Methods and compositions for copy number information-based tissue origin analysis

Abstract

Provided herein is a method of analyzing nucleic acid to detect DNA containing cell type or tissue type specific epigenetic variable regions, such as differentially methylated regions that are also copy number variants.Also provided herein is a method for determining the likelihood that a subject has a disease or condition, such as cancer.The method includes capturing at least a set of epigenetic target regions of DNA from a blood sample or a subsample thereof, contacting the DNA with a plurality of target-specific probes specific to members of the set of epigenetic target regions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 213,050, filed June 21, 2021, which is incorporated by reference herein in its entirety for all purposes.

[0002] FIELD OF THEINVENTION The present disclosure provides methods and compositions for the analysis of nucleic acid such as DNA.In some embodiments, the nucleic acid is derived from a subject who has or is suspected to have a disease or disorder such as cancer.In some embodiments, the nucleic acid includes nucleic acid derived from cancer cells.In some embodiments, the nucleic acid includes cell type or tissue type specific differential methylation regions or fragments and copy number variants. [Background technology]

[0003] Introduction and Abstract Cancer causes millions of deaths annually worldwide. Early detection of cancer can result in improved outcomes, as early cancers tend to be more susceptible to treatment.

[0004] Inappropriate control of cell proliferation is a hallmark of cancer. Cancer is usually caused by the accumulation of mutations in an individual's normal cells, at least some of which result in inappropriate control of cell division. Such mutations generally include single nucleotide variants (SNVs), gene fusions, insertions and deletions (indels), transversions, translocations, and inversions. Cancer may also exhibit the accumulation of epigenetic changes, including modifications of cytosine (e.g., 5-methylcytosine, 5-hydroxymethylcytosine, and other more oxidized forms) and the association of chromatin proteins and transcription factors with DNA.

[0005] Biopsy represents a traditional approach to detect or diagnose cancer, in which cells or tissues are extracted from a potential cancer site and analyzed for associated phenotypic and / or genotypic characteristics. Biopsy has the drawback of being invasