lloviu virus, Lujo Virus, Machupovirus, Magboi virus, Marburg virus Marburg virus, Marburg virus, measles virus, Melakavirus, Measles Nanglevirus, Middle East Respiratory Syndrome Coronavirus, Minioptras Bat Coronavirus Virus 1, Minioptras bat coronavirus HKU8, monkeypox virus, Mononga Hiravirus, Mujuvirus, Mumpsvirus, Nipahvirus, Norwalkvirus S, orbivirus, parainfluenza virus, parvovirus B19, phytole Viruses, bat coronavirus HKU5, poliovirus, swine adenovirus S, Prospect Hill virus, Calub virus, Rabies virus, Ravn virus Respiratory rash virus, Reston virus, Reticuloendotheliosis virus, Horseshoe bat Coronavirus HKU2, rhinovirus, roseolovirus, Ross River virus, Tavirus, Rousette bat coronavirus HKU9, rubella virus, Sarehmannia - Viruses, Sabia virus, Sangassou virus, bat coronavirus S512, Ceran virus, Severe Acute Respiratory Syndrome virus, Schoepp's papillomavirus, Monkey foam virus, Sin Nombre virus, smallpox, Souchong virus, Sudanese virus Bola virus, Sudan virus, Thai forest Ebola virus, Thai forest virus Rus, Tanzania virus, Thottapalayam virus irus), Topografov virus, tremowy Rus, Tula virus, turkey coronavirus, turkeypox virus, bamboo bat virus Nile virus HKU4, varicella-zoster virus, varicella-zoster virus, West Nile virus Woodchuck hepatitis virus, yellow fever virus, Zika virus or Zaire Evolu virus It is an illus.

[0091] Some non-exclusive examples of pathogens include viruses, bacteria, prions, fungi, and parasites. Examples include substances, protozoa, and microorganisms. Some non-exclusive examples of pathogens include A Canthamoeba (Acanthamoeba), mites (Acari), Acinetobacter - Acinetobacter baumannii, actinomycete Actinomyces israelii, Actinomyces... Actinomyces gerencseriae, Propionibacterium Propionibacterium propionic us), Actinomycetoma, Eumycetoma (E Umycetoma, Adenoviridae, Alphawi Alphavirus, Anaplasma genus, Anaplasma • Phagocytophilum (Anaplasma phagocytophilum), A Ancylostoma braziliense Ancylostoma duodenale, Necator americanus, Angiostro Angiostrongylus costaricensis Anisakis sis, Arachnida ixodidae Ixodidae, Argasidae, hemolytic alkanobacteria Terrier (Arcanobacterium haemolyticum), order Protoacanthoptera (Archiacanthocephala), Moniliformis Moniliformis ( Moniliformis moniliformis), family Arenaviridae (Aren Ascaris lumbricoides (aviridae), roundworm Species of the genus (Ascaris), roundworm (Ascaris lumbricoides), As Aspergillus genus, Astroviridae family ae), Babesia genus (Babesia) polymorphic Babesia (B. divergens), Babe Sia Bigemina, Babesia Equi, Babesi B. microfti, B. duncani, Babesia genus, Bacillus anthracis, Bacillus cereus, Bacteroides genus Balamuthia mandrillari s), Balantidium coli, Henselae (Bar Tonella henselae, Baylisascari genus s), raccoon roundworm (Baylisascaris procyonis), human tapeworm (Bertiella mucronata), Bertiella sutderii (Bertie Illa studeri), BK virus, Blastocystis s), human blastocystis (Blastocystis hominis), blast Mrs. Blastomyces dermatitidis, 100 days Bordetella pertussis, Borrelia burgdorferi (B Orrelia burgdorferi), a species of the genus Borrelia, Borrelia, Brucella, Br Brugia malayi, Brugia timori, Bunya Viridae family, Burkholderia c. epacia), a species of the genus Burkholderia, glanders (Bu rkholderia mallei), Burkholderia pse udomallei), Caliciviridae, Campylobacter Campylobacter, Candida albicans Candida albicans, a species of the genus Candida, class Cestoda. , Taenia multiceps, Chlamydia trachoma Chlamydia trachomatis, Chlamydia trachomatis rachomatis), Neisseria gonorrhoeae, pneumonia Chlamydia (Chlamydophila pneumoniae), psittacosis (Chlamydophila psittaci), bed bug family (Cimicid) ae), bed bugs (Cimex lectularius), liver fluke (Clonorc his sinensis; Clonorcchis vibellini errini), Clostridium botulinum, Clostridium botulinum Clostridium difficile, well Clostridium perfringens, Clostridium perfringens (Tridium perfringens), Clostridium genus The species of tetanus bacterium (Clostridium tetani), *Coccidioides*. Coccidioides immitis, Coccidioides posadasi (Coccidioides posadasii), Cocliomia hominiborax ( Cochliomyia hominivorax, Colorado tick fever virus (CTF) V), Coronaviridae family, Corynebacterium diphtheriae Bacterium diphtheriae, Coxiella (Q fever coxiella) burnetii), Crimean-Congo hemorrhagic fever virus, Cryptococcus neoforma Cryptococcus neoformans, Cryptosporidium (C Cryptosporidium, the genus Cryptosporidium Cyclospora cayetane (Cyclospora cayetane) NSIS, Cytomegalovirus, Demodex... Folliculorum (Demodex folliculorum) / brev canis, dengue virus (DEN-1, DEN-2, DEN-3) (and DEN-4), Flavivirus, Human botfly (Der matobia homini), spear-shaped fluke (Dicrocoelium dendri) Dioctocybe (ticum), dinuclear amoeba (Dientamoeba fragilis), dioctocybe Dioctophyme renale, genus Diph yllobothrium), Diphyllobothrium la tum), Dracunculus medinensis, Ebola virus Echinococcus (EBOV), Echinococcus genus, Echinococcus (Echinococcus) nococcus granulosus), Echinococcus m Echinococcus ultilocularis, Echinococcus vogeli, Dormouse E. oligarthrus, Ehrlichia shafensis hia chaffeensis, Ehrlichia e Ehrlichia genus, Entamoeba histolytica (wingii), Entamoeba histolytica Entamoeba histolytica, Entamoeba histolytica tica), pinworm (Enterobius vermicularis), enterobiu Enterobius gregorii, Enterococcus genus (E Enterococcus, Enterovirus genus, Entero Virus, Coxsackie A virus, Enterovirus 71 (EV71), Epidermophyton floccosum , Trichophyton rubrum, Trichophyton rubrum Yton mentagrophytes), Epstein-Barr virus (EBV), Escherichia coli O157:H7, O111, and O104 :H4, Fasciola hepatica, Giant liver fluke (Fasciola) gigantica), Fasciolopsis buski, filari Superfamily Filarioidea, Family Filoviridae (Flaviviridae), Flaviviridae, Fonseca Fonsecaea pedrosoi, Francisel tularensis Fusobacterium la tularensis, Fusobacterium genus, Geotrichum candidum, enteroflagellate ( Giardia intestinalis, Giardia la mblia), Gnathostoma spinigerum, Gnathostoma rigens (Gnathostoma hispidum), Group A Streptococcus (Treptococcus), Staphylococcus, Guanari Tovirus, Haemophilus ducreyi, Haemophilus influenzae, Halicephalobium Halicephalobus gingivalis, Heartra Helicobacter pylori, Hepadnaviridae family, Hepatitis A virus, Hepatitis B virus Viruses, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Hepaviridae (H Herpes simplex virus 1 and 2 (HSV-1 and HSV-) 2) Herpesviridae family, Histoplasma capsulata Histoplasma capsulatum, HIV (Human Immunodeficiency Virus) Russ, Hortaea werneckii, Hitobokau Virus (HBoV), Human Herpesvirus 6 (HHV-6), Human Herpesvirus 7 (HHV-7), human metapneumovirus (hMPV), human papillomavirus (H PV), human parainfluenza virus (HPIV), small tapeworm (Hymenolep is nana), miniature tapeworm (Hymenolepis diminuta), isospo Isospora belli, JC virus, Junin virus, Kinge La Kingella kingae, Clebsierra granulomatis Klebsiella granulomatis, Lassa virus, Legionella nematode Pneumophila (Legionella pneumophila), Leishmania (L Leptospira, Ling Listeria monocytogenes (Listeria monocytogenes) (genes), Loa loa filarial worm, lymphocytic choriomeningitis virus Machupovirus (LCMV), Malassezia genus, Mansone La streptocerca (Mansonella streptocerca), Marlboro Measles virus, flu virus, Metagonimus yokagawai ), Microsporidia phylum, Middle East Respiratory Syndrome (Coronavirus) Navirus, molluscum contagiosum virus (MCV), monkeypox virus, Mucor a Mucormycosis, Entomophthorales Entomophthora disease, mumps virus, Mycobacterium leprae Mycobacterium leprae, Mycobacterium lepromatosis rium lepromatosis), Mycobacterium tuberculosis (Mycobacterium tuberculosis) Mycobacterium ulcerans (Mycobacterium ulcerans) Mycoplasma pneumonitis (Mycoplasma ulcerans), Mycoplasma pneumonitis (iae), Naegleria fowleri, Neisseria gonorrhoeae (N eisseria gonorrhoeae), meningococcus (Neisseria men Nocardia asteroides Nocardia species, Oestroidea dea), Calliphoridae, Sarcophagidae gidae), Onchocerca volvulus, Thai liver fluke ( Opisthorchis viverrini), cat liver fluke (Opisthorch is felineus), liver fluke (Clonorchis sinensis), or Tomyxoviridae, Papillomaviridae Apicillomaviridae, South American spore-forming fungi (Paracoccidioid) Paragonimus africanus (Paragonimus brasiliensis) us africanus); Paragonimus cariensis Paragonimus kell icotti); Paragonimus skrj abini); Paragonimus uterobiraris obilateralis), Westerman's lung fluke (Paragonimus wes) Paramyxovirus (termani), a species of the genus Paragonimus, paramyxovirus Paramyxoviridae family, parasitic diptera fly larva, parvoviridae family (P Pasteurella genus (Pasteurella arvoviridae), parvovirus B19 la), human lice (Pediculus humanus), human head lice (Pe diculus humanus capitis), body lice (Pediculu) s humanus corporis), pubic lice (Phthirus pubis) Picornaviridae, Piedria hortae (P Plasmodium falciparum (Platypleura japonica), Plasmodium falciparum (Platypleura japonica) Plasmodium vivax, Plasmodium ciparum, Plasmodium ovaceum Plasmodium ovale curtisi, oval-shaped larynx Plasmodium ovale wallikeri, quaternary fever Plasmodium malariae, Plasmodium vivax (Pl Asmodium knowlesi, Plasmodium genus, Pneumocystis jirovecii, Poly Ovirus, Polyomaviridae family, Poxvirus Family Poxviridae, Genus Prevotella, PRNP, Ke Lice (Pthirus pubis), human flea (Pulex irritans), Rabies virus, Reoviridae family, Respiratory syncytial virus (RSV), Retroviridae, Rhabdoviridae Rhinosporidium ridae), Rhinosporidium seeberi Rhinovirus (Eri), Rhinovirus genus, Rhinovirus, Coronavirus Rickettsia akari, the genus Rickettsia Rickettsia prowazekii (Typhus ttsia) Rickettsia rickettsii, rash fever Rickettsia typhi, Rift Valley fever virus, Rotavirus S, rubella virus, Sabia, Salmonella (Salmonella en typhus (Typhi), subspecies enterica, serotype typhi, Salmonella genus, Sarcocystis bobicanis Sarcocystis bovihominis, Sarcocystis suihominis Sarcoptes suihominis, Sarcoptes scabiei SARS coronavirus (cabiei), genus Schistosoma. Schistosoma haematobium, Japanese schistosomiasis Tumor (Schistosoma japonicum), Cystosoma mansoni (Sc histosoma mansoni and Schistosoma intercalatum (Schist Schistosoma intercalatum (Mekong schistosomiasis) Shigella (Ekongi), a species of the genus Schistosoma, a species of Shigella Spirometra erella, Sinnonbull virus, Spirometra mansoni (Diphyllobothrium meristemum) naceieuropaei), Sporothrix schönqi Staphylococcus genus (Schenckii), Treptococcus agalactiae ), Streptococcus pneumoniae, Streptococcus pyogenes (Streptococcus pyogenes), Strongyloid es stercoralis), tapeworm (Taenia), tapeworm (Taenia) saginata), Taenia solium, Bacteriaceae, Enterobacteriaceae (Enterobacteriaceae), California Thelazia Thelazia callipae (a californiensis), Oriental eye worm da), Togaviridae, Canine roundworm (Toxocara c anis), feline roundworm (Toxocara cati), Toxoplasma gondii (Toxoplasma gondii) (Plasma gondii), Treponema pallidum um), Trichinella spiralis, Trichinella britobi (Trichinella britovi), Tricinella Nelsoni (Trichin (ella nelsoni), Tricinella nati va), Trichobilharzia regen ti), Schistosomatidae, Trichomonas vaginalis (Tric) Homonas vaginalis, Trichophyton genus ), purple fungus (Trichophyton rubrum), Trichophyton tonsuran Trichophyton tonsurans, Beiger's hair follicle-forming bacteria (Trich osporon beigelii), whipworm (Trichuris trichiura) ), whipworm (Trichuris trichiura), whipworm (Trichuris trichiura), whipworm (Trichuris trichiura), whipworm (Trichuris trichiura) Trypanosoma brucei (vulpis) ), Trypanosoma cruzi, sand flea (T Ureaplasma penetrans, Ureaplasma urealithicum Varicella urealyticum), varicella-zoster virus (VZV), smallpox (Vari) Variola major, Variola minor, Venezuelan encephalitis Virus, Vibrio cholerae, West Nile virus, Bancroft Wuchereria bancrofti, Wuchereria bancrofti Hereria bancrofti, Brugia malayi Yellow fever virus, Yersinia enterococcus Yersinia olitica, the bacterium that causes plague (Yersinia pestis), and pseudotuberculosis (Yersinia olitica). One example is the fungus Yersinia pseudotuberculosis.

[0092] How to create an oligonucleotide collection This disclosure specifically targets or detects target populations (e.g., microbial or pathogenic nucleic acids). To produce, a collection of oligonucleotides for use in the methods provided herein. Multiple means are provided for preparing oligonucleotides. In some cases, oligonucleotides The method for creating the collection is based on bioinformatics, computation, and hybridization. Alternatively, it could be a method based on digestion.

[0093] In some cases, background population nucleic acids such as host (e.g., human) genomic DNA. Using this, oligonucleotides containing host and non-host (e.g., non-human, pathogen) sequences are used. Host sequences can be removed from a population of ocides. This method allows for the removal of host sequences (e.g., humans). and oligonucleotides that can bind to non-host nucleic acids Chido's collection of different species (for example, 5'-NNNNNNNNNNNNN-3' (N is A, We offer a collection of oligonucleotide randommers (such as C, G, or T). This may include, in some cases, the host genomic DNA being converted into heterologous oligonucleotides. Under conditions that promote the binding of host genomic DNA to complementary sequences in the collection, oligonucleotides This method can be introduced into heterogeneous collections of creotides. Deplete oligonucleotides bound to host genomic DNA from a heterogeneous collection. This may include the following. Typically, in such a method, the host nucleic acid (e.g., host genome nucleic acid) Oligonocytes are supplied in excess for heterogeneous collections. Depletion occurs when oligonucleotides are depleted. Hybridized or conjugated oligonucleotides from a collection of different creotides To remove, to hybridize or destroy the bound oligonucleotide, Or by preferentially isolating unbound oligonucleotides, as provided herein. It can be completed by the method described.

[0094] Figure 3 shows a pictorial example of preparing a collection of oligonucleotides (370). This method includes the step of preparing a heterogeneous collection (310) of oligonucleotides. Obtain. In some cases, heterologous collections of oligonucleotides are nucleic acid labeled, chemically labeled. Alternatively, it may include an oligonucleotide labeled with optical labeling (320). This method is From a heterogeneous collection of nucleotides, background population nucleic acids (e.g., host nucleic acids) Nucleotides having sequences identical, nearly identical, complementary, or nearly complementary to human nucleic acids, etc. This may include a step of depleting oligonucleotides containing the domain. A heterogeneous collection of oligonucleotides, with background population nucleic acids being oligonucleotides Complementary or near-phase nucleotides of oligonucleotides in an heterogeneous collection of ocide To hybridize or specifically bind to complementary domains, background population Depletion by contacting nucleic acids (340) (e.g., human genome DNA) (360) This is achieved. The hybridization reaction involves a denaturation step and / or a regeneration step. It may include heating nucleic acids to denature them during a reaction. Alternatively, it can be subjected to stepwise heating (for example, 95°C for 10 seconds and 65°C for 3 minutes). To regenerate nucleic acids, the nucleic acids are heated at 36°C for a certain period of time, for example, several hours or several weeks. It can be debatable. In some cases, background population nucleic acids are the host (33 0) Derived or isolated from nucleic acid. In some cases, background population nucleic acids are nucleic acid targets. Labeled with an identifier, chemical label or optical label (350). In some cases, this method is used in batches. The step of denaturing at least a portion of the cluster nucleic acids, for example by heat, is further include.

[0095] In some cases, one or more blocker oligonucleotides hybridize It may be present during the 360 ​​step and before the hybridization step. Alternatively, they may be included in the process. In some examples, oligonucleotides are DNA, RNA, P Includes NA, LNA, BNA, or any combination thereof. Blocker oligonucleotides Otide may be complementary to the sequence of oligonucleotides outside the nucleotide domain. Therefore, it does not hybridize to the nucleotide domain, but rather, it does not hybridize to other nucleotides present on the same chain. Blocker oligonucleotides can be designed to "block" the nucleotides. The presence of the sequence outside the nucleotide domain is the background of the oligonucleotide. This could help reduce the likelihood of binding to a population (e.g., human genome DNA). Blocker oligonucleotides are effective against the background population of oligonucleotides. This can enhance the binding specificity of the oligonucleotide collection. In specific examples, a heterogeneous collection of oligonucleotides can be used as a blocker oligonucleotide. (For example, 0.5X PBS, blocker oligonucleotides, RNase inhibitors) It can hybridize with genomic DNA in a buffer solution.

[0096] Typically, the starting point for creating the collection of oligonucleotides provided herein The heterogeneous collection of oligonucleotides used as a group was randomly generated. A collection of nucleic acid sequences (for example, 5'NNNNNNNNNNNNN-3' (where N is A, C is C) It is a collection of oligonucleotide randommers (such as G or T). In some cases, heterologous collections of oligonucleotides are obtained (for example, by DNA synthesis) They can be synthesized. In some cases, heterogeneous collections of oligonucleotides are It does not contain artificially fragmented nucleic acids. In some cases, heterologous oligonucleotides The selection is for approximately 5, 10, 20, and 30 possible sequences for each nucleotide domain. 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 9 8, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, Oligon containing nucleotide domains, where 99.8%, 99.9%, or 100% are present. Contains creotides. In some cases, heterogeneous collections of oligonucleotides are nucleotides. For the domain of Otido, the maximum number of possible sequences is 5, 10, 20, 30, 40, 50, 60. 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 Or it contains oligonucleotides that have nucleotide domains, which are present in 100% of cases. In some cases, heterologous collections of oligonucleotides are related to the domain of the nucleotides. For the possible sequences, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100 Contains oligonucleotides that include nucleotide domains, which are present in %.

[0097] In some cases, heterogeneous collection of oligonucleotides is in the background population nucleus. A sequence identical, nearly identical, complementary, or nearly complementary to the nucleic acid from the acid or the target population. It contains oligonucleotides that include a nucleotide domain having the following characteristics. A heterologous collection of ligonucleotides is identical to one or more background populations. , containing nucleotide domains having nearly identical, complementary, or nearly complementary sequences It contains oligonucleotides. In some cases, heterogeneous collections of oligonucleotides Identical, nearly identical, complementary, or nearly identical to one or more background populations. It includes oligonucleotides containing nucleotide domains that have non-complementary sequences. In some cases, heterogeneous collection of oligonucleotides is one or more targets. Nucleotide domains having sequences identical, nearly identical, complementary, or nearly complementary to the population. Contains oligonucleotides. In some cases, heterogeneous collection of oligonucleotides The group may be identical, nearly identical, complementary, or nearly identical to one or more target groups. This includes oligonucleotides containing nucleotide domains having non-complementary sequences.

[0098] In some cases, one or more oligonucleotides in a heterogeneous collection Nucleotides are not labeled; in some cases, heterologous collections of oligonucleotides. One or more oligonucleotides within are labeled. In some cases, one or more are labeled. Numerous oligonucleotides are labeled, for example, with nucleic acid labels, chemical labels, or optical labels. In some cases, one or more oligonucleotides are conjugated onto a solid support. In some cases, one or more oligonucleotides are conjugated onto a solid support. Do not allow it to happen. In some cases, the label is placed at the 5' or 3' end of the oligonucleotide. Alternatively, it can be attached to the inside of oligonucleotides. In some cases, oligonucleotides One or more oligonucleotides in a heterogeneous collection of creotides are two or more labels It is marked by recognition.

[0099] Background population nucleic acids are in excess of heterogeneous oligonucleotide collections. It can be provided as follows: The ratio of the background population to oligonucleotides is approximately 0.1; 0.2;0.3;0.4;0.5;0.6;0.7;0.8;0.9;1.0;1.1; 1.2;1.3;1.4;1.5;1.6;1.7;1.8;1.9;2.0;2.5; 3.0;3.5;4.0;4.5;5.0;5.5;6.0;6.5;7.0;7.5; 8.0;8.5;9.0;9.5;10;11;12;13;14;15;16;17; 18;19;20;25;30;35;40;45;50;55;60;65;70;7 5;80;85;90;95;100;200;300;400;500;600;70 0;800;900;1000;2000;3000;4000;5000;6000; It may be 7000; 8000; 9000; or 10000. The ratios of the background populations are, at most, 0.1; 0.2; 0.3; 0.4; 0.5; 0. 6;0.7;0.8;0.9;1.0;1.1;1.2;1.3;1.4;1.5;1. 6;1.7;1.8;1.9;2.0;2.5;3.0;3.5;4.0;4.5;5. 0;5.5;6.0;6.5;7.0;7.5;8.0;8.5;9.0;9.5;10 ;11;12;13;14;15;16;17;18;19;20;25;30;35; 40;45;50;55;60;65;70;75;80;85;90;95;100; 200;300;400;500;600;700;800;900;1000;200 0;3000;4000;5000;6000;7000;8000;9000;or It could be 10,000. The ratio of the background population to oligonucleotides is small. 0.1;0.2;0.3;0.4;0.5;0.6;0.7;0.8;0.9;1 .0;1.1;1.2;1.3;1.4;1.5;1.6;1.7;1.8;1.9;2 .0;2.5;3.0;3.5;4.0;4.5;5.0;5.5;6.0;6.5;7 .0;7.5;8.0;8.5;9.0;9.5;10;11;12;13;14;15 ;16;17;18;19;20;25;30;35;40;45;50;55;60; 65;70;75;80;85;90;95;100;200;300;400;500 ;600;700;800;900;1000;2000;3000;4000;500 It can be 0; 6000; 7000; 8000; 9000; or 10000. (Oligon) The ratio of the background population to the creotide may be saturated. The ratio of the background population to the thiosulfate population may be unsaturated. The ratio can be expressed in terms of concentration, molars, etc. Alternatively, it can be calculated from the perspective of mass.

[0100] In some cases, nucleic acids are single-stranded. In some cases, double-stranded nucleic acids are converted into single-stranded nucleic acids. Nucleic acids can be denatured using heat. In some cases, nucleic acids can be denatured by heat, approximately 35,4 0, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93 Heat to 94, 95, 96, 97, 98 or 99°C. In some cases, the nucleic acids are heated to approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 60 minutes Heat during heating. In some cases, use a chemical denaturant (e.g., acid, base, solvent, carboxylate) to denature the nucleic acid. Nucleic acids are denatured using tropic agents (salts).

[0101] Single-stranded nucleic acid samples can be regenerated or hybridized in some cases. Hybridize single-stranded nucleic acid samples with heterogeneous collections of oligonucleotides. In some cases, heterogeneous collections of oligonucleotides block oligonucleotides. Hybridize with nucleotides. In some cases, regenerate at least a portion of the single-stranded nucleic acid. This causes hybridization. In some cases, nucleic acids are approximately 0, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 ,22,23,24,25,26,27,28,29,30,31,32,33,34, Regenerate at 35, 36, 37, 40, 45, 50, 55, 60, 65, 68 or 70°C. This allows for hybridization. In some cases, nucleic acids are regenerated or hybridized on ice. In some cases, nucleic acids are regenerated or hybridized at room temperature. In some cases, Nucleic acids are approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 also Alternatively, 60 minutes, or 2, 3, 4, 5, 10, 15, 20, 22, 24, 30, 40, 4 6, 48, 50, 60, 70, 72, 80 or 96 hours of playback or hybridization. Temperature, buffer composition, reaction time, and concentration affect the degree of hybridization. It can have an effect. In some cases, nucleic acids are regenerated in the presence of trimethylammonium chloride. ru.

[0102] Removing hybridized nucleic acids from a heterogeneous collection of oligonucleotides Depletion can be completed by the methods provided herein. The ground group is chemically labeled and removed, thereby hybridized oligo Nucleotides can be removed. The background population is conjugated onto magnetic beads. The hybridized oligonucleotides are removed by using a magnet to remove them. It is possible to label the background population with nucleic acid tags. This refers to nucleic acid molecules that are conjugated to a solid support or tagged with chemical tags. They can be joined or hybridized into columns. Hybridized oligonucleotides D includes size separation (e.g., gel electrophoresis, capillary electrophoresis, etc.) and affinity separation (e.g., For example, by DNA pull-down assay, chromatography, or other methods. It can be removed. In some cases, hybridized oligonucleotides can be gelled. Using separation methods such as electrophoresis, capillary electrophoresis, or chromatography, Non-hybridized oligonucleotides and hybridized oligonucleotides They can be isolated based on size differences. In some cases, they hybridize The background population nucleic acids that are not present are not removed.

[0103] In some cases, oligonucleotides hybridized to background population nucleic acids Depletion can be completed by inactivating oligonucleotides. Inactivation can be performed, for example, by inactivating oligonucleotides. The creotide was chemically bound to a labeled background population nucleic acid, and then unbound. By amplifying using only oligonucleotides, the background population nucleic acids can be enhanced. This prevents the hybridizing oligonucleotide from acting as a primer. .

[0104] In some cases, oligonucleotide collections are bioinformatics or computational tools. The system is designed to target specific populations (e.g., pathogenic nucleic acids, non-human nucleic acids) using a specific method. It is calculated and then synthesized according to the design. In some cases, the synthesis method is DNA synthesis. This method involves creating a database of randomly generated oligonucleotides of a certain length. This method may include the step of obtaining. This method involves one or more background populations (e.g., To determine the nucleotide regions present in the human genome nucleic acid using bioinformatics or computation. This may include the step of selecting the oligonucleotides that are of a certain length. The background of (for example, 10, 11, 12, 13, 14 or 15 nucleotides) Such domains of nucleotides have little or no cluster sequence Sequences related to this were calculated from a randomly generated heterogeneous collection of oligonucleotides. It can be subtracted from the calculation.

[0105] Synthesis of non-host oligonucleotide collections After identifying the non-host oligonucleotide sequence, the collection of non-host oligonucleotides Synthesis can be achieved through countless methods. For example, non-host oligonucleotides. To produce oligonucleotides of a desired length (e.g., 13mer), a nucleo They can be synthesized individually by adding tides in a stepwise manner.

[0106] In some examples, ultramer oligonucleotides are provided herein. Synthesize a collection of oligonucleotides. Generally, ultramer oligonucleotides Each of the dots is separated by one or more spacer nucleotides, forming a ligament. It is a long oligonucleotide containing numerous combined oligonucleotide sequence units. To generate a set of oligonucleotides from a single ultramer oligonucleotide. This allows for the cleavage or separation of spacer nucleotides. Figure 9A shows a typical example. A general design of ultramer oligonucleotides is shown. Here, individual oligonucleotides The sequence is due to deoxyuracil nucleotide (U) acting as a spacer nucleotide. It is separated as follows: Ultramer oligonucleotides are separated from uracil-DNA glycosyl -ase (UDG) (removes uracil residues from DNA by cleaving N-glycosidic bonds) (to cleave) and to the base-free site (e.g., aprine / apyrimidine site or AP site) It is digested by the dual action of endonucleases with specific properties for each, thereby individually It is possible to generate oligonucleotides (for example, 13-mer oligonucleotides). In some cases, ultramer oligonucleotides are used to remove endonuclease IV. It is hydrolyzed using endonuclease VII. A typical reaction is shown in Figure 10A. Figure 10B shows the ultramer oligonucleotides mediated by UDG and endonuclease VII. This shows an example of digestion reaction products after the digestion of nucleotides.

[0107] Ultramer oligonucleotides are formed from individual oligonucleotides within the same ultramer chain. It is possible to design units to share the same degeneracy. Degeneracy is generally a single This refers to the number of unique sequences that can be generated from a variable sequence. This can be achieved by inserting one or more mixed bases at specific positions within the same location. Mixed bases can be any one of the bases in a set (for example, the four standard bases: A, C, G). It can refer to either one of the T bases. For example, in a standard coding system, it can refer to "N". A compound base can be any one of the bases A, C, G, or T. Similarly, a "D" mixed salt The base can be A, G, or T; the mixed base "V" can be A, C, or G; the mixed base "B" can be A, C, or G. A compound base can be C, G, or T; a mixed base can be A, C, or T; a W "Mixed bases can be A or T; "S" mixed bases can be C or G; "K" mixed bases A compound base can be C or G; a mixed base marked "M" can be A or C; a mixed salt marked "Y" The base can be C or T; and the mixed base "R" can be A or G. Under the numbering system, four possible sequences are generated, as shown in Figures 9B and 11A. Therefore, an oligonucleotide sequence containing one N mixed base has a degeneracy of 4. Similarly, as shown in Figures 9C and 11A, 16 possible sequences are generated. Therefore, an oligonucleotide sequence containing two N mixed bases can be reduced to 4 × 4 = 16. It will have a severe effect. As shown in Figure 9D, eight possible sequences can be generated, An oligonucleotide containing one N mixed base and one W, S, K, M, Y, or R mixed base. The creotide sequence will have a degeneracy of 4 × 2 = 8. Six possible sequences can be generated. Therefore, an oligonucleotide sequence containing one R mixed base and one D mixed base is It would have a degeneracy of 2 × 3 = 6.

[0108] If the oligonucleotide sequences within an ultramer share the same degeneracy, then the ultramer Each oligonucleotide sequence within is likely represented by an equal number after ultramer digestion. For example, each oligonucleotide sequence within the ultramer has a degeneracy of 4. In this case, four different unique oligonucleotides per oligonucleotide sequence unit. Each of them will be present in roughly equal amounts in the resulting synthesis pool. However, However, for example, an ultramer has one oligonucleotide unit with a degeneracy of 2 degrees and If it contains nine oligonucleotide units with a degeneracy of 4 degrees, then one unit with a degeneracy of 2 degrees The two oligonucleotides from are then used in the resulting synthesis and digestion pool. For each of the 36 oligonucleotides from 9 4-degree units, each of the following was performed: It is overexpressed by approximately 5%. Therefore, it has oligonucleotide units that share degeneracy. Lutramer is a collection of oligonucleotides having a uniformly distributed and unique sequence. It can bring about.

[0109] Non-host oligonucleotide sequences are computationally identified and grouped according to their degeneracy. This is possible. Degenerate arrays are arrays that differ only by a certain number of positions, for example, position 1 Identification is achieved by computationally sorting all sequences that are identical except for 2. It is possible to convert multiple degenerate sequences into a single variable sequence having one or more mixed bases. The number and types of mixed bases required for classification can determine the degree of degeneracy. Figure 11 shows This shows the computational classification of non-host oligonucleotides by degeneracy. Figure 11A shows 1, 2 and This shows a typical oligonucleotide having three "N" mixed base sites. Figure 11B shows The stogram shows the bucketing of non-human 13mers based on degeneracy. First column, Figure 11B. The degeneracy is "2" (these are single mixed bases that represent two possible bases (e.g., W Contains mixed bases (S mixed bases, K mixed bases, M mixed bases, Y mixed bases, or R mixed bases). The second column shows the number of non-human 13mers (meaning to do so). The second column shows the degeneracy of 3 degrees (e.g., For example, it shows the number of non-human 13mers (containing D, V, B, or H mixed bases). The remaining columns are This indicates the number of non-human 13mers with degeneracy of 4, 6, 8, 9, 12, or 16 degrees. Example For example, a non-human 13mer with a degeneracy of 4 is one N mixed base or W, S, K, M It may contain any two mixed bases selected from Y and R.

[0110] Representatives of each degenerate group of non-host (e.g., non-human) oligonucleotides (e.g., 13mer ) as shown in Figure 9, other representatives of the same degeneracy discussed elsewhere in this specification. It can be combined with other materials to form ultramer oligonucleotides. Then, the design is Ultramer oligonucleotides are used by conventional nucleic acid synthesis methods and service providers, for example. It can be synthesized by IDT.

[0111] Ultra containing oligonucleotide units (or sequences) having shared degeneracy The digestion of ma can be done in one of several different steps. In total, we combine groups of ultramers that contain units that all share the same degree of degeneracy, and then... Then, digestion. For example, in order to maintain equimolar concentrations of the obtained oligonucleotides, Multiple ultramer oligonucleotides having oligonucleotide sequences with the same degeneracy Rheotides can be combined and digested at equimolar concentrations. Another method involves ultrama - First, digest them individually, and then the resulting oligonucleotide units after such digestion They can be combined in equimolar concentrations.

[0112] Ultramer containing oligonucleotide units (or sequences) with different degeneracy The digestion of can be carried out in one of several different steps. In some cases, A group of ultramers, each containing units of different degeneracy, are combined, and then digested. For example, in order to obtain equimolar concentrations of the obtained oligonucleotides, each may be different. Multiple ultramer oligonucleotides having degenerate oligonucleotide sequences They can be combined and digested in the appropriate ratio. Another method is to first use ultramer. They are digested individually, and then the resulting oligonucleotide units are appropriate after such digestion. By combining them in ratios, equimolar concentrations of the resulting oligonucleotides can be obtained.

[0113] The ultramer oligonucleotides provided herein can be any number of oligonucleotides. It may contain nucleotide units (or sequences). For example, ultramer oligonucleotides may contain 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100 or 200 or more oligonu It may contain creotide units or sequences. In some cases, oligonucleotide units or The sequence consists of 5 to 100 nucleotides (for example, 5, 10, 11, 12, 13, 14, 15, A certain length, such as the length of 16, 17, 18, 19, 20 or more nucleotides. It contains a nucleotide domain having 1. Preferably, the nucleotide domain is 1 It is a 3-mer. In some embodiments, each domain of the nucleotide is one or more, two or more or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 Contains one or more, eleven or more, twelve or more, or thirteen or more mixed bases. Several implementation forms In this state, the mixed bases are N(A, C, G, T), D(A, G, T), V(A, C, G), B( C, G, T), H(A, C, T), W(A, T), S(C, G), K(G, T), M(A From the group consisting of C), Y(C, T), R(A, G), and any combination thereof Selected. In some embodiments, oligonucleotide sequences have the same degeneracy. In some embodiments, the oligonucleotide sequence is 2, 3, 4, 6, 8, 9, 12, 1 6, 18, 24, 27, 32, 36, 48, 54, 64, 72, 81, 96, 108, 1 It has degeneracy levels of 28, 144, 162, 192, 216, 243, or 256.

[0114] Individual oligonucleotides, for example, T4 polynucleotide kinase (T4 PNK) This can be done using or chemically biotinylation as shown in Figure 12. This step may be particularly useful when surface immobilization or purification of magnetic beads is required. Example For example, by combining biotinylated oligonucleotides with a sample, non-host oligonucleotides in the sample can be detected. It can be hybridized to liganonucleotides. It can be coated with streptavidin. The beads are added to the sample and used to pull down the non-host oligonucleotide. Alternatively, it can be isolated.

[0115] The ultramer method for generating oligonucleotides provided herein is It has several advantages. In some cases, such methods are highly efficient for oligonucleotides. A diverse collection (for example, a highly diverse collection of 13-mer oligonucleotides) This method can reduce the number of synthesis runs required to produce the product. Furthermore, it reduces synthesis costs and decreases the synthesis of excess oligonucleotides. Minimize the manual step of mixing the creotide, or obtain high-yield oligonucleotides. This makes synthesis possible.

[0116] Use of oligonucleotide collections In some cases, as shown in Figures 1 and 2, the collection of oligonucleotides Primers for PCR amplification, cDNA synthesis, sequencing, or primer extension reactions. Alternatively, it can be used as a scavenger. In some cases, oligonucleotides Mix the action (150) with the nucleic acid sample (140). In some cases, the nucleic acid sample The sample is a biological sample, for example, a blood sample (120) from a host (110) or Derived from plasma (130). Often using a collection of oligonucleotides. When priming amplification, cDNA synthesis, sequencing, or primer extension, oligonucleotides are used. Creotides and sample nucleic acids are processed by DNA polymerase, reverse transcriptase, and RNA polymerase. It can be combined with appropriate polymerization enzymes such as ze. In some cases, nucleic acid samples If RNA is present, the collection of oligonucleotides is from the RNA template (150). It can be used to prime DNA for synthesis. In some cases, nucleic acid samples are D If NA is present, the oligonucleotide collection will be used in the primer extension reaction (170). It can be used. Primer extension reactions can be used, for example, in nucleic acid labeling, nucleic acid tags, barcodes (for example) (e.g., sample barcode), universal primer sequence, primer binding site (for example) However, this is not limited to just that, but also DNA sequencing primer binding sites, sample barcode sequences Determinative primer binding site and amplification primer that meets the requirements of various sequencing platforms Sequencer (for sequencing or barcode reading, including MAR binding sites) Compatibility sequence, sequence-determining platform-attached sequence, sequence-determining adapter sequence or adapter By adding a pter, an overhang sequence can be added to the nucleic acids of the target population. In some cases, the primer extension reaction is used in the target population (e.g., non-mammals, non-hybrids). Sequence determination adapters can only be attached to nucleic acids (microorganisms or pathogens). In some cases, a collection of oligonucleotides is used to amplify the nucleic acids of the target population. It can be used as a primer for PCR reactions. In some cases, labeling The target population can be labeled using a collection of oligonucleotides. Next, the nucleic acids of the target labeled population are, for example, hybridized. It can be isolated by the - method or the pull-down method.

[0117] A pool of non-host oligonucleotides of any specific length can be prepared from DNA or RNA. When attempting to enrich non-host sequences in the prepared library, they may be different. For example, compared to removing an N-mer that is completely complementary to the host exome, After removing N-mers that are perfectly complementary to the genome, the larger of all possible N-mers... It is acceptable for some fractions to remain. A larger fraction of the non-host exome is used against the non-host genome. It can be probed, and in some cases, it may potentially offer higher sensitivity. It can. Furthermore, the same number of oligonucleotides can be used to probe the non-host genome and exome. When attempting to utilize rheotides, a larger number of oligonucleotides that are not complementary to the host exome may be used. When rheotide selects which non-complementary oligonucleotides are included in the pool, This may provide additional versatility and enable the selection of oligonucleotides with desired sequence characteristics. ru.

[0118] In some cases, as shown in Figure 2, a collection of oligonucleotides is used to form nucleic acid It can be used for drag-down. For example, collection of oligonucleotides (250 ) is used as bait to capture the target population from nucleic acid samples (240). It is possible to label a collection of oligonucleotides or Conjugate to a solid support. Oligonucleotides in a collection of oligonucleotides Rheotide is used to enhance the nucleic acids of the target complementary or nearly complementary population within the sample (260). It can hybridize or specifically bind. In some cases, labeled oligonucleotides A collection of nucleotides functions as primers for PCR amplification. In some cases, the PCR amplification product is pulled down. In some cases, the oligonucleotide Using labels on the oligonucleotides in the collection, the oligonucleotide collection The nucleic acids of the target hybridized or bound population (e.g., biotin - Avidin, biotin-streptavidin, or nucleic acid hybridization interactions (by) isolating. In some cases, nucleic acid samples are circulating nucleic acids such as circulating cell-free nucleic acids. Includes. In some cases, nucleic acid samples are augmented, such as nucleic acid sequencing libraries. Contains broadened, purified, or isolated nucleic acids.

[0119] In some cases, methods for enriching non-host (e.g., pathogen) sequences after library preparation. For example, a collection of oligonucleotides can be used for nucleic acid pulldown. In some cases, a collection of labeled oligonucleotides (e.g., labeled with biotin) A collection of scientifically labeled oligonucleotides is a single-stranded DNA or cDNA It can be hybridized into a library. In some cases, polymerase is used. For example, under high-fidelity conditions, the oligonucleotides hybridized along the library fragments The cleotide can be elongated. Elongated hybridized oligonucleotide This is then drawn using a labeling binding partner such as streptavidin for biotin labeling. It can be extracted. In some cases, the concentrated library can be extracted by, for example, PCR. It can be amplified. The advantage of this method is that the host nucleic acid is selected in an open environment. This includes methods of enriching DNA or cDNA. In some cases, the enrichment method is used for DNA or cDNA. It can be used in libraries. In some cases, positive selection of non-host library fragments. The selection leads to improved dynamics and thermodynamics. In some cases, standard library analysis is used. By applying the method after production, multiple samples that could be individually barcoded can be batch processed. This will become possible.

[0120] In some cases, the target population (e.g., non-host) in nucleic acid samples such as circulating nucleic acids The array, (a) A step of preparing a nucleic acid sample, wherein the nucleic acid sample is collected in a background. A step comprising the nucleic acid of a group (e.g., a host) and the nucleic acid of the target population; (b) Mix the nucleic acid sample with the oligonucleotide collection, thereby mixing This is a step to obtain a compound, and the collection of oligonucleotides is a nucleotide domestic A step comprising including an oligonucleotide having , (c) Contact the collection of oligonucleotides in the mixture with the nucleic acid sample. This is a method of contact that separates the nucleic acids of a target group in a mixture into nucleotide domains. It is bound to, thereby priming or capturing the nucleic acids of the target population. Pu and It can be primed or captured by this method.

[0121] The primed or captured nucleic acids can be further analyzed or processed. In that case, the primed or captured nucleic acid is superior in the reaction (e.g., PCR reaction). It can be amplified beforehand. In some cases, the primed or captured nucleic acid can be used. Sequencing assays (e.g., next-generation sequencing assays, high-throughput assays) Sequencing assays, high-volume parallel sequencing assays, nanopore sequencing assays Sequence determination can be performed by performing a SEY or Sanger sequencing assay. It is possible. In some cases, the priming or captured nucleic acid (for example, pull down up (By performing a sieve) preferentially isolate the sample. In some cases, the primer extension reaction is performed. (For example, to prime or attach nucleic acid labels to captured nucleic acids) This is done on priming or captured nucleic acids. In some cases, priming or captured The nucleic acid is RNA, and the polymerization reaction is primed (for example by reverse transcriptase) or This is performed on captured RNA nucleic acids.

[0122] In some cases, the target population (e.g., non-host) in nucleic acid samples such as circulating nucleic acids The array of (a) A step of preparing a nucleic acid sample, wherein the nucleic acid sample is collected in a background. A step comprising the nucleic acid of a group (e.g., a host) and the nucleic acid of the target population; (b) Mix the nucleic acid sample with the oligonucleotide collection, thereby mixing This is a step to obtain a compound, and the collection of oligonucleotides is a nucleotide domestic A step comprising including an oligonucleotide having , (c) Contact the collection of oligonucleotides in the mixture with the nucleic acid sample. This is a method of contact that separates the nucleic acids of a target group in a mixture into nucleotide domains. The step of combining them; (d) Sequencing assays (e.g., next-generation sequencing assays, high-through assays) Put sequencing assays, high-volume parallel sequencing assays, nanopore sequencing By performing a sequencing assay or Sanger sequencing assay, the target Steps to sequence the nucleic acids of the bound population and The sequence can be determined by this. In some cases, before sequencing, the target result It preferentially amplifies the nucleic acids of a combined population in a reaction (e.g., PCR reaction). In some cases... Before sequencing, the nucleic acids of the target binding population can be analyzed (for example, by performing a pull-down assay). (and) preferentially isolate. In some cases, primer extension reaction before sequencing. (For example, to attach a nucleic acid label to the bound nucleic acid) to the nucleic acids of the target binding population This is done. In some cases, the nucleic acid of the target binding population is RNA. In some cases, Polymerization reaction (e.g., by reverse transcriptase) on the nucleic acids of the target binding population, which are RNA. To do it.

[0123] The oligonucleotide collection is brought into contact with nucleic acid samples such as circulating nucleic acids. Therefore, a portion of the background population (e.g., host) nucleic acids are incorporated into the nucleotide domain. It can be combined. In some cases, contact can combine background population nucleic acids. Approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 4, 15, 20, 25, 30, 35, 40, 45 or 50% of the nucleotide domain It binds to the background population nucleic acid. In some cases, contact can reduce the background population nucleic acid by up to 0.1 , 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 25, 30, 35, 40, 45 or 50% are attached to the nucleotide domain. In some cases, contact can reduce the background population nucleic acid by at least 0.1. 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, Attach 20, 25, 30, 35, 40, 45, or 50% of the nucleotides to the domain. ru.

[0124] Oligonucleotide collections may be provided in excess of the sample nucleic acid. The ratio of oligonucleotides to sample nucleic acids is approximately 0.1;0.2;0.3;0.4;0 .5;0.6;0.7;0.8;0.9;1.0;1.1;1.2;1.3;1.4;1 .5;1.6;1.7;1.8;1.9;2.0;2.5;3.0;3.5;4.0;4 .5;5.0;5.5;6.0;6.5;7.0;7.5;8.0;8.5;9.0;9 .5;10;11;12;13;14;15;16;17;18;19;20;25;3 0;35;40;45;50;55;60;65;70;75;80;85;90;95 ;100;200;300;400;500;600;700;800;900;100 0;2000;3000;4000;5000;6000;7000;8000;900 It can be 0 or 10000. The ratio of oligonucleotide to sample nucleic acid is maximum 0.1;0.2;0.3;0.4;0.5;0.6;0.7;0.8;0.9;1.0; 1.1;1.2;1.3;1.4;1.5;1.6;1.7;1.8;1.9;2.0; 2.5;3.0;3.5;4.0;4.5;5.0;5.5;6.0;6.5;7.0; 7.5;8.0;8.5;9.0;9.5;10;11;12;13;14;15;16 ;17;18;19;20;25;30;35;40;45;50;55;60;65; 70;75;80;85;90;95;100;200;300;400;500;60 0;700;800;900;1000;2000;3000;4000;5000;6 It could be 000;7000;8000;9000;or 10000. The ratio of oligonucleotides to oligonucleotides should be at least 0.1;0.2;0.3;0.4;0.5; 0.6;0.7;0.8;0.9;1.0;1.1;1.2;1.3;1.4;1.5; 1.6;1.7;1.8;1.9;2.0;2.5;3.0;3.5;4.0;4.5; 5.0;5.5;6.0;6.5;7.0;7.5;8.0;8.5;9.0;9.5; 10;11;12;13;14;15;16;17;18;19;20;25;30;3 5;40;45;50;55;60;65;70;75;80;85;90;95;10 0;200;300;400;500;600;700;800;900;1000;2 000;3000;4000;5000;6000;7000;8000;9000;ma It can be 10000. The ratio of oligonucleotides to sample nucleic acids is saturated. It is acceptable. The ratio of oligonucleotides to sample nucleic acids may be unsaturated. Ratios can be calculated from the perspective of concentration, moles, or mass.

[0125] Oligonucleotide collections are provided in excess of the nucleic acids of the target population. It is possible. The ratio of oligonucleotides to nucleic acids in the target population is approximately 0.1;0.2;0 .3;0.4;0.5;0.6;0.7;0.8;0.9;1.0;1.1;1.2;1 .3;1.4;1.5;1.6;1.7;1.8;1.9;2.0;2.5;3.0;3 .5;4.0;4.5;5.0;5.5;6.0;6.5;7.0;7.5;8.0;8 .5;9.0;9.5;10;11;12;13;14;15;16;17;18;19 ;20;25;30;35;40;45;50;55;60;65;70;75;80; 85;90;95;100;200;300;400;500;600;700;800 ;900;1000;2000;3000;4000;5000;6000;7000; It could be 8000; 9000; or 10000. The original value for nucleic acids of the target population. The ratio of gonucleotides is up to 0.1;0.2;0.3;0.4;0.5;0.6;0.7; 0.8;0.9;1.0;1.1;1.2;1.3;1.4;1.5;1.6;1.7; 1.8;1.9;2.0;2.5;3.0;3.5;4.0;4.5;5.0;5.5; 6.0;6.5;7.0;7.5;8.0;8.5;9.0;9.5;10;11;12 ;13;14;15;16;17;18;19;20;25;30;35;40;45; 50;55;60;65;70;75;80;85;90;95;100;200;30 0;400;500;600;700;800;900;1000;2000;3000 ;4000;5000;6000;7000;8000;9000;or 10000 It's possible. The ratio of oligonucleotides to nucleic acids in the target population is at least 0.1. 0.2;0.3;0.4;0.5;0.6;0.7;0.8;0.9;1.0;1.1; 1.2;1.3;1.4;1.5;1.6;1.7;1.8;1.9;2.0;2.5; 3.0;3.5;4.0;4.5;5.0;5.5;6.0;6.5;7.0;7.5; 8.0;8.5;9.0;9.5;10;11;12;13;14;15;16;17; 18;19;20;25;30;35;40;45;50;55;60;65;70;7 5;80;85;90;95;100;200;300;400;500;600;70 0;800;900;1000;2000;3000;4000;5000;6000; It could be 7000; 8000; 9000; or 10000. The nucleic acid of the target population The ratio of oligonucleotides to nucleic acids may be saturated. The ratio of oligonucleotides may be unsaturated. The ratio may be expressed in terms of concentration, moles, or mass. It can be calculated from there.

[0126] In some cases, nucleic acids, such as circulating nucleic acids or sequenceable libraries, are single-stranded. In some cases, double-stranded nucleic acids are denatured into single-stranded nucleic acids. Nucleic acids can be denatured using heat. This can be done. In some cases, nucleic acids can be divided into approximately 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 also Heat to 99°C. In some cases, nucleic acids are about 0.1, 0.2, 0.3, 0.4, 0 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, Heat for 15, 20, 25, 30, 40, 50, or 60 minutes. In some cases, nucleic acids Nucleic acids are denatured using chemical denaturing agents (e.g., acids, bases, solvents, chaotropic agents, salts). do.

[0127] Regenerate single-stranded nucleic acid samples, such as single-stranded circular nucleic acids or sequenceable libraries. Alternatively, hybridization is possible. In some cases, single-stranded nucleic acid samples can be used as an alternative. It hybridizes with a collection of oligonucleotides. In some cases, oligonucleotides Hybridize the Otid collection with blocker oligonucleotides. How many In that case, at least a portion of the single-stranded nucleic acid is regenerated or hybridized. In this case, nucleic acids are approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 2 7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 40, 45, 50 Regenerate or hybridize at 55, 60, 65, 68, or 70°C. In some cases, nucleic acids are regenerated or hybridized on ice. In other cases, nucleic acids are regenerated at room temperature. Raw or hybridized. In some cases, nucleic acids are about 0.1, 0.2, 0.3. 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 60 minutes, or 2, 3, 4, 5 , 10, 15, 20, 22, 24, 30, 40, 46, 48, 50, 60, 70, 72, Regenerate or hybridize for 80 or 96 hours. Temperature, buffer composition, reaction time... The concentration can affect the degree of hybridization. In some cases, trim Nucleic acids are regenerated in the presence of ammonium chloride.

[0128] Self-hybridization methods Further priming, capturing, or enriching of target populations within nucleic acid samples Methods and compositions are also provided herein. In some cases, the population to be targeted is self- Hybridization methods, such as concentration-based hybridization kinetics, are used. It is concentrated by this method. Nucleic acid hybridization kinetics is the hybridizing chain It depends on the concentration. Generally, high concentrations of nucleic acids hybridize faster than low concentrations of nucleic acids. In complex nucleic acid populations, abundant nucleic acids (e.g., background population) are less abundant nucleic acids ( For example, it hybridizes faster than the target population. Using this concentration dependence, After partial hybridization of a group of nucleic acids, at least a portion of the double-stranded nucleic acids are removed. By either or isolating at least a portion of the single-stranded nucleic acid, the amount of abundant nucleic acid is reduced. This allows for the concentration of rare nucleic acids in the sample. As a result, the background population (For example, human) nucleic acid levels can be reduced, and the target population (for example, non-human, The amount of nucleic acid (from microorganisms or pathogens) can be concentrated.

[0129] In some cases, background population DNA (e.g., human DNA) and the target DNA A sample of DNA containing population DNA (e.g., non-human or pathogen DNA) (e.g., Circulating DNA (or circulating cell-free DNA) is obtained. The nucleic acids in the sample are denatured to obtain single-stranded D NA samples can be prepared. In some cases, the nucleic acids in the sample are single-stranded D It is NA and does not require denaturation. In some cases, then background population DNA Because it is present at a higher concentration than the target DNA population, background population DNA The sample was subjected to a defined loose vein, with the expectation that it would hybridize faster than the target population DNA. The sample is regenerated in the cava for a defined period of time. Then, the sample is treated with a double-strand specific nuclease. The sample is subjected to conditions that remove double-stranded DNA, thereby removing the background DNA from the sample. Ground population DNA can be preferentially removed. In some cases, RNA is also included. The sample is combined with a mixture of single-stranded background population (e.g., human) exome sequences. Combine. In some cases, play the sample for a defined period to get richer exomes. The sequence allows for hybridization to RNA. Then, the DNA-RNA double strand is formed. This removes background population (e.g., human) RNA from the sample, thereby prioritizing it. Remove it first.

[0130] In some cases, nucleic acids, such as circulating nucleic acids, are single-stranded. In some cases, double-stranded nucleic acids are... It denatures into single-stranded nucleic acids. In some cases, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 of the nucleic acid. , 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% are single-stranded. In some cases, nucleic acids can be up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 , 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90%, 95%, 96%, 97%, 98%, 99%, or 100% are single-stranded. In some cases, Nucleic acids of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96%, 97%, 98%, 99%, or 100% are single-stranded. Nucleic acids can be denatured by heat. This can be done. In some cases, nucleic acids can be approximately 35, 40, 45, 50, 55, 60, 65, 7 0, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or Heat to 99°C. In some cases, the nucleic acids are heated to a maximum of 35, 40, 45, 50, 55, 60°C. , 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, Heat to 98 or 99°C. In some cases, the nucleic acids are heated to at least 35, 40, 45°C. 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 9 5. Heat to 96, 97, 98 or 99°C. In some cases, the nucleic acid is about 0.1, 0 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 Heat for 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 60 minutes. In some cases, nucleic acids can be up to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 Heat for 30, 40, 50, or 60 minutes. In some cases, the nucleic acid is at least 0. 1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, Add for 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 60 minutes. Heat. In some cases, use chemical denaturants (e.g., acids, bases, solvents, chaotropes) to denature nucleic acids. Nucleic acids are denatured using denaturants (such as salts).

[0131] Regenerating or hybridizing single-stranded nucleic acid samples, such as single-stranded circulating nucleic acids, is possible. Yes, it is possible. In some cases, it is possible to regenerate or hybridize at least a portion of a single-stranded nucleic acid. In some cases, the single-stranded nucleic acid is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, Regenerate or hybridize 85, 90, 95, 96, 97, 98, 99 or 100% To cause. In some cases, single-stranded nucleic acids can be up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, Play 80, 85, 90, 95, 96, 97, 98, 99 or 100% or hybrid To soybean. In some cases, at least 1, 2, 3, 4, 5, 6, 7 single-stranded nucleic acids. 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, Play at 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% or hybridize. In some cases, nucleic acids are approximately 0, 1, 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 Regenerate at 35, 36, 37, 40, 45, 50, 55, 60, 65, 68 or 70°C. or hybridize. In some cases, nucleic acids are 0, 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 3 Regenerate at 4, 35, 36, 37, 40, 45, 50, 55, 60, 65, 68, or 70°C. Or hybridize. In some cases, the nucleic acids are 0, 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ,20,21,22,23,24,25,26,27,28,29,30,31,32, 33, 34, 35, 36, 37, 40, 45, 50, 55, 60, 65, 68 or 70 Regenerate or hybridize at °C. In some cases, regenerate or hybridize nucleic acids on ice. To re-dise. In some cases, nucleic acids are regenerated or hybridized at room temperature. In the case of Tsuka, nucleic acids are approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 60 minutes, or 2, 3, 4, 5, 10, 15, 20, 22, 24, 3 0, 40, 46, 48, 50, 60, 70, 72, 80 or 96 hours of playback or high Breed them. In some cases, nucleic acids up to 0.1, 0.2, 0.3, 0.4, 0 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 60 minutes, or 2, 3, 4, 5, 10, 1 5, 20, 22, 24, 30, 40, 46, 48, 50, 60, 70, 72, 80 or Allow to regenerate or hybridize for 96 hours. In some cases, the nucleic acid is at least 0. 1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 60 minutes, Or 2, 3, 4, 5, 10, 15, 20, 22, 24, 30, 40, 46, 48, 50 Allow to regenerate or hybridize for 60, 70, 72, 80, or 96 hours. Temperature, gentle The composition of the reaction fluid, reaction time, and concentration can affect the degree of hybridization. In some cases, nucleic acids are regenerated in the presence of trimethylammonium chloride.

[0132] During regeneration, single-stranded nucleic acids hybridize or re-anneal to double-stranded nucleic acids. This can be done. In some cases, double-stranded nucleic acids are double-stranded DNA, double-stranded RNA, or DNA- It is a double-stranded RNA. At least a portion of the double-stranded nucleic acid is removed to generate a nucleic acid enrichment population. It is possible. In some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9 strands of double-stranded nucleic acid. , 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, Remove 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100%. In some cases, the maximum number of double-stranded nucleic acids is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 2 0, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 Remove 90, 95, 96, 97, 98, 99, or 100%. In some cases, two At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 of the main stranded nucleic acid, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 9 Remove 5, 96, 97, 98, 99, or 100%.

[0133] In some cases, at least a portion of the double-stranded nucleic acid is removed to generate an enriched population of nucleic acids. In some cases, at least a portion of single-stranded nucleic acids is isolated to generate an enriched population of nucleic acids. In some cases, at least a portion of the double-stranded nucleic acid is subjected to gel electrophoresis or capillary electrophoresis. Removed using separation methods such as pneumatophoresis. In some cases, at least a portion of single-stranded nucleic acids. These are isolated using separation methods such as gel electrophoresis or capillary electrophoresis. In that case, at least a portion of the double-stranded nucleic acid is removed using one or more nucleases. In some cases, nucleases act on double-stranded nucleic acids. Aces act on double-stranded DNA. In some cases, nucleases act on two strands of DNA-RNA. It acts on the chain. In some cases, nucleases do not act on single-stranded nucleic acids. In general, nucleases do not act on single-stranded DNA. In some cases, nucleases act on single-stranded DNA. It does not act on RNA. Some non-specific examples of nucleases include double-strand specific nucleases. Clease (DSN) (e.g., derived from Kamchatka crab), thermally unstable DSN-TL, BAL-31, double-strand specific DNase (e.g., Northern shrimp, Pandarus boreham) (derived from Pandalus borealis), exonuclease III Endonucleases secreted by the tropical house mosquito (Culex quinquefa) Examples include (derived from sciatus). Temperature, buffer composition, reaction time, concentration and nucleation The ratio of ase to substrate can affect nuclease specificity or activity. For example, Hokko Substrate preference of double-stranded specific DNases derived from red shrimp is double-stranded in the presence of magnesium. Although it has been reported to be single-stranded DNA, nucleases are single-stranded D in the presence of calcium. It becomes active against NA (Nilsen et al. "The Enzyme and d the cDNA Sequence of a Thermolabile an d Double-Strand Specific DNase from Nort hern Shrimps (Pandalus borealis) PLOS On (e 2010). Nuclease combinations can be used in series or parallel. In some cases, nucleic acids are purified after nuclease treatment and the buffer composition is replaced. Removes nucleases, nucleotides, and / or short nucleic acid fragments.

[0134] In some cases, a sample containing nucleic acids is compared to a single-stranded background population (e.g., human). ) Combine with a mixture of nucleic acids. Regenerate the sample for a specified period to obtain a richer background. This allows the undone population nucleic acid to hybridize with the nucleic acid in the sample. Then, two This removes the main strand of nucleic acid, thereby eliminating the background population (e.g., human) in the sample. Nucleic acids are preferentially removed. In some cases, the sample contains RNA. In some cases, Double-stranded nucleic acids are DNA-RNA double strands. In some cases, background population nucleic acids This is the exome sequence. In some cases, depletion occurs when the background population nucleic acid is sampled. Hybridize or specifically hybridize to complementary or nearly complementary background populations within the group. To enable binding, a sample containing nucleic acids is brought into contact with a background population of nucleic acids. This is achieved. In some cases, this method uses at least one background population nucleic acid. The process further includes the step of modifying the part, for example, by heat. In some cases, the backgrain Nucleic acid sequences in a population can be identified bioinformatically or computationally. In some cases... Background population nucleic acids are synthesized. In some cases, background population nucleic acids It is DNA.

[0135] Background population nucleic acids may be provided in excess of the sample nucleic acid. The ratio of round population nucleic acids to sample nucleic acids is approximately 0.1;0.2;0.3;0.4;0.5;0 .6;0.7;0.8;0.9;1.0;1.1;1.2;1.3;1.4;1.5;1 .6;1.7;1.8;1.9;2.0;2.5;3.0;3.5;4.0;4.5;5 .0;5.5;6.0;6.5;7.0;7.5;8.0;8.5;9.0;9.5;1 0;11;12;13;14;15;16;17;18;19;20;25;30;35 ;40;45;50;55;60;65;70;75;80;85;90;95;100 ;200;300;400;500;600;700;800;900;1000;20 00;3000;4000;5000;6000;7000;8000;9000;Also This can be 10,000. The ratio of background population nucleic acid to sample nucleic acid is at most 0.1; 0.2;0.3;0.4;0.5;0.6;0.7;0.8;0.9;1.0;1.1; 1.2;1.3;1.4;1.5;1.6;1.7;1.8;1.9;2.0;2.5; 3.0;3.5;4.0;4.5;5.0;5.5;6.0;6.5;7.0;7.5; 8.0;8.5;9.0;9.5;10;11;12;13;14;15;16;17; 18;19;20;25;30;35;40;45;50;55;60;65;70;7 5;80;85;90;95;100;200;300;400;500;600;70 0;800;900;1000;2000;3000;4000;5000;6000; It could be 7000; 8000; 9000; or 10000. Background population nucleus The ratio of acid to sample nucleic acid should be at least 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7;0.8;0.9;1.0;1.1;1.2;1.3;1.4;1.5;1.6; 1.7;1.8;1.9;2.0;2.5;3.0;3.5;4.0;4.5;5.0; 5.5;6.0;6.5;7.0;7.5;8.0;8.5;9.0;9.5;10;1 1;12;13;14;15;16;17;18;19;20;25;30;35;40 ;45;50;55;60;65;70;75;80;85;90;95;100;20 0;300;400;500;600;700;800;900;1000;2000; 3000; 4000; 5000; 6000; 7000; 8000; 9000; or 10 It can be 000. The ratio of the background population to the sample nucleic acid may be saturated. Good. The ratio of the background population to the sample nucleic acid may be unsaturated. The ratio is It can be calculated from the standpoint of concentration, moles, or mass.

[0136] In some cases, at least a portion of the double-stranded nucleic acid is removed to generate an enriched population of nucleic acids. The background population nucleic acids are chemically labeled and removed, thereby obtaining double-stranded nucleic acids. It can be removed. The background nucleic acid population is conjugated onto magnetic beads. It can be removed with a magnet, thereby removing double-stranded nucleic acids. (Background) Nucleic acid aggregates can be labeled with nucleic acid labels. The nucleic acid labels are conjugated onto a solid support. It binds to or hybridizes with nucleic acid sequences that are tagged with chemical labels. Double-stranded nucleic acids can be separated by size (e.g., gel electrophoresis, capillary electrophoresis). Electrophoresis, affinity separation (e.g., pull-down assay, chromatography), or It can be removed by other methods. In some cases, double-stranded nucleic acids can be removed by gel electrophoresis. Using separation methods such as capillary electrophoresis or chromatography, single-stranded nuclei They can be isolated based on the difference in size between the acid and the double-stranded nucleic acid. In some cases, high Do not remove background population nucleic acids that have not bred.

[0137] Nucleosome depletion leads to enrichment. Another example of a concentration method provided herein is the nucleosome depletion method. Human cell-free DNA has a length periodicity of approximately 180 base pairs, as shown in Figure 6. This suggests that the majority of cell-free DNA consists of histones associated with nucleosomes. Bacterial DNA does not exhibit a specific length periodicity, as shown in Figure 6. Alternatively, by depleting nucleosome-associated DNA, the target population can be enriched. We can provide the law.

[0138] In some cases, free DNA (e.g., non-nucleosomal DNA or non-nucleosomal DNA) A method for separating nucleosome DNA or nucleosome-associated DNA from nucleosome-associated DNA. The methods include electrophoresis and isokinetic electrophoresis, which separate based on mass and / or net charge. Porous filters that separate based on shape and / or size, and filters that separate based on charge. Ion exchange column (Nucleosomes have histones with positively charged tails, DN A has a negatively charged backbone, and also immune host nucleosomes and associated DNA. It contains antibodies specific to the host histones that are depleted. In some cases, a portion of the host nucleic acid Nucleosomes are associated, while some host nucleic acids are not.

[0139] In some cases, as shown in Figure 7, one or more antibodies are used to target histones Alternatively, nucleosomes can be immune-depleted. In some cases, antibodies (750) , specifically in the background population (e.g., host) histones or nucleosomes (710) They can be heterogeneous. In some cases, antibodies are specific to mammalian histones or nucleosomes. They can be heterogeneous. In some cases, antibodies are specific to human histones or human nucleosomes. It may be. In some cases, one or more antibodies are specific to one or more histones. They are different. In some cases, histones, even histone mutants, have histone modifications. It may be done. In some cases, immune depletion affects the target population (e.g., non-hosts). Rheosome DNA (730), target non-nucleosome DNA population (740) The background population (e.g., host) may retain non-nucleosomal DNA (720). In some cases, immunodepletion can be treated with immunoprecipitation, chromatin immunoprecipitation, or bulk binding of antibodies. This may include affinity chromatography using column-immobilized antibodies. In some cases, one or more antibodies are immobilized on the column. In some cases, one or more antibodies are immobilized. Several antibodies (for example, anti-immunoglobulins conjugated on beads or attached to columns) It is removed (using brin antibodies). In some cases, one or more antibodies are monoclonal. It is -nal. In some cases, one or more antibodies are one or more histones It targets the C-terminus. In some cases, one or more antibodies target one or more histocytes. Target the N-terminus of the ton.

[0140] Non-limiting examples of histones, histone varieties, and histone modification types include histone H2 AN terminus, histone H2A solvent-exposed epitope, monomer on Lys9 in histone H3 Chillation, dimethylation of Lys9 in histone H3, dimethylation of Lys56 in histone H3 Limethylation, phosphorylation of Ser14 in histone H2B, Se in histone H2A.X Phosphorylation on r139, H2A-H2B acidic patch motif, histone H1, histone H 1.0, Histone H1.1, Histone H1.2, Histone H1.3, Histone H1.4, Histone H1.5, histone H1.oo, sperm cell-specific linker histone H1-like protein Histone H1t, Histone H1t2, Histone H1FNT, Histone H2A type 1-B / E (e.g., histone H2A.2, histone H2A / a, histone H2A / m) Histone H2A type 2-A (e.g., histone H2A.2, histone H2A / 0), Histone H2A type 1-D (e.g., histone H2A.3, histone H2A / g), Histone H2A type 1 (e.g., H2A.1, histone H2A / p), histone H2A type Ip2-C (e.g., histone H2A-GL101, histone H2A / q), histone H 2A type 1-A (for example, histone H2A / r), histone H2A type 1-C (for example) For example, histone H2A / l, histone H2A type 1-H (e.g., histone H2A / s ), Histone H2A type 1-J (e.g., Histone H2A / e), Histone H2A type Type 2-B, Histone H2A Type 3, Histone H2AX (e.g., H2a / x, Histone H2A.X, histone H2A.Z, H2A / z, H2A.Z.1, H2A.Z.2)) Histone H2A.V (e.g., H2A.F / Z), histone H2A.J (e.g., H2a / j), mH2A1, mH2A2, histone H2A-Bbd type 1 (for example, H2A b - Body deficiency, H2A.Bbd), histone H2A-Bbd type 2 / 3 (e.g., H2A Barr body deficiency, H2A.Bbd), core histone macro H2A.1 (e.g., histone macro Chlorophyll H2A1, mH2A1, histone H2A.y, H2A / y, medulloblastoma antigen MU-MB- 50.205, macro H2A), core histone macro H2A.2, histone H2A, his Histone H2A.J, Histone H2B, Histone H2B Type 1-C / E / F / G / I (Example) histone H2B.1A, histone H2B.a, H2B / a, histone H2B.g, H 2B / g, histone H2B.h, H2B / h, histone H2B.k, H2B / k, his H2B.l, H2B / I), H2BE, Histone H2B Type 1-H, Histone H2B Type 1-A (e.g., testicular histone H2B, TSH2B.1, testicular-specific histone) Histone H2B, TSH2B), Histone H2B type 1-B (for example, Histone H2B.1, Histone H2B f, H2B / f), Histone H2B type 1-D (e.g., HIRA phase) Interacting proteins 2, histone H2B, histone H2B, histone H2B, histone H2B, histone H2B / b, his Histone H2B type 1-J (e.g., histone H2B.1, histone H2B.r, H2B / r)), Histone H2B type 1-O (e.g., Histone H2B.2, Histone H2B. Histone H2B type 2-E (e.g., Histone H2B-GL105) Histone H2B.q, H2B / q), Histone H2B type 1-H (for example, histone H2B.j, H2B / j), Histone H2B type 1-M (e.g., Histone H2B. e, H2B / e), histone H2B type 1-L (e.g., histone H2B.c, H2B) / c), histone H2B type 1-N (e.g., histone H2B.d, H2B / d), Histone H2B type FS (e.g., histone H2B.s, H2B / s), estimated his Tons of H2B type 2-C (e.g., histone H2B.t, H2B / t), histone H2B Type 1-K (e.g., H2B K, HIRA-interacting protein 1), histone H2B Type 2-F, Histone H2B Type 2-E, Histone H2B Type 3-B (for example, H Histone H2B type 12), histone H2B type FM (e.g., histone H2B.s, H2B) ( / s), estimated histone H2B type 2-D, histone H2B type WT (for example, H2B histone family member (W testis-specific), histone 1H2bn isoform CRA_b, Histone H2B Type 1-N, H2B Histone Family Member M, His Histone H2B type FM, Histone H2B type 1-J, H2BFWT, Histone H3, Histone H3.1 (for example, histone H3 / a, histone H3 / b, histone H3 / c, Histone H3 / d, histone H3 / f, histone H3 / h, histone H3 / i, histone H3 / j, histone H3 / k, histone H3 / l), histone H3.2 (for example, histone Histone H3 / m, histone H3 / o, histone H3.3C (for example, histone H3.5), Histone H3.1t (e.g., H3 / t, H3t, H3 / g), histone H3.3, his Ton H3-like centromere protein A (e.g., centromere autoantigen A, centromere Protein A (CENP-A), histone H3.3 (cDNA FLJ57905), Histone H3.4, Histone H3.5, Histone H3.X, Histone H3.Y, Histone H 4. Histone H4-like protein type G, HIST1H4J protein, human (Homo Histone family members H4 and H5 are examples. .

[0141] This method can deplete nucleosome-associated DNA in the background population. The nucleosome-associated DNA of the ground population was approximately 5, 6, 7, 8, 9, 10, 15, 20 , 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, Deplete 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% It is possible to select up to 5, 6, or 7 nucleosome-associated DNA samples from the background population. 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 9 It can be depleted to 90% or 100%. Nucleosome assembly of the background population. DNA at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 9 It can be depleted to 4, 95, 96, 97, 98, 99, or 100%.

[0142] Enrichment by removing or isolating DNA at specific length intervals. In some cases, the enrichment method provided herein involves cell-free DNA at specific length intervals. Removing DNA of a specific length interval and / or cell-free DNA of a specific length interval This involves isolating DNA of specific length intervals, such as A. For example, a cell-free DNA sample In this case, the ratio of host to non-host cell-free DNA may be minimal. A range of cell-free DNA length may exist, and this can change significantly with some predictiveness. If you are interested in analyzing non-host DNA in cell samples, consider the relationship between non-host DNA and host DNA. By enriching the DNA length range with a more favorable NA ratio, non-host DNA can be enhanced against host DNA. The main DNA can be enriched. For example, Figure 6 shows that the top bar represents host or human DNA. Reflecting this, the bar below represents non-host DNA, comparing DNA lengths in human-derived samples. The plot shows that in most cases, the association of human DNA and histones is approximately 175 base pairs long. The first size fragment that results, as well as a fragment that is about 147 base pairs long (i.e., a single hiss) This results in (reflecting the DNA length wrapped around the core). As shown in Figure 6, The graph shows the size distribution of human DNA, which similarly exhibits maxima and maxima for other fragment lengths. .

[0143] The host-to-nonhost DNA ratio is low, for example, in the size range shown, for example, approximately Choose a length of less than 120bp, approximately 240-280bp, or approximately 425-475bp. By doing so, non-host DNA can be relatively enriched. Therefore, the present disclosure According to certain aspects, cell-free nucleic acid samples enrich non-host nucleic acids more than host nucleic acids. This process concentrates relatively short fragments. In most cases, the concentration process is performed on fragments approximately 10 base pairs to 3 base pairs in length. 00 base length, approximately 10 base length to approximately 200 base length, approximately 10 base length to approximately 175 base length, approximately 10 salt Base length ~ approximately 150 bases, approximately 10 bases ~ 120 bases, approximately 10 bases ~ approximately 60 bases, approximately 30 base pairs to approximately 300 base pairs, approximately 30 base pairs to approximately 200 base pairs, approximately 30 base pairs to approximately 175 base pairs Base length, approximately 30 bases to approximately 150 bases, approximately 30 bases to 120 bases, or approximately 30 salt Fragments with a length of approximately 60 base pairs are concentrated. The upper limit of the selection process and The lower limit selection can target either the lower or upper limit of the size selection described above. To ensure understanding, the size selection above is intended to list the exact sizes. Rather, the range of the above includes the normal size distribution around the fragment size described. This focuses on is selection. For example, if you select a given fragment size range, you can enumerate the results. If so, the dominant size range within the concentrated sample falls within that range, for example, concentrated sample at least 50%, at least 60%, at least 70%, at least 80% of the fragments inside Or in some cases, at least 90% are recognized as reflecting the size range being enumerated. In other cases, the reflected fragment in the concentrated sample is within the upper and lower limits of the listed range. In effect, approximately 30% or less outside, 20% or less above the upper and lower limits of the range, 10% or less and so on. In the case of Tsuka, it is recognized if it contains fragments that are 5% or less on the outside.

[0144] This method involves the DNA and background of the target population (e.g., pathogen). A sample of nucleic acid containing the DNA of a population (e.g., human) (e.g., a sample of circulating DNA) This is obtained. In some cases, DNA at specific length intervals is used as background population DNA. By concentrating and depleting the sample, the DNA of the target population is... This allows for preferential enrichment. In some cases, DNA of a specific length interval can be concentrated with the target. The DNA of a certain population is concentrated and isolated from the sample, thereby determining the target population. DNA can be preferentially enriched. In some cases, D at specific length intervals. Methods for removing and / or isolating NA include electrophoresis (e.g., gel electrophoresis or galvanic electrophoresis). Pyrally electrophoresis, chromatography (e.g., liquid chromatography), cell-free nucleus Acid purification (e.g., silica membrane column, buffer optimization) and / or mass, size and / or includes mass spectrometry that separates based on net charge.

[0145] In some cases, cell-free nucleic acid purification is performed using silica membranes (e.g., QIAamp Mini columns). (Silica membrane columns such as QIAamp Circulat) or commercially available kits (e.g., QIAamp Circulat) This can be done using a Nucleic Acid Kit. Generally, Cellular nucleic acid purification involves four steps: lysis, binding, washing, and elution.

[0146] The lysis step releases nucleic acids from proteins, lipids, and / or vesicles, and DNase and / or inactivate RNases, under denaturing conditions, at high temperatures, and / or protein This can be carried out in the presence of enzyme K. The dissolution step is performed in buffer ACL and / or pro- Teinase K can be used.

[0147] The binding step allows nucleic acids to be bound or adsorbed onto a silica membrane. TEP contains buffer ACB or approximately or at least approximately 35% by volume, 40% by volume, 45 units. If % of volume, 50% of volume, 55% of volume, 60% of volume, 65% of volume, 66% of volume, 70% of volume The binding buffer contains 75% by volume of alcohol (e.g., isopropanol), for example, A binding buffer containing approximately 40% or 66% isopropanol can be used. In some cases, during cell-free nucleic acid purification, further steps are taken compared to the manufacturer's recommended protocol. Use a commercially available buffer solution (e.g., buffer solution ACB). For example, in some cases, add The additional volume of commercially available buffer used is approximately the volume specified in the manufacturer's recommended protocol. or at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, These are 4.9, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 times. In this case, from 1 mL of silica membrane column or sample (e.g., serum or plasma) Approximately or at least approximately 1.5, 1.8, 2.0, 2.5, 3.0, 3 per solution. 5, 4.0, 5.0, 5.3, 5.4, 5.5, 6.0, 7.0, 8.0, 9.0, 10 0.0, 11.0, 12.0, 13.0, 14.0, 15.0 or 16.0 mL of binding loosening solution Use a buffer solution or buffer ACB.

[0148] The cleaning step can remove residual contaminants and may include multiple cleaning steps. This can be done. The washing step involves buffer ACW1, buffer ACW2, ethanol and / or The volume is approximately or at least about 50% by volume, 55% by volume, 56.8% by volume, 60% by volume, 6% by volume. 5% by volume, 66% by volume, 69.8% by volume, 70% by volume, 75% by volume, 80% by volume, 85% by volume Volume%, 86% volume%, 87% volume%, 87.4% volume%, 88% volume%, 89% volume%, 90% volume% , 95% by volume, 96% by volume, 97% by volume, 98% by volume, 99% by volume or 100% by volume Washing buffer containing alcohol (e.g., ethanol), e.g., approximately 56.8%, approximately 69.8% Use a washing buffer containing 100% ethanol, approximately 87.4%, approximately 89%, or approximately 100%. It is possible. In some cases, ethanol can refer to 96-100% ethanol. In some cases, ethanol is not denatured alcohol. In some cases, ethanol is denatured alcohol. It does not contain tanol or methyl ethyl ketone. In some cases, the washing step is buffer. The first washing step involves using ACW1 (for example, 600 μL per silica membrane column), and then... Second washing step with ACW2 buffer (e.g., 750 μL per silica membrane column) and a third washing step with ethanol (e.g., 750 μL per silica membrane column) May include the manufacturer's recommended protocol during cell-free nucleic acid purification. Compare with additional ethanol (e.g., anhydrous ethanol) in a commercially available buffer (e.g., Qi Add to ACW1 buffer (ACW2 buffer). For example, in some cases, The volume of additional ethanol added is per 600 μL or 750 μL of commercially available buffer. , approximately or at least approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0 , 9.0 or 10.0 mL. In some cases, additional ethanol is added. The volume is up to 0.5, 0.6, or 0.7 per 600 μL or 750 μL of commercially available buffer. , 0.8, 0.9, 1.0, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1. 6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4 The volume is 0.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 mL.

[0149] In some cases, during cell-free nucleic acid purification, further steps are taken compared to the manufacturer's recommended protocol. Add guanidinium chloride to a commercially available buffer (e.g., Qiagen ACW1 buffer). For example, in some cases, the amount of additional guanidinium chloride added is commercially available. Per 600 μL of buffer, approximately or at least approximately 0.1, 0.2, 0.3, 0.31, 0 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1 These are 0.4, 1.5, 1.6, 1.75, 1.8, 1.9, or 2.0 g. In addition, the amount of further guanidinium chloride added is the same as the amount of commercially available buffer or washing buffer 60 Maximum values ​​per 0 μL: 0.1, 0.2, 0.3, 0.31, 0.32, 0.33, 0.34 , 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.5, 0.6, 0. 7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1. The amounts are 75, 1.8, 1.9, or 2.0 g. In some cases, the washing buffer is the washing buffer. Per 600 μL of solution, approximately or at least approximately 0.1, 0.2, 0.3, 0.31, 0.3 2, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4 , 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 Contains 1.5, 1.6, 1.75, 1.8, 1.9, or 2.0 g of guanidinium chloride. It is possible.

[0150] In some cases, during cell-free nucleic acid purification, further steps are taken compared to the manufacturer's recommended protocol. Ethanol (e.g., anhydrous ethanol) and further guanidinium chloride are commercially available loose ethanol. Add to a buffer solution (e.g., Qiagen ACW1 buffer). In some cases, wash stage The first washing buffer contains guanidinium chloride and approximately 89.0% ethanol. The first washing step involves a second washing step with a second washing buffer containing approximately 87.4% ethanol. The process may include a cleaning step and a third washing step with ethanol. Each washing buffer should be at least approximately 60%, 65%, 66%, 69.8%, 70%, and 75%. Contains 80%, 85%, 86%, 87%, or 87.4% ethanol.

[0151] The elution step allows nucleic acids to be released from the silica membrane. A buffer is used for elution. AVE can be used.

[0152] For example, Qiagen Circulating Nucleic Acid (CNA) The kit manufacturer's recommended protocol can be modified by the following modifications: a) Use 3 times the volume of ACB buffer, (b) Use ACW1 buffer according to the manufacturer's recommendations Prepare the solution by adding 1.75 mL of anhydrous ethanol and chloride per 600 μL of ACW1 buffer. Supplement with 0.36 g of guanidinium, and (c) prepare ACW2 buffer according to the manufacturer's recommendations. Prepare the solution and supplement with 1.05 mL of anhydrous ethanol per 750 μL of ACW2 buffer.

[0153] In addition to other nucleosome target depletion methods described elsewhere in this specification, nucleic acid Methods for selecting / isolating ISIS are well known in the art. For example, gel electrophoresis, gel Nucleic acids of a desired length are obtained using chromatography methods such as exclusion chromatography. It can be selectively isolated. Furthermore, using a bead-based charge separation method, the desired Nucleic acids within a size range can be selectively isolated. For example, Beckman Coul SPRI bead systems available from ter, and for example, New England The above can be easily achieved using the AMPure bead system available from Biolabs. Size selection of cell-free DNA that can amplify non-host DNA relative to sea urchin host DNA. Purification can be performed.

[0154] In some cases, size selection concentration is at least about 1.5 times, at least about 2 times, and less At least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times , at least about 30 times, at least about 40 times, at least about 50 times, at least about 100 The ratio of non-host DNA to host DNA is 2:1 and in some cases, about 500:1 or more. This can lead to an increase in non-host DNA compared to host DNA in a 1:100 ratio. If present in a cell-free sample at a ratio of 1:50, the enrichment resulting in a 2x increase produces a 1:50 ratio. It will likely increase. In some cases, the increase in the ratio of non-host DNA to host DNA is about 1.5 times. 2x, 3x, 4x or 5x, and about 10x, 20x, 30x, 40x, 50x, 10 It can be 0 times, 500 times, or more.

[0155] It is possible to remove DNA at intervals of one or more lengths. In some cases, one The intervals of several lengths are approximately 140 base pairs, 145 base pairs, 150 base pairs, and 155 base pairs. Base pair, approximately 160 base pairs, approximately 165 base pairs, approximately 170 base pairs, approximately 175 base pairs, approximately 180 salt Base pair, approximately 185 base pairs, approximately 190 base pairs, approximately 195 base pairs, approximately 200 base pairs, approximately 205 salts The number of base pairs and one or more multiples of approximately 210 base pairs can be selected. In this case, the multiples are, for each occurrence, multiples of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. They are selected independently. For example, approximately 180 base pairs are multiples of 1 for approximately 180 base pairs. Approximately 360 base pairs is a multiple of 2 for approximately 180 base pairs. In some cases, one or Multiple interval lengths are approximately 180 base pairs, 360 base pairs, 540 base pairs, and 720 base pairs. Alternatively, it could be approximately 900 base pairs.

[0156] DNA of one or more length intervals can be isolated. In some cases, one Or multiple length intervals are approximately 10 base pairs, approximately 20 base pairs, approximately 30 base pairs, approximately 40 base pairs, approximately 50 base pairs, approximately 60 base pairs, approximately 70 base pairs, approximately 80 base pairs, approximately 90 base pairs, approximately 100 bases Pairs, approximately 110 base pairs, approximately 120 base pairs, approximately 130 base pairs, approximately 140 base pairs, approximately 150 bases Pairs, approximately 160 base pairs, approximately 170 base pairs, maximum approximately 10 base pairs, maximum approximately 20 base pairs, maximum approximately 3 0 base pairs, up to approximately 40 base pairs, up to approximately 50 base pairs, up to approximately 60 base pairs, up to approximately 70 base pairs , up to approximately 80 base pairs, up to approximately 90 base pairs, up to approximately 100 base pairs, up to approximately 110 base pairs, Approximately 120 base pairs, maximum approximately 130 base pairs, maximum approximately 140 base pairs, maximum approximately 150 base pairs, maximum Approximately 160 base pairs, a maximum of approximately 170 base pairs, and also approximately 70 base pairs, approximately 75 base pairs, and approximately 80 Base pairs, approximately 85 base pairs, approximately 90 base pairs, approximately 95 base pairs, approximately 100 base pairs, and approximately 105 salts One or more multiples of the base pair can be selected. In some cases, the multiple is, for each occurrence, The numbers are independently selected from multiples of 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19. For example, approximately 90 base pairs is a multiple of 1, and approximately 270 base pairs is a multiple of 90. The intervals are multiples of 3 for each base pair. In some cases, one or more length intervals are approximately 90. The base pairs are approximately 270 base pairs, 450 base pairs, 630 base pairs, or 810 base pairs. obtain.

[0157] The method allows for the isolation or removal of one or more DNA sequences at intervals of length. Approximately 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 DNA molecules at fixed length intervals. 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 9 3, 94, 95, 96, 97, 98, 99, or 100% can be isolated or removed. It is possible. Up to 5, 6, 7, 8, 9, 10, 15, 20, 25, 3 DNA at specific length intervals. 0, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 Isolate or remove 92, 93, 94, 95, 96, 97, 98, 99 or 100% It is possible to have at least 5, 6, 7, 8, 9, 10, 15 DNA at specific length intervals. , 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% It can be isolated or removed.

[0158] Exosomes for background population depletion and / or enrichment of target population Another example of the enrichment method provided herein is for the target population (e.g., non-host population). To deplete or concentrate the sample of nucleic acids, biological samples obtained from the host are used. This includes targeting exosome nucleic acids within the sample. Exosomes and other extracellular micronutrients. These cells are secreted by cells and are present in the biological fluid. They are directly exposed at the plasma membrane. They can be released by buds or through the body pathways of the polysporoplasm.

[0159] In some cases, nucleic acids of the target population (e.g., pathogens, non-humans) are exosomes. They may be distributed asymmetrically, either internally or externally. In some cases, the background population Nucleic acids (e.g., human) are distributed asymmetrically inside or outside exosomes. When nucleic acids of a group (e.g., non-host, pathogen) are present outside the exosome, The method involves extracting nucleic acids from a target population outside the exosome from a sample. This may include removing sosomes. Similarly, background populations (e.g., host or When nucleic acids (in humans) are present within exosomes, this method involves the target outside the exosome and This may include removing exosomes to concentrate the nucleic acid population. Before isolating exosomes, such as circulating nucleic acids, from the biological sample, To isolate or remove from something.

[0160] The target population (e.g., microorganisms or pathogens) has nucleic acids located inside exosomes. In this case, this method is used to concentrate the nucleic acids of the target population inside the exosome. This may include capturing chromosomes. The nucleic acids of the target population are leukocytes (e.g., macrophages). If present in the phagocytic sac or processed by white blood cells (e.g., macrophages), This method involves immunoprecipitation of leukocyte-derived exosomes with antibodies, etc. Xosomes can be preferentially isolated. Similarly, background populations (e.g., If the host (or human) nucleic acid is located outside the exosome, this method will be used for the exosome. To concentrate the nucleic acids of the target population within the sample, exosomes are extracted from the sample. It may include the following.

[0161] In some cases, background population nucleic acids are not paired inside or outside the exosome. They are distributed in a specific manner. For example, human nucleic acids such as human cell-free nucleic acids are found inside exosomes and It may be distributed asymmetrically to the outside. Generally, the majority of human cell-free RNA in plasma is It is likely found within exosomes due to the abundance of RNases in plasma. Circulating nucleic acids Direct extraction of cell-free RNA using the kit is necessary for high-quality RNA degradation. This may not occur. Exosome isolation yields intact mRNA, 18S and 28S receptacles. It can produce high-quality RNA, including bosomal RNA. The majority of human cell-free mRNA is exoso It may be present within the room. Therefore, in some cases, the method provided herein is sample To deplete human cell-free mRNA from the sample, exosomes are depleted. This may include the fact that, in general, exosomes probably package the cytosol. Therefore, it is possible that the majority of non-human cellular DNA in plasma is not present in exosomes. Therefore, in some cases, the methods provided herein can be used to obtain human cell-free D from a sample. This may involve enriching the exosomes in the sample to deplete NA.

[0162] In some cases, this method involves plasma, isolated exosomes and / or exosomes. This may include determining the ratio of nucleic acids from the target population to the background population in depleted plasma. For example, this method involves plasma, isolated exosomes and / or exosome depletion. This may include determining the ratio of microorganisms to human nucleic acids in plasma. In some cases, this method may include Microorganisms in plasma, isolated exosomes and / or exosome-depleted plasma and human D This may include determining the ratio of NA. In some cases, this method may involve plasma, isolated extracts To determine the ratio of microorganisms to human RNA in sososomal and / or exosome-depleted plasma. This method may include plasma, isolated exosomes and / or This determines the amount, percentage, or concentration of nucleic acids in the target population in exosome-depleted plasma. This may include doing so.

[0163] The nucleic acids of the target population are present in white blood cells (e.g., macrophages), or white blood When processed by cells (e.g., macrophages), this method is effective for leukocyte-derived exosphorus. By immunoprecipitation with antibodies, etc., leukocyte-derived exosomes are preferentially isolated. This is possible. Microorganisms or pathogenic nucleic acids are present in white blood cells or processed by white blood cells. If so, this method involves immunoprecipitation of leukocyte-derived exosomes with antibodies. This method allows for the preferential isolation of exosomes derived from blood cells. In some cases, this method is effective. This includes enriching exosomes derived from mammalian leukocytes (e.g., macrophages). It is possible. In some cases, this method is derived from human leukocytes (e.g., macrophages). This may include enriching exosomes. In some cases, this method involves host leukocytes (for example) This may include enriching exosomes derived from macrophages. While some macrophages re-enter the bloodstream, most do not, so this method is not suitable for deep tissue infection. Information about the disease can be accessed.

[0164] Exosomes can be isolated or removed by this method. Approximately 5,6 exosomes , 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 9 8, 99, or 100% of exosomes can be isolated or removed. Up to 5,600 exosomes can be isolated or removed. , 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 9 8, 99, or 100% of the exosomes can be isolated or removed. 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 9 7, 98, 99, or 100% can be isolated or removed from the exosomes in the plasma. As some non-limiting examples of kits and protocols available for isolation from This is the Exo-Spin Blood Exosome Purification Kit (Cell Guidance Sy stems), exoRNeasy serum / plasma kit (Qiagen), whole exosomes Isolation reagents (Life Technologies), ExoQuick (System Biosciences), Exo-Flow exosome immunopurification (System B iosciences), ME kit (New England Peptide), V n96 peptide (New England Peptide), PureExo exo exosome isolation kit (101 Bio) and plasma / serum circulation and exosomal RNA purification One example is the kit (Norgen Biotek Corp).

[0165] Target host DNA depletion In some cases, depletion of host nucleic acids in cell-free samples is due to either the host or non-host nucleic acids being present. A specific sequence, sequence structure, nucleic acid characteristic, or modification that can be specific to one of the host nucleic acids. Targeting can be utilized. For example, against one of the host or non-host nucleic acids. A specific arrangement as a mechanism for binding, extracting, settling, digesting, or removing or selecting Serial motifs, structures, base modifications, etc., can be targeted. For example, in many cases, human... Nucleic acids may contain base modifications that are not reflected in non-host or pathogen-associated nucleic acids. Modifications include methylation patterns such as cytosine methylation. Lucytosine can be found in CpG islands of higher organisms. Several eukaryotic pathogens... It exists, but its presence is considerably higher in vertebrates compared to other organisms.

[0166] In some cases, methylation motif-specific endonucleases (e.g., McRB) Using C, FspEI, LpnPI, MspJI endonucleases, these Thai Modifications of the protein can be targeted. These endonucleases typically perform digestion. This requires two closely spaced base modifications. Separation is possible for any length between 50 bp and 3 kbp. For example, at least 50bp, at least 60bp, at least 70bp, at least 8 0 bp, at least 90 bp, at least 100 bp, at least 200 bp, at least Also 300bp, at least 400bp, at least 500bp, at least 600bp, At least 700bp, at least 800bp, at least 900bp, at least 1k bp, at least 1.5kbp, at least 2kbp, at least 2.5kbp or less It can be at least 3kbp. Most are 147-175bp long, so this is human c This can result in poor digestion efficiency of fDNA fragments during the preparation of sequencing libraries. In the ligation step, one of the two adapters provided is used for partner base repair. This can be improved by introducing embellishments. For example, in the original human cfDNA fragment Even if only one methylcytosine is present, the P5 sequence-supporting adapter can be used to support ligase To stimulate post-McrBC digestion, CpG islands contain methylcytosine. This is possible. Therefore, in some cases, the "helper" modified base can be used in the sequence library. By including a methylated base in the adapter sequence used when preparing the sequence, It can be artificially introduced into the library. Therefore, host-derived library elements can be pre-programmed. While digesting, it allows non-host-derived library elements to proceed with amplification and sequencing. To achieve this, the digestion step is used after the adapter ligation to the sequence fragment. It should be possible.

[0167] Alternative or additional methods selectively isolate sequences longer than the required separation distance. Alternatively, to prioritize digestion, spacing may be required between two or more such parts. Recognition sequences of pairs separated by more than 50 bases can be used. For example, (e.g., 50 McrBC endonucleases capable of recognizing two methylated cytosines (approximately 3 kbp) In this case, a methylated base can be provided to the adapter sequence attached to either end. As a result, fragments that maintain more than 50 bases between methylated bases on opposing adapters It is digested by McrBC endonuclease and as described elsewhere in this specification. As such, the concentration of non-host fragments is greater for shorter nucleic acid fragments. They probably will.

[0168] Combined enrichment method The aforementioned enrichment schemes are described individually, but any or all of the above methods may be used in accommodation. In enriching non-host DNA in a sample relative to the main DNA, various combinations are used. It will be recognized that it can be used. For example, a cell-free sample, first, For example, it can be used in DNA purification schemes based on size selection using the SPRI bead system. Next, chromatography size selection scheme, nucleosome immunoprecipitation scheme It can be used for any one or more of these purposes.

[0169] In this case as well, as mentioned above, in some cases, one or multiple steps of non-host DNA Concentration increases the ratio of non-host DNA to host DNA in the sample by approximately 2 to 10,000 times. An increase may be brought about. In some cases, the ratio can be at least twice, at least three times, and 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, and at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, At least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 30 times, at least 40 times , at least 50 times, at least 60 times, at least 70 times, at least 80 times, less 90 times, at least 100 times, at least 1000 times, at least 5000 times or In some cases, it can be increased by at least 10,000 times.

[0170] Molecular barcoding of samples using nucleic acid spike-in Samples can be barcoded using nucleic acid barcode spike-in. The code may be one or more lengths. In some cases, nucleic acid barcodes are one Ligonucleotides, double-stranded longmers, PCR products and / or nucleotides It can be rasmid. In some cases, nucleic acid barcodes are DNA, RNA, PNA, LN It can be A, BNA, or any combination thereof. In some cases, nucleic acid bursage. The dot is one or more background populations, for example, the human genome or pathogen genome. It may contain sequences that do not exist. In some cases, nucleic acid barcodes are one or more pairs It may contain sequences that are not present in the elephant population.

[0171] In some cases, the barcode is different from the label on the outside of the sample tube, It is part of the. In some cases, the barcode is any, including before the addition of the biological sample. It can be added at any stage (for example, by PCR or sequencing). It can be read. In some cases, barcodes are used to track samples. Cross-contamination can be detected and / or the reagents can be tracked. In some cases, Using codes can reduce sample confusion. In some cases, nucleic acid barcodes are used. By using this method to increase the total nucleic acid concentration, it is possible to increase the recovery of low-concentration samples. In some cases, barcodes are used to compare a known input with a measured output. Alternatively, a reference standard (e.g., a normalized oligonucleotide) can be proposed for comparison or normalization. By providing this, the sample input can be estimated. In some cases, barcode Using this, library complexity, sample loss, sensitivity, and / or size bias By measuring this, performance can be improved through quality control and development.

[0172] In some cases, the sample (e.g., biological sample or nucleic acid sample) is normalized. It can be spiked with normalized oligonucleotides. In some cases, normalized oligonucleotides Rheotides are used to monitor the efficiency of DNA manipulation, purification, and / or amplification steps. This can be done. For example, sequencing and reading of the target population (e.g., non-host or pathogen). Compare the absolute number of normalized oligonucleotide reads recovered with the sample The difference in molecular manipulation efficiency between different molecules can be normalized. In some cases, normalization is possible. For the purpose of barcoding ligonenucleotides, or for both normalization and barcoding purposes. It can be used for this purpose.

[0173] Strategic Capture of Target Areas The collection and methods of oligonucleotides provided herein are applicable to the region Oligonucleotypes containing nucleic acid sequences identical, nearly identical, complementary, or nearly complementary to the region It may further include. Some non-limiting examples of the regions covered include pathogenic genes. (For example, Clostridium diffic IL (pathogenicity gene locus); antimicrobial agent resistance marker; antibiotic resistance marker; antimicrobial Antiparasitic agent resistance markers; beneficial genotyping regions (e.g., Derived primarily from humans, microorganisms, pathogens, bacteria, viruses, fungi, or parasites); two or more microorganisms A sequence common to organisms, pathogens, bacteria, viruses, fungi and / or parasites; host geno Non-host sequence incorporated into the m; masking non-host sequence; non-host mimic sequence; masking host Sequences; host mimic sequences; and one or more microorganisms, pathogens, bacteria, viruses, and fungi. Alternatively, sequences specific to parasites may be included. In some cases, the target region is non-inhabitant. They can be present in the genomes of bacteria, viruses, pathogens, fungi, or microorganisms. In some cases... The target region may be located in the host, mammal, or human genome. By containing identical, nearly identical, complementary, or nearly complementary nucleic acid sequences, the genotype is the same. Detection of resistance to antimicrobial agents, antibiotics, antivirals, and antiparasitic agents. Enhanced detection and / or pathogen detection sensitivity may be possible. The target area is the same as, or nearly the same as, the area of ​​interest. Identical, complementary, or nearly complementary nucleic acid sequences, for example, bioinformatics or computationally It can be chemically synthesized by design and / or DNA synthesis. Nucleic acid sequences are nucleated by the nucleus. For acid amplification or detection, to prime cDNA synthesis from RNA templates, sequencing For the sake of consistency, in the primer extension reaction, for DNA / RNA hybridization , and / or can be used as a primer for DNA / RNA pulldown. Cut.

[0174] Antibiotic resistance markers are one or more mutations that confer antibiotic resistance, monosodium methyl saline (MSR) This may include polymorphisms, genes, or gene products. Antibiotic resistance markers are not limited to these. However, antibiotic leakage, antibiotic inactivation, antibiotic target modification, antibiotic target protection, antibiotic One or more mechanisms such as reduced penetration to substance target substitution and / or antibiotics. Antibiotic resistance can be conferred through the structure. In some cases, antibiotic resistance markers are cross-reactive. Tridium difficile (C. difficile), carbapenem-resistant Enterobacteriaceae (CRE) Drug-resistant gonorrhea (cephalosporin resistant), multidrug-resistant Acinetobacter, drug-resistant Campione Lobacter, fluconazole-resistant Candida (fungus), broad-spectrum β-lactamase Enterobacteriaceae (ESBL), vancomycin-resistant enterococci (VRE), multidrug-resistant Pseudomonas aeruginosa, drugs Drug-resistant non-typhoid Salmonella, drug-resistant Salmonella typhi, drug-resistant Sigella, methicillin-resistant Yellow Bacteria St. plague (MRSA), drug-resistant Streptococcus pneumoniae, drug-resistant tuberculosis (MDR and XDR), multidrug Medicinal-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus (VRSA), erythromycin Seen in clindamycin-resistant Streptococcus A group or clindamycin-resistant Streptococcus B group It may be released. In some cases, antibiotic resistance markers are acridine dyes, aminocoumyl Antibiotics, aminoglycosides, aminonucleoside antibiotics, β-lactams, diamino Pyrimidines, elfamicin, fluoroquinolones, glycopeptide antibiotics, lincosamines Lipopeptide antibiotics, macrocyclic antibiotics, macrolides, nucleoside antibiotics, Arsenic antibiotics, oxazolidinone antibiotics, peptide antibiotics, phenicol, pleurol Lomucilin antibiotics, polyamine antibiotics, rifamycin antibiotics, streptogramin Antibiotics, sulfonamides, sulfones, tetracycline derivatives or any of these Antibiotic resistance to combinations may be conferred. In some cases, antibiotic resistance markers These include β-lactams, penicillins, aminopenicillins, early-generation cephalosporins, and β-lactams. Combinations of kutamase inhibitors, broad-spectrum cephalosporins, carbapenems, flu Oloquinolone, aminoglycoside, tetracycline, glycycrine, polymyxin, pe Nishilin, methicillin, erythromycin, gentamicin, ceftazidime, vancomycin Syn, levofloxacin, imipenem, linezolid, ceftriaxone, cephthaloline Or, it may confer antibiotic resistance to any combination of these.

[0175] Non-limiting examples of antibiotic resistance markers include aac2ia, aac2ib, and aac 2ic, aac2id, aac2i, aac3ia, aac3iia, aac3iib, aac3iii, aac3iv, aac3ix, aac3vi, aac3viii, aa c3vii, aac3x, aac6i, aac6ia, aac6ib, aac6ic, a ac6ie, aac6if, aac6ig, aac6iia, aac6iib, aad9 , aad9ib, aadd, acra, acrb, adea, adeb, adec, am ra, amrb, ant2ia, ant2ib, ant3ia, ant4iia, ant 6ia, aph33ia, aph33ib, aph3ia, aph3ib, aph3ic , aph3iiia, aph3iva, aph3va, aph3vb, aph3via, aph3viia, aph4ib, aph6ia, aph6ib, aph6ic, aph 6id, arna, baca, bcra, bcrc, bl1_acc, bl1_ampc , bl1_asba, bl1_ceps, bl1_cmy2, bl1_ec, bl1_f ox, bl1_mox, bl1_och, bl1_pao, bl1_pse, bl1_s m, bl2a_1, bl2a_exo, bl2a_iii2, bl2a_iii, bl2 a_kcc, bl2a_nps, bl2a_okp, bl2a_pc, bl2be_ct xm, bl2be_oxy1, bl2be_per, bl2be_shv2, bl2b_ rob, bl2b_tem1, bl2b_tem2, bl2b_tem, bl2b_tl e、bl2b_ula、bl2c_bro、bl2c_pse1、bl2c_pse3、 bl2d_lcr1、bl2d_moxa、bl2d_oxa10、bl2d_oxa1 、bl2d_oxa2、bl2d_oxa5、bl2d_oxa9、bl2d_r39、 bl2e_cbla、bl2e_cepa、bl2e_cfxa、bl2e_fpm、b l2e_y56、bl2f_nmca、bl2f_sme1、bl2_ges、bl2_ kpc、bl2_len、bl2_veb、bl3_ccra、bl3_cit、bl3 _cpha、bl3_gim、bl3_imp、bl3_l、bl3_shw、bl3_ sim、bl3_vim、ble、blt、bmr、cara、cata10、cata 11、cata12、cata13、cata14、cata15、cata16、ca ta1、cata2、cata3、cata4、cata5、cata6、cata7、 cata8、cata9、catb1、catb2、catb3、catb4、catb 5、ceoa、ceob、cml_e1、cml_e2、cml_e3、cml_e4、 cml_e5、cml_e6、cml_e7、cml_e8、dfra10、dfra1 2、dfra13、dfra14、dfra15、dfra16、dfra17、dfr a19、dfra1、dfra20、dfra21、dfra22、dfra23、df ra24、dfra25、dfra25、dfra25、dfra26、dfra5、d fra7、dfrb1、dfrb2、dfrb3、dfrb6、emea、emrd、e mre、erea、ereb、erma、ermb、ermc、ermd、erme、e rmf、ermg、ermh、ermn、ermo、ermq、ermr、erms、e rmt、ermu、ermv、ermw、ermx、ermy、fosa、fosb、f osc、fosx、fusb、fush、ksga、lmra、lmrb、lnua、l nub, lsa, maca, macb, mdte, mdtf, mdtg, mdth, md tk, mdtl, mdtm, mdtn, mdto, mdtp, meca, mecr1, m efa、mepa、mexa、mexb、mexc、mexd、mexe、mexf、m exh、mexi、mexw、mexx、mexy、mfpa、mpha、mphb、m phc、msra、norm、oleb、opcm、opra、oprd、oprj、o prm、oprn、otra、otrb、pbp1a、pbp1b、pbp2b、pbp 2、pbp2x、pmra、qac、qaca、qacb、qnra、qnrb、qnr s、rosa、rosb、smea、smeb、smec、smed、smee、sme f、srmb、sta、str、sul1、sul2、sul3、tcma、tcr3、 tet30、tet31、tet32、tet33、tet34、tet36、tet3 7、tet38、tet39、tet40、teta、tetb、tetc、tetd、 tete、tetg、teth、tetj、tetk、tetl、tetm、teto、 tetpa、tetpb、tet、tetq、tets、tett、tetu、tetv 、tetw、tetx、tety、tetz、tlrc、tmrb、tolc、tsnr 、vana、vanb、vanc、vand、vane、vang、vanha、van hb、vanhd、vanra、vanrb、vanrc、vanrd、vanre、v anrg, vansa, vansb, vansc, vansd, vanse, vansg , vant, vante, vang, vanug, vanwb, vanwg, vanx a, vanxb, vanxd, vanxyc, vanxye, vanxyg, vanya , vanyb, vanyd, vanyg, vanz, vata, vatb, vatc, v atd, vate, vgaa, vgab, vgba, vgbb, vph, ykkc and YKKD is one example.

[0176] concentrated The nucleic acids of the target population can be concentrated by the method described herein. In Tsuka's case, the nucleic acids of the target population are approximately 5%; 10%; 15%; 20%; 25%; 30% ;35%;40%;45%;50%;55%;60%;65%;70%;75%;80% ;85%;90%;95%;100%;150%;200%;250%;300%;35 0%;400%;450%;500%;550%;600%;650%;700%;75 0%;800%;850%;900%;950%;1000%;2000%;3000% ;4000%;5000%;6000%;7000%;8000%;9000%;100 00%;20000%;30000%;40000%;50000%;60000%;7 0000%;80000%;90000%;100000%;200000%;3000 00%;400000%;500000%;600000%;700000%;8000 00%;900000%;1000000%;2000000%;3000000%;4 000000%;5000000%;6000000%;7000000%;80000 00%;9000000%;10000000%;20000000%;3000000 0%;40000000%;50000000%;60000000%;7000000 Concentrate to 0%, 80,000,000%, 90,000,000%, or 10,000,000%. It is possible to reduce the nucleic acids of the target population by up to 5%, 10%, 15%, or 2%. 0%;25%;30%;35%;40%;45%;50%;55%;60%;65%;7 0%;75%;80%;85%;90%;95%;100%;150%;200%;25 0%;300%;350%;400%;450%;500%;550%;600%;65 0%;700%;750%;800%;850%;900%;950%;1000%;2 000%;3000%;4000%;5000%;6000%;7000%;8000% ;9000%;10000%;20000%;30000%;40000%;50000 %;60000%;70000%;80000%;90000%;100000%;20 0000%;300000%;400000%;500000%;600000%;70 0000%;800000%;900000%;1000000%;2000000%; 3000000%;4000000%;5000000%;6000000%;7000 000%;8000000%;9000000%;10000000%;2000000 0%;30000000%;40000000%;50000000%;6000000 0%;70000000%;80000000%;90000000%;or 1000 It can be concentrated to 00000%. In some cases, the nucleic acids of the target population can be reduced. 5%; 10%; 15%; 20%; 25%; 30%; 35%; 40%; 45%; 50%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; 95%; 100% ;150%;200%;250%;300%;350%;400%;450%;500% ;550%;600%;650%;700%;750%;800%;850%;900% ;950%;1000%;2000%;3000%;4000%;5000%;6000 %;7000%;8000%;9000%;10000%;20000%;30000% ;40000%;50000%;60000%;70000%;80000%;9000 0%;100000%;200000%;300000%;400000%;50000 0%;600000%;700000%;800000%;900000%;10000 00%;2000000%;3000000%;4000000%;5000000%; 6000000%;7000000%;8000000%;9000000%;1000 0000%;20000000%;30000000%;40000000%;5000 0000%;60000000%;70000000%;80000000%;9000 It can be concentrated to 0000% or 100000000%. For example, if the sample is It contains nucleic acids from the target population, which is 5% of the total nucleic acid population, and 10% of the total nucleic acid population. When concentrated to contain % of the target population's nucleic acids, the nucleic acids of the target population are 1 It is concentrated to 00%. In some cases, the nucleic acids of the target population are concentrated to approximately 1.5 times; 2 times; 2 .5x;3x;3.5x;4x;4.5x;5x;5.5x;6x;6.5x;7x;7 .5x;8x;8.5x;9x;9.5x;10x;15x;20x;25x;30x; 35x; 40x; 45x; 50x; 55x; 60x; 65x; 70x; 75x; 80x; 85x;90x;95x;100x;150x;200x;250x;300x;350 times; 400 times; 450 times; 500 times; 550 times; 600 times; 650 times; 700 times; 750 times; 800 times; 850 times; 900 times; 950 times; 1000 times; 2000 times; 3000 times; 4000x; 5000x; 6000x; 7000x; 8000x; 9000x; 1000 0x;20000x;30000x;40000x;50000x;60000x;70 000x;80000x;90000x;100000x;200000x;30000 0x;400000x;500000x;600000x;700000x;80000 It can be concentrated 0 times; 900,000 times; 1,000,000 times. In some cases, the target The nucleic acids of the resulting group are multiplied by up to 1.5 times; 2 times; 2.5 times; 3 times; 3.5 times; 4 times; 4.5 times; 5x; 5.5x; 6x; 6.5x; 7x; 7.5x; 8x; 8.5x; 9x; 9.5x; 10x;15x;20x;25x;30x;35x;40x;45x;50x;55x; 60x;65x;70x;75x;80x;85x;90x;95x;100x;150 times;200 times;250 times;300 times;350 times;400 times;450 times;500 times;550 times; 600 times; 650 times; 700 times; 750 times; 800 times; 850 times; 900 times; 950 times;1000 times;2000 times;3000 times;4000 times;5000 times;6000 times;70 00x;8000x;9000x;10000x;20000x;30000x;400 00x;50000x;60000x;70000x;80000x;90000x;1 00000x;200000x;300000x;400000x;500000x;6 00000x; 700000x; 800000x; 900000x; 1000000x dark It can be reduced. In some cases, the nucleic acids of the target population can be reduced by at least 1.5 times; 2 times;2.5 times;3 times;3.5 times;4 times;4.5 times;5 times;5.5 times;6 times;6.5 times;7 times;7.5 times;8 times;8.5 times;9 times;9.5 times;10 times;15 times;20 times;25 times;3 0x;35x;40x;45x;50x;55x;60x;65x;70x;75x;8 0x; 85x; 90x; 95x; 100x; 150x; 200x; 250x; 300x; 350x; 400x; 450x; 500x; 550x; 600x; 650x; 700x; 750x; 800x; 850x; 900x; 950x; 1000x; 2000x; 300 0x;4000x;5000x;6000x;7000x;8000x;9000x;1 0000x; 20000x; 30000x; 40000x; 50000x; 60000x ;70000x;80000x;90000x;100000x;200000x;30 0000x;400000x;500000x;600000x;700000x;80 It can be concentrated 0000 times; 900000 times; 1000000 times. For example, a sample The nucleic acid contains nucleic acids from the target population, which is 5% of the total nucleic acid population. When concentrated to contain 10% of the nucleic acids of the target population, the nuclei of the target population The acid is twice as concentrated.

[0177] The background population can be depleted by the methods described herein. In some cases, background population nucleic acids were approximately 5, 6, 7, 8, 9, 10, 15, 20. 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 9 Deplete 0, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%. It is possible. In some cases, background population nucleic acids can be up to 5, 6, 7, 8, 9. 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 7 5, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or It can be 100% depleted. In some cases, the background population nucleic acids can be reduced. Tomo 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 5 5, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96 It can be depleted to 97%, 98%, 99%, or 100%. For example, if the sample is nucleic acid It contains background population nucleic acids representing 50% of the total population, and 10% of the total nucleic acid population. If the background population is depleted to contain % of the nucleic acids, then the background population The group nucleic acid is 80% depleted. In some cases, the background population nucleic acid is approximately 1.5 times greater. ;2x;2.5x;3x;3.5x;4x;4.5x;5x;5.5x;6x;6.5x ;7x;7.5x;8x;8.5x;9x;9.5x;10x;15x;20x;25x ;30x;35x;40x;45x;50x;55x;60x;65x;70x;75x ;80x;85x;90x;95x;100x;150x;200x;250x;300 times; 350 times; 400 times; 450 times; 500 times; 550 times; 600 times; 650 times; 700 750x; 800x; 850x; 900x; 950x; 1000x; 2000x; 3 000x; 4000x; 5000x; 6000x; 7000x; 8000x; 9000x ;10000x;20000x;30000x;40000x;50000x;6000 0x; 70000x; 80000x; 90000x; 100000x; 200000x; 300000x; 400000x; 500000x; 600000x; 700000x; It can be concentrated 800,000 times; 900,000 times; 1,000,000 times. In this case, the background population nucleic acid can be multiplied by up to 1.5 times; 2 times; 2.5 times; 3 times; 3.5 times; 4 times;4.5 times;5 times;5.5 times;6 times;6.5 times;7 times;7.5 times;8 times;8.5 times;9 times;9.5 times;10 times;15 times;20 times;25 times;30 times;35 times;40 times;45 times;5 0x;55x;60x;65x;70x;75x;80x;85x;90x;95x;1 00x;150x;200x;250x;300x;350x;400x;450x;5 00x;550x;600x;650x;700x;750x;800x;850x;9 00x;950x;1000x;2000x;3000x;4000x;5000x;6 000x;7000x;8000x;9000x;10000x;20000x;300 00x;40000x;50000x;60000x;70000x;80000x;9 0000x;100000x;200000x;300000x;400000x;50 0000x;600000x;700000x;800000x;900000x;10 It can be concentrated 00000 times. In some cases, the background population nucleic acid is reduced. At least 1.5 times; 2 times; 2.5 times; 3 times; 3.5 times; 4 times; 4.5 times; 5 times; 5.5 times; 6x; 6.5x; 7x; 7.5x; 8x; 8.5x; 9x; 9.5x; 10x; 15x; 20x; 25x; 30x; 35x; 40x; 45x; 50x; 55x; 60x; 65x; 70x;75x;80x;85x;90x;95x;100x;150x;200x;2 50x;300x;350x;400x;450x;500x;550x;600x;6 50x; 700x; 750x; 800x; 850x; 900x; 950x; 1000x; 2000x; 3000x; 4000x; 5000x; 6000x; 7000x; 8000 times;9000 times;10000 times;20000 times;30000 times;40000 times;5000 0x;60000x;70000x;80000x;90000x;100000x;2 00000x;300000x;400000x;500000x;600000x;7 It can be concentrated 00,000 times; 800,000 times; 900,000 times; 1,000,000 times. For example, a sample may contain background nucleic acids representing 50% of the entire nucleic acid population. And, it is depleted to contain 10% of the background population nucleic acids of the entire nucleic acid population. If present, the background population nucleic acid is five times more depleted.

[0178] sample The methods and compositions provided herein are derived from a control (e.g., a human host) It is useful for detecting nucleic acids in a variety of samples. Non-specific examples include blood, plasma, serum, whole blood, mucus, saliva, cerebrospinal fluid, synovial fluid, and lavage fluid. Examples include urine, tissue biopsy, cell samples, skin samples, and stool. The samples are circulating. Nucleic acids, including circulating nucleic acids (e.g., circulating This may include the following: In some cases, the sample obtained from the subject undergoes further processing. For example, a sample can be processed to extract DNA or RNA, and this is the true purpose. The analysis can be performed using the methods provided in the detailed document.

[0179] In some cases, nucleic acid samples are biological samples, isolated nucleic acid samples, or circulating samples. It may be a sample such as a purified nucleic acid sample containing nucleic acids, such as nucleic acids. Nucleic acid samples may contain host and non-host sequences, such as human and non-human sequences. In some cases, nucleic acids in nucleic acid samples, such as nucleic acid sequencing libraries, are used. For example, it is amplified by a PCR amplification reaction. In some cases, the nucleic acids in the nucleic acid sample are Artificial fragmentation (e.g., by sonication, shearing, enzymatic digestion, or chemical fragmentation) It is not done. In some cases, nucleic acid fragmentation is unnecessary because the nucleic acid length is relatively short. In some cases, nucleic acids in nucleic acid samples are artificially fragmented. Circulating nucleic acid samples are, Contains biological samples, isolated nucleic acid samples, or circulating nucleic acids such as circulating cell-free nucleic acids. This could be a sample such as a purified nucleic acid sample. In some cases, it could be a circulating cell-free nucleic acid sample. This includes biological samples, isolated nucleic acid samples, or circulating cell-free DNA or circulating cell-free DNA. This could be a sample such as a purified nucleic acid sample containing circulating cell-free nucleic acids such as RNA. In some cases, single-stranded nucleic acid samples are biological samples, isolated nucleic acid samples, or single-stranded nucleic acid samples. A purified nucleic acid sample containing single-stranded nucleic acids, such as circulating nucleic acids or circulating single-stranded nucleic acids without cells. This could be a sample of something like a lu.

[0180] In some cases, nucleic acids are DNA, RNA, cDNA, mRNA, cRNA, dsDNA , ssDNA, miRNA, circulating nucleic acid, circulating DNA, circulating RNA, cell-free nucleic acid, cell-free D This can be NA, cell-free RNA, circulating cell-free DNA, circulating cell-free RNA, or genomic DNA. In some cases, circulating nucleic acids are circulating DNA, circulating RNA, cell-free nucleic acids, or cell-free circulating nucleic acids. These are nucleic acids. In some cases, cell-free nucleic acids include cell-free DNA, cell-free RNA, and circulating cell-free DNA. It can be NA or circulating cell-free RNA. In some cases, circulating cell-free nucleic acids are circulating cell-free. This could be DNA or circulating cell-free RNA.

[0181] In some cases, nucleic acids in the sample do not need to be labeled; in some cases, the nucleus Acids are labeled, for example, with nucleic acid labels, chemical labels, or optical labels. In some cases, nucleic acids The nucleic acid is conjugated onto a solid support. In some cases, the label is placed at the 5' or 3' end of the nucleic acid. It can be attached to the edge or inside the nucleic acid. In some cases, two or more nucleic acids can be attached. Mark with signs.

[0182] Nucleic acids in the sample may be tagged with nucleic acid labels. In some cases, nucleic acid labels are used. It may include one or more of the following: barcode (e.g., sample barcode), universal barcode Primer sequence, primer binding site (for example, not limited to that, but DNA sequence) Determinative primer binding site, sample barcode sequence determination primer binding site and various A sequencing or (For barcode reading), sequencer-compatible sequences, sequencing platform Attaching sequence, sequencing adapter sequence, or adapter. Nucleic acid labeling is (e.g., ly It can be attached to nucleic acids (by gate or synthetic design).

[0183] Nucleic acid labels can include chemical labels. Some non-limiting examples of chemical labels include: Biotin, avidin, streptavidin, radiolabeling, polypeptides and polymers Examples include: Nucleic acid labels can include optical labels. Some non-limiting optical labels Examples include fluorophores, fluorescent proteins, dyes, and quantum dots. The consciousness can be conjugated to a solid support. Several non-limiting aspects of the solid support Examples include beads, magnetic beads, polymers, slides, tips, surfaces, plates, and chips. Examples include channels, cartridges, microfluidic devices, and microarrays. Nucleic acids It is possible to conjugate it onto a solid support for affinity chromatography. Yes, it is possible. In some cases, each nucleic acid has a different label. In some cases, each nucleic acid is the same. It has a label. In some cases, the nucleic acid in the sample is conjugated onto a solid support. stomach.

[0184] Sequence determination method In some cases, a concentrated population of nucleic acids is sequenced. In some cases, as specified herein The described method further includes the step of performing a sequencing assay. Amplification or capture The nucleic acids of the captured target population are subjected to sequencing assays, particularly high-throughput sequencing assays. Sequencing assays, next-generation sequencing platforms, high-volume parallel sequencing platforms Rat home, nanopore sequencing assay, Sanger sequencing, or this technology By performing another sequencing assay known in the field, etc., It can be identified by the following method. Several non-limiting sequencing assays Examples include high-throughput sequencing assays and next-generation sequencing platforms. Home, Mass Parallel Sequencing Platform, Nanopore Sequencing and Sanga One example is sequencing. Some non-exclusive examples of types of sequencing machines include Illumina, Roche 454, Ion Torrent and Nanopo re is one example.

[0185] The process methods described herein also include, for example, distinguishing between host sequences and non-host sequences. A method for selecting information science data that can be applied to the obtained sequence data, including a host nucleus This can be used in conjunction with other methods for distinguishing between acid sequences and non-host nucleic acid sequences. An example of such a process is that the whole disclosure is for all purposes and is therefore referenced by this. The published U.S. Patent Application No. 2015-0133391 is incorporated herein by reference. Examples include those listed in the specifications.

[0186] Purpose This method and composition involve one or more non-host species (e.g., microorganisms, pathogens, bacteria, To analyze biological samples from a host infected with a virus, fungus, or parasite. Useful. The methods and compositions provided herein are for diseases or disorders, particularly microorganisms. Or specifically for the detection, prediction, diagnosis, or monitoring of diseases or disorders caused by pathogens. Useful for circulating cell-free DNA or Certain types of samples obtained from infected hosts, such as samples containing circulating cell-free RNA It is useful for detecting target groups within a group.

[0187] This method and composition can also be used to enable the identification of the genotype of pathogens in a sample. Yes, it is possible. This method and composition are also particularly useful for detecting changes in the genome of microorganisms or pathogens. These are useful for, for example, detecting antibiotic-resistant strains of bacteria or for treating diseases. It is possible to track changes that affect the pathogenicity of pathogens, particularly viruses and bacteria.

[0188] In other cases, this method and composition may be used, for example, to address one or in a microbiome. The presence of multiple microorganisms can be monitored. For example, using this method and composition, It is possible to monitor the presence of the microbiome in healthy or uninfected hosts. This method and composition can be used to identify microbial cells in a sample containing multiple microorganisms. It can also monitor the situation.

[0189] The methods and compositions provided herein also include one or more non-host organisms (e.g., The hypothetical absence of nucleic acids derived from microorganisms, pathogens, bacteria, viruses, fungi, or parasites. It can also enable the detection or diagnosis or monitoring of diseases, disorders, or infections without hypothesis. Thus, the methods provided herein can target specific targets (e.g., one or more). Screening for specific nucleic acid sequences (proteins or antibodies) and testing selected targets Unlike other diagnostic methods that are limited to the methods provided herein, in some cases the methods provided herein may differ. The unhypothetical properties of the composition may be used for the detection of rare infections, or for the identification of two or more diseases or disorders. Detection of outbreaks, identification of the source of infection, or multiple diseases with similar or common symptoms. Alternatively, it can make it easier to identify the disability.

[0190] In some specific examples, this method involves performing one or more of the following steps in any order or Possible combinations include: (a) subjects or patients who have or are suspected of having a pathogen infection. (b) preparing nucleic acid samples from the person; (b) providing nucleic acid samples as provided herein A collection of oligonucleotides, particularly those selectively enriched to bind to non-host sequences. (c) Step of contacting the sample and oligonucleotide collection; (c) sample and oligonucleotide collection A collection of nucleotides in a nucleic acid sample (d) a step of subjecting the conditions to promote hybridization with nucleic acid molecules; Nucleic acids hybridized to a collection of creotides were subjected to amplification assays and producing assays. (e) A step of performing an assay such as a sequencing assay or a 2D assay; and (e) To detect specific pathogens in a sample, the sequences of hybridized nucleic acids are analyzed. Step. Typically, the number of specific pathogens detected is around 10, 20, 30, 40, or more than 50. There are many more, including other different pathogens, as further described herein. Pathogen detection is typically used for infectious diseases, infectious disorders, infections, or other diseases or disorders. For example, it can enable the detection, prognosis, monitoring, or diagnosis of cancer. In addition, such detection facilitates the staging of diseases or disorders, or pathogenic infections. This can provide an indicator of the degree of this.

[0191] In some cases, the methods described herein involve the distribution of at least one target population. Further includes detecting sequences (e.g., pathogenic or other non-host or non-human sequences). In some cases, the methods described herein involve the distribution of at least five populations. Further including determining the column. In some cases, the methods described herein are less This further includes determining five non-mammalian sequences. In some cases, as described herein The methods described involve at least one non-mammalian from each of at least five non-mammalian species. Further includes detecting sequences. In some cases, the methods described herein are less This further includes determining at least five non-human sequences. In some cases, as described herein. The method involves extracting at least one non-human sequence from each of at least five non-human species. Further including detection. In some cases, the methods described herein are at least Further includes detecting one bacterial sequence and at least one viral sequence. In the case of Tsuka, the method described herein applies to two or more points in time, for example, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 5, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 , further including taking the time 95 or 100. In some cases described herein The methods include taking time points before and after treatment, such as antimicrobial agents.

[0192] In some cases, the methods described herein are about 1;2;3;4;5;6;7;8; 9;10;15;20;25;30;35;40;45;50;60;70;80;90 ;100;200;300;400;500;1000;5000;10000;500 Further includes detecting at least one nucleic acid derived from 00 or 100,000 species. In some cases, the methods described herein may be up to 1;2;3;4;5;6;7; 8;9;10;15;20;25;30;35;40;45;50;60;70;80; 90;100;200;300;400;500;1000;5000;10000;5 Further detecting at least one nucleic acid derived from 0000 or 100000 species Included in some cases the methods described herein include at least 1;2;3;4;5 ;6;7;8;9;10;15;20;25;30;35;40;45;50;60;7 0;80;90;100;200;300;400;500;1000;5000;10 Detects at least one nucleic acid derived from 000;50000; or 100000 species. This further includes: In some cases, the species is a non-host species. In some cases, the species is non-mammalian. It is an animal species. In some cases, the species is a species other than a human. In some cases, the species is a microorganism. , bacteria, viruses, fungi, retroviruses, pathogens or parasites. In addition, the methods described herein are approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 20 At least 0, 300, 400, 500, or 1000 bacterial or viral species The method described herein further includes detecting a single nucleic acid. In some cases, the method described herein , up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 4 0, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 Alternatively, it is possible to detect at least one nucleic acid derived from 1000 bacterial or viral species. These include, in some cases, the methods described herein include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 bacteria or Further includes detecting at least one nucleic acid derived from a virus species. In addition, the methods described herein are approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 20 At least 0, 300, 400, 500, or 1000 bacterial and viral species The method described herein further includes detecting a single nucleic acid. In some cases, the method described herein , up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 4 0, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 Alternatively, to detect at least one nucleic acid derived from 1000 bacterial and viral species. These include, in some cases, the methods described herein include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 bacteria and Further includes detecting at least one nucleic acid derived from a virus species. In addition, the methods described herein include approximately 1 bacterial species and 1 virus species, 2 bacterial species and 2 viruses. Virus species, 3 bacterial species and 3 virus species, 4 bacterial species and 4 virus species, 5 bacterial species and 5 virus species, 6 bacterial species and 6 virus species, 7 bacterial species and 7 virus species, 8 bacterial species and 8 virus species, 9 bacterial species and 9 virus species, 10 bacterial species and 10 virus species, 1 5 bacterial species and 15 virus species, 20 bacterial species and 20 virus species, 25 bacterial species and 2 5 virus species, 30 bacterial species and 30 virus species, 35 bacterial species and 35 virus species, 4 0 bacterial species and 40 virus species, 45 bacterial species and 45 virus species, 50 bacterial species and 5 0 virus species, 60 bacterial species and 60 virus species, 70 bacterial species and 70 virus species, 8 0 bacterial species and 80 virus species, 90 bacterial species and 90 virus species, 100 bacterial species and 100 virus species, 200 bacterial species and 200 virus species, 300 bacterial species and 300 virus Virus species, 400 bacterial species and 400 virus species, 500 bacterial species and 500 virus species or at least one nucleic acid derived from 1000 bacterial species and 1000 viral species This further includes dispensing. In some cases, the methods described herein may contain up to one bacterial species. and 1 virus species, 2 bacterial species and 2 virus species, 3 bacterial species and 3 virus species, 4 species Bacterial species and 4 virus species, 5 bacterial species and 5 virus species, 6 bacterial species and 6 virus species, 7 bacterial species and 7 viral species, 8 bacterial species and 8 viral species, 9 bacterial species and 9 viruses species, 10 bacterial species and 10 virus species, 15 bacterial species and 15 virus species, 20 bacterial species and 20 virus species, 25 bacterial species and 25 virus species, 30 bacterial species and 30 viruses Species, 35 bacterial species and 35 virus species, 40 bacterial species and 40 virus species, 45 bacterial species and 45 virus species, 50 bacterial species and 50 virus species, 60 bacterial species and 60 viruses 70 bacterial species and 70 virus species, 80 bacterial species and 80 virus species, 90 bacterial species and 90 virus species, 100 bacterial species and 100 virus species, 200 bacterial species and 200 Virus species, 300 bacterial species and 300 virus species, 400 bacterial species and 400 viruses species, 500 bacterial species and 500 virus species, or 1000 bacterial species and 1000 viruses Further includes detecting at least one nucleic acid derived from the species. In some cases, this The methods described in the specification include at least one bacterial species and one virus species, two bacterial species and two Virus species, 3 bacterial species and 3 virus species, 4 bacterial species and 4 virus species, 5 bacterial species and and 5 virus species, 6 bacterial species and 6 virus species, 7 bacterial species and 7 virus species, 8 bacterial species and 8 virus species, 9 bacterial species and 9 virus species, 10 bacterial species and 10 virus species, 15 bacterial species and 15 virus species, 20 bacterial species and 20 virus species, 25 bacterial species and 25 virus species, 30 bacterial species and 30 virus species, 35 bacterial species and 35 virus species, 40 bacterial species and 40 virus species, 45 bacterial species and 45 virus species, 50 bacterial species and 50 virus species, 60 bacterial species and 60 virus species, 70 bacterial species and 70 virus species, 80 bacterial species and 80 virus species, 90 bacterial species and 90 virus species, 100 bacterial species and 100 virus species, 200 bacterial species and 200 virus species, 300 bacterial species and 300 Virus species, 400 bacterial species and 400 virus species, 500 bacterial species and 500 viruses A species, or at least one nucleic acid derived from 1000 bacterial species and 1000 virus species This further includes detection.

[0193] In some cases, the methods described herein are used to determine whether the infection is active or latent. This includes determining the following. In some cases, gene expression quantification can detect and predict active infections. , a method of diagnosis or monitoring may be provided. In some cases, the method described herein This includes detecting active infections. In some cases, it involves testing gene expression in the target population. It can be quantified through gene expression or sequencing. In some cases, gene expression quantification This may provide a method for detecting, predicting, diagnosing, or monitoring latent infections. In some cases, The methods described herein include detecting latent infectious diseases.

[0194] Representative diseases and disorders include any disease or disorder related to infection, such as sepsis. pneumonia, tuberculosis, HIV infection, hepatitis (e.g., hepatitis A, B, or C), human Papillomavirus (HPV) infection, Chlamydia infection, Syphilis infection, Ebola infection, Staphylococcus aureus infection or influenza Enza is included. The methods provided herein include drug-resistant microorganisms, including multidrug-resistant microorganisms. Particularly useful for detecting infections caused by [unspecified factor]. Some non-exclusive examples of diseases and disorders. Examples include Alzheimer's disease, amyotrophic lateral sclerosis, anorexia nervosa, anxiety disorders, asthma, Atherosclerosis, attention deficit hyperactivity disorder, autism, autoimmune diseases, bipolar disorder, cancer, chronic fatigue Syndrome, chronic obstructive pulmonary disease, Crohn's disease, coronary heart disease, dementia, depression, type 1 diabetes, Type 2 diabetes, dilated cardiomyopathy, epilepsy, Guillain-Barré syndrome, irritable bowel syndrome, lower back pain, Lupus, metabolic syndrome, multiple sclerosis, myocardial infarction, obesity, obsessive-compulsive disorder, panic Stroke, Parkinson's disease, psoriasis, rheumatoid arthritis, sarcoidosis, schizophrenia, stroke Thromboangiitis obliterans, Tourette's syndrome, vasculitis, plague, tuberculosis, anthrax, sleeping sickness, dysentery Toxoplasmosis, ringworm, candidiasis, histoplasmosis, Ebola, Acinetobacter Infectious diseases, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (acquired immunity) (Insufficiency syndrome), amoebic infection, anaplasmosis, anthrax, alkanobacterium hemorrhage Cam (Arcanobacterium haemolyticum) infection, Argentina Tsin hemorrhagic fever, roundworm infection, aspergillosis, astrovirus infection, babesiosis, cele Bacterial infections, bacterial pneumonia, bacterial vaginosis (BV), Bacteroides infections, Balantidote infections Symptoms, Bilithus calis infection, BK virus infection, black sand hair, human blastocystis Infections, blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, botulism (and infants) Botulism, Brazilian hemorrhagic fever, brucellosis, bubonic plague, Burkholderia infection Buruli ulcer, calicivirus infection (norovirus and sapovirus), Campylobacter Bacteriosis, candidiasis (moniliosis; thrush), cat scratch disease, cellulitis, Chagas. Diseases (American trypanosomiasis), chancroid, chickenpox, chikungunya fever, chlamydia, thyroiditis Chlamydophila pneumoniae infection (Chlamydophila pneumoniae) Taiwanese Acute Respiratory Syndrome (TWAR), cholera, black bacilli infection, liver fluke infection, Clostridium erythrorhizosis Dium difficile infection, coccidioidomycosis, Colorado tick fever (CTF), common cold ( Acute viral nasopharyngitis (acute coryza), Creutzfeldt-Jakob disease (CJD), Mia-Congo hemorrhagic fever (CCHF), cryptococcosis, cryptosporidiosis, cutaneous larvae Transient disease (CLM), cyclosporiasis, cysticercosis, cytomegalovirus infection, dengue fever, Dinuclear amebiasis, diphtheria, diphyllobothrium erythropoiesis, dung worm infection, Ebola hemorrhagic fever, echinococcus Ehrlichiosis, enteric pinworm infection, enterococcal infection, enterovirus Infections, typhus, erythema infectiosum (fifth disease), roseola (sixth disease), hypertrophic fluke infection, liver Leech disease, fatal familial insomnia (FFI), filariasis, Clostridium perfringens food poisoning, freedom Life-sustaining amoebic infection, Fusobacterium infection, gas gangrene (Clostridium myonecrosis) ), geotrichumosis, Gerstmann-Sträussler-Scheinker syndrome (GSS), Giardiasis, glanders, gnathostomiasis, gonorrhea, inguinal granuloma (Donovan's disease), Group A Streptococcus infection Diseases, Group B Streptococcal infection, Haemophilus influenzae infection, Hand, foot, and mouth disease (HFMD), Hantawi Russian pulmonary syndrome (HPS), Heartland virus disease, Helicobacter pylori (Heli cobacter pylori) infections, hemolytic uremic syndrome (HUS), and renal symptomatology. Blood fever (HFRS), hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex Histoplasmosis, hookworm infection, human bocavirus infection, human ehrlichiosis, human granuloma Granulocyte anaplasmosis (HGA), human metapneumovirus infection, human monocytic ehrlichrysum Alopecia, human papillomavirus (HPV) infection, human parainfluenza virus infection, Membranoid tapeworm infection, Epstein-Barr virus-induced mononucleosis (Mono), influenza (Cold), isosporiasis, Kawasaki disease, keratitis, Kingella ki NGAE infection, Kuru disease, Lassa fever, Legionnaires' disease, Legionnaires' disease (Pontiac fever), leishmaniasis, leprosy, leptospirosis, listeriosis Lyme disease (Lyme borreliosis), lymphangiofilariasis (elephantiasis), lymphocytic choriomeningitis Flammation, malaria, Marburg hemorrhagic fever (MHF), measles, Middle East respiratory syndrome (MERS), Melioidosis (Whitmore's disease), meningitis, meningococcal disease, Yokogawa's fluke infection, microsporidia, infectious Molluscum contagiosum (MC), monkeypox, mumps, typhus rothen (rash fever), mycoplasma pneumonia, mycomas Myiasis, neonatal conjunctivitis (neonatal ophthalmitis), (new) variant Creutzfeldt-Jakob disease (v CJD, nvCJD), nocardiosis, onchocerciasis (river blindness), paracoccidiopathies Idiasis (South American blastomycosis), lung fluke infection, pasteurellosis, head lice infestation (A (Body lice), body louse infestation (body lice), pubic louse infestation (pubic lice, pubic lice) Lami), pelvic inflammatory disease (PID), pertussis (whooping cough), plague, pneumococcal infection, ni Pneumocystis pneumonia (PCP), pneumonia, poliomyelitis, Prevotella infection, primary amoebic Meningoencephalitis (PAM), progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, RSV infection Infections, linosporidiasis, rhinovirus infection, rickettsial infection, rickettsial smallpox Rift Valley fever (RVF), Rocky Mountain rash fever (RMSF), rotavirus infection, Rubella, salmonellosis, SARS (Severe Acute Respiratory Syndrome), scabies, schistosomiasis, sepsis, Shigella infection (bacterial dysentery), shingles (herpes zoster), smallpox, sporotropic acid Cummosis, staphylococcal food poisoning, staphylococcal infection, strongyloidiasis, subacute sclerosing panencephalitis, syphilis, Tapeworm infection, tetanus (trismus), tinea folliculitis (barber's pruritus), tinea capitis (ringworm of the scalp), corpus Tinea (tinea corporis), tinea cruris (jock itch), tinea manuum (tinea manuum), tinea versicolor, tinea pedis (athlete's foot) Tystomatitis, onychomycosis (nail fungus), tinea versicolor (black catfish), toxocariasis (ocular larval migrans (O LM), Toxocariasis (Visceral Larval Migrans (VLM)), Trachoma, Trinocucliasis Trichomoniasis, trichomoniasis, whipworm disease (whipworm infection), tuberculosis, tularemia, typhus Ureaplasma urealyticum Infectious diseases, valley fever, Venezuelan encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile Fever, white sandy hair (ringworm), Yersinia pseudotuberculosis infection, Yersiniasis, yellow fever, and zygomycosis These are some examples.

[0195] As used herein, the term "or" means nonexclusive unless otherwise indicated. It is used to indicate that something is the same as, for example, "A or B" means "although A is the same as, This includes "not B", "B rather than A", and "A and B".

[0196] As used herein, the term “about” refers to a number or range of numbers. The mentioned numerical value or range of values ​​is an approximation within experimental variation (or within statistical experimental error). This means that the number or range of numbers is, for example, 1% of the stated number or range of numbers. It can vary by up to 15%. In the example, the term "approximately" means ±1% of the stated number or value. It indicates 0%.

[0197] [Examples] [Example 1] Preparation of cell-free RNA from patient whole blood samples Whole blood is taken from patients suspected of having an infectious disease, and dextrose acid citrate is used. Place the blood into an ACD (Aerochemical Collagen) blood collection tube. Take out a portion of the blood (1.5 mL) from the ACD blood collection tube. Place in a 1.5 mL microcentrifuge tube. Add 10 μL of standardized oligonucleotide to the blood, and Mix thoroughly. Centrifuge the mixture at 1600g for 10 minutes at 4°C, and remove the supernatant (normalized blood). Take out 550 μL of plasma and place it in a new microcentrifuge tube. Bring the standardized plasma to 4°C. Then, centrifuge at 16000g for 10 minutes. Remove the supernatant ("standardized cell-free plasma"), Transfer to a new tube and store at -80°C.

[0198] Thaw the standardized cell-free plasma at room temperature for 10 minutes. Thaw the standardized cell-free plasma at 4°C. Place at 16000g and centrifuge for 10 minutes to remove debris. Cell-free RNA is then processed by the manufacturer. Follow the instructions for Plasma / Serum Circulation and Exoso mal RNA Purification Kit(Slurry Format)( Isolate cell-free RNA using Norgen Biotek Corp. Store at °C.

[0199] [Example 2] Preparation of non-human oligonucleotide collections by computational design and synthesis The approximately 6.7 × 10⁶ possible different domains of a nucleotide have a length of 13 nucleotides. From this theoretical sequence pool, any 13 nucleotides found in human DNA If we discard the nucleotide sequence, we have approximately 2.3 × 10⁶ unique sequences of 13 nucleotides, i.e., all 3.5% of the body remains. From these 2.3 × 10⁶ non-human 13 nucleotide sequences The following criteria apply: 1) uniformity of melting temperature, 2) sufficient sequence complexity, and 3) known pathogens. Based on the abundance of binding sites in the body, 4) and the distribution of binding sites among known pathogens, non We select approximately 1 × 10⁶ sequences to include in the human sequence pool. A set of 13 nucleotide sequences, with an additional non-human sequence of 14-20 nucleotides in length. By adding this, the coverage of target areas such as strategic pathogen sequences is improved, and these p The factors considered for the mer are: 1) melting temperature, 2) sequence complexity, and 3) abundance of binding sites in known pathogens. 4) Design based on the distribution of binding sites between known pathogens. 13-20 nucleotide length. The entire pool of nucleotide domains is called the complete non-human sequence pool. Each 13-20 nucleotide sequence in the molecule contains one or more of the following nucleic acid labels. Adds an additional 5' sequence of approximately 15-25 nucleotides in length: 1) DNA sequencing pry 1) Mer binding site, 2) Sample barcode, 3) Sample barcode sequencing primer binding site 4) Amplified primer binding site that meets the requirements of various sequencing platforms. This collection of oligonucleotides is called a non-human primer pool. Human primer pools are chemically synthesized. Alternatively, a pool of completely non-human sequences is chemically synthesized. Then, one or more nucleic acid labels are added (e.g., by ligation) to non-human plastics. Form an immersion pool.

[0200] [Example 3] Non-human collection of oligonucleotides using hybridization-based methods Preparation Approximately 67 million different nucleotide sequences (for example, N is A, C, G, or T). The 5'-NNNNNNNNNNNNN-3') is labeled with one or more of the following nucleic acid labels. Chemically synthesized with a bonded 15-25 nucleotide overhang containing: 1) DNA 1) Sequencing primer binding site, 2) Sample barcode, 3) Sequencing of sample barcode 4) Amplified primer binding site and suitable for various sequencing platform requirements Lymer binding site. This heterogeneous collection of oligonucleotides is hybridized. Buffer solution (0.5 × PBS, 24 μM blocker oligonucleotide, RNase inhibitor) (The agent) contains 95% biotinylated human single-stranded genomic DNA (gDNA) fragments with a 1000-fold excess mass. Hive for a fixed amount of time, such as 10 seconds at ℃, 3 minutes at 65℃, and several hours to several weeks at 36℃. Reduce the size. At the end of the incubation period, add the human gDNA fragment to the fragment and then add the hives. These strips are removed by streptavidin beads along with the reduced probe. If a probe does not bind to human gDNA, add 14-20 nucleotides to the remaining pool. By supplementing the length of additional non-human sequences, the coverage of target regions such as strategic pathogen sequences is improved. To improve these primers, consider 1) melting temperature, 2) sequence complexity, and 3) known pathogens. The design is based on the abundance of binding sites and 4) the distribution of binding sites among known pathogens. The collection of oligonucleotides is called a non-human primer pool.

[0201] [Example 4] Preparation of non-human collections of highly degenerate oligonucleotides in high yield. Non-human 13mers were used to generate all possible 13mer sequences and then placed into the reference human genome. It is determined computationally by removing what appears. Then, the non-human 13mer is degenerate. Classified by degree. The histogram in Figure 11B shows the non-human 13mer based on degeneracy. This shows ketization.

[0202] Next, we use the same degenerate non-human 13-mer variable oligonucleotide sequence units in ultrama - Classified as an oligonucleotide. Degenerate oligonucleotide sequence contained in ultramer The number of units can be based on the length of each unit and the ultramer length that can be reliably synthesized. Figure 9 shows some common designs of such ultramer oligonucleotides. Each degenerate 13mer can be separated by deoxyuracil nucleotide (U). Next, the designed ultramer oligonucleotides are synthesized using conventional nucleic acid synthesis methods and It can be synthesized by a service provider, such as IDT.

[0203] Next, the ultramer oligonucleotide is subjected to uracil-DNA glycosylase (UD G) and specificity at the debase site (e.g., aprine / apyrimidine site or AP site) Individual degenerate non-human 13-mer oligonucleotides are extracted by an endonuclease possessing the properties of a certain type. This digestion can be performed on all degenerate 13mers having the same degeneracy. The ultramer oligonucleotide can be tested in the same tube. Typical reactions The response is shown in Figure 10.

[0204] Next, each degenerate 13mer is transformed into, for example, T4 polynucleotide kinase (T4 PN It can be biotinylated using K) or chemically as shown in Figure 12. This step is optional, for example, if surface immobilization or purification of magnetic beads is required. Other probes besides biotin (e.g., digoxigenin, fluorescent probes, etc.) can be used here. It can be applied.

[0205] [Example 5] Preparation of enriched sequencing libraries using non-human oligonucleotide collections Cell-free RNA from Example 1 was processed in hybridization buffer (0.5×PBS, 2 4 μM blocker oligonucleotide (RNase inhibitor), 65°C for 10 seconds Refrigerate the non-human primer pool (as described in Example 2 or 3) at °C for 3 minutes and overnight at 36 °C. (Prepared to be used) and hybridized. First strand cDNA synthesis master mix (reversed) Regeneration enzyme, blocked second-chain synthesis oligonucleotide, dNTP, RNase inhibitor Add (and a suitable buffer) at 36°C, then raise the temperature to 42°C for 90 minutes. This enables the synthesis of the first strand of cDNA. The mixture is then incubated at 70°C for 10 minutes. The reverse transcriptase is then inactivated, and the mixture is kept at 4°C.

[0206] The first strand cDNA product is purified using a commercially available kit. The second strand cDNA is purified using the previous method. During the step, the fixed sequences attached to the 5' and 3' ends of the first strand of cDNA are hybridized. It is generated by PCR using primers that isolate the strand. The following nucleic acid labels may be added by further rounds of PCR. Possible: 1) DNA sequencing primer binding site, 2) Sample barcode, 3) Sample Rubercode sequencing primer binding sites, and 4) various sequencing platform requirements The amplification primer binding site must be suitable for the application. The final cDNA library must be prepared according to the manufacturer's instructions. Purification is performed using a commercially available kit according to the instructions.

[0207] [Example 6] DNA or cDNA libraries using non-human oligonucleotide collections Preparation of enriched sequencing libraries for non-human sequences. Prepare a sequencing-ready library. Use a non-human primer pool in Example 2 or 3. Prepare as described and promote 5' biotinylation of individual oligonucleotides in the pool. The conditions for advancement are applied. Then, 1 pmol of 5'-biotinylated non-human primer pool is added. Hybridization / polymerization buffer (buffer, nucleotide, blocker oligonucleotide) Add to a sequence-determinable library of 500ng (Ochid). In some cases, DN A. Oligonucleotypes with molecules that enhance the rate or specificity of hybridization reactions. Pre-incubation of non-host nucleic acid fragments such as RecA or MutS Capture can be improved. Denaturate the DNA at 95°C for 10 minutes. Non-human primers. Hybridize the 5' exonuclear to a sequencing library at 50°C for 4 hours. Add the strand-substituting DNA polymerase lacking the enzyme to the mixture. Incubate the mixture at 55°C for 15 minutes. Cuvate and add non-human primers of at least 25 bases, or in subsequent steps To extend the double-stranded DNA to a length sufficient for stable double-stranded DNA hybridization. Bijin beads are added to the mixture to bind DNA fragments. Captured DNA on the beads. Wash the fragments. The captured library DNA fragments are then subjected to DNA sequencing library decomposition. A specific primer and a DNA fragment captured on a bead are attached to the adapter at the end of one end. It is used as a template and amplified using a standard PCR amplification method. The enriched library... This is purified using standard DNA purification methods.

[0208] The library was collected using the KAPA DNA Library Quantification Kit (KAPA Biosy). Quantitative analysis was performed using stems, and NextSeq 500(Il) according to the manufacturer's instructions. Prepare for sequencing using Lumina. Sequencing is performed using 150 cycles of single-end readings. It consists of a manual selection process and 8-cycle barcode reading.

[0209] The sequence readings are computationally mapped to the genome of the pathogen, and one or more nucleic acids Identify one or more sources.

[0210] [Example 7] Human nucleosome association using anti-human histone antibodies and anti-immunoglobulin antibodies D NA depletion Cell-free DNA is obtained by either centrifugation or albumin and immunoglobulin removal. It is prepared by the following method. In either method, first, 1 mL of human plasma is left at 4°C for 16 minutes. Centrifuge at 00g for 10 minutes. In the case of centrifugation, collect the supernatant (950μL) and purify. Transfer to a test tube and centrifuge again at 16000g for 10 minutes at 4°C. Cell-free DN Collect 900 μL of the supernatant containing A and transfer it to a new test tube. Albumin and immunoglobulin In the brin removal method, the supernatant (950 μL) from the initial centrifugation is collected, and human albumin is added. and human immunoglobulin-binding columns (e.g., Alb from GE Healthcare) umin and I...

Claims

1. 1. A method for priming or capturing a sequence in a nucleic acid sample from a host, comprising: (a) providing a cell-free nucleic acid (cfNA) sample from said host; (b) mixing the nucleic acid sample from the host with nucleic acids of one or more regions of interest specific to one or more pathogenicity loci; antimicrobial resistance markers; antibiotic resistance markers; antiviral resistance markers; antiparasitic resistance markers; informative genotyping regions; sequences common to two or more microorganisms, pathogens, bacteria, viruses, fungi, or parasites; non-host sequences integrated into the host genome; masking non-host sequences; non-host mimicking sequences; masking host sequences; host mimicking sequences; and sequences specific to one or more microorganisms, pathogens, bacteria, viruses, fungi, or parasites, thereby obtaining a mixture; (c) contacting nucleic acids in the one or more regions of interest with the nucleic acid sample in the mixture, wherein said contacting causes nucleic acids in the nucleic acid sample to bind to nucleic acids in the one or more regions of interest, thereby priming or capturing said nucleic acids; A method comprising:

2. 10. The method of claim 1, further comprising performing a nucleic acid amplification reaction using one or more nucleic acids specific to one or more pathogenicity loci; antimicrobial resistance markers; antibiotic resistance markers; antiviral resistance markers; antiparasitic resistance markers; informative genotyping regions; sequences common to two or more microorganisms, pathogens, bacteria, viruses, fungi, or parasites; non-host sequences integrated into the host genome; masking non-host sequences; non-host mimicking sequences; masking host sequences; host mimicking sequences; and sequences specific to one or more microorganisms, pathogens, bacteria, viruses, fungi, or parasites.

3. 3. The method of claim 2, wherein the nucleic acid amplification reaction comprises a polymerase chain reaction, reverse transcription, transcription-mediated amplification, or ligase chain reaction.

4. 10. The method of claim 1, further comprising isolating nucleic acids specific to one or more pathogenicity loci; antimicrobial resistance markers; antibiotic resistance markers; antiviral resistance markers; antiparasitic resistance markers; informative genotyping regions; sequences common to two or more microorganisms, pathogens, bacteria, viruses, fungi, or parasites; non-host sequences integrated into the host genome; masking non-host sequences; non-host mimicking sequences; masking host sequences; host mimicking sequences; and sequences specific to one or more microorganisms, pathogens, bacteria, viruses, fungi, or parasites.

5. The method of claim 1 , further comprising performing a sequencing assay.

6. The method of claim 5, wherein the sequencing assay is a high-throughput sequencing assay.

7. The method of claim 1 , wherein the host is a human.

8. 2. The method of claim 1, wherein the cfNA is cell-free DNA.

9. 10. The method of claim 1, wherein the sample is selected from the group consisting of whole blood, plasma, serum, mucus, saliva, cerebrospinal fluid, synovial fluid, lavage fluid, urine, and stool.

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

11. 2. The method of claim 1, wherein the antibiotic resistance marker comprises any of bl2be ctxm, b12 kpc, b13 imp, b13 vim, meca, mecr1 vana, vanb, and combinations thereof.

12. 10. The method of claim 1, wherein the one or more antibiotic resistance markers comprises meca.

13. 2. The method of claim 1, wherein the one or more antibiotic resistance markers comprises mecr1.

14. 1. A method for enriching cell-free microbial non-host nucleic acid sequences in a nucleic acid sample from a host, comprising: (a) providing a nucleic acid sample from a host, the nucleic acid sample from the host comprising non-living host nucleic acids and non-living microbial non-host nucleic acids associated with nucleosomes, the sample being selected from the group consisting of blood, plasma, serum, saliva, cerebrospinal fluid, synovial fluid, and lavage fluid; (b) removing at least a portion of the non-living host nucleic acid associated with nucleosomes, thereby enriching the cell-free microbial non-host nucleic acid in the nucleic acid sample from the host. The method comprising:

15. 15. The method of claim 14, further comprising performing a sequencing assay.

16. 15. The method of claim 14, wherein the nucleic acid sample from the host comprises at least five microbial non-host nucleic acid sequences, and further comprising detecting the at least five microbial non-host nucleic acid sequences.

17. The method of claim 14, wherein the host is a human.

18. The method of claim 14 , wherein the host nucleic acid is associated with a human nucleosome.

19. 15. The method of claim 14, wherein the removing in step (b) comprises performing electrophoresis or isotachophoresis.

20. 15. The method of claim 14, wherein the removing in step (b) comprises using a porous filter or an ion exchange column.

21. 15. The method of claim 14, wherein the removal in step (b) comprises using one or more antibodies specific for one or more histones.

22. 22. The method of claim 21, wherein the one or more histones are selected from the group consisting of a histone H2A N-terminus, a histone H2A solvent-exposed epitope, a monomethylation on Lys9 in histone H3, a dimethylation on Lys9 in histone H3, a trimethylation on Lys56 in histone H3, a phosphorylation on Ser14 in histone H2B, and a phosphorylation on Ser139 in histone H2A.X.

23. 22. The method of claim 21, wherein the one or more antibodies are immobilized on a column.

24. 22. The method of claim 21, further comprising removing one or more antibodies.

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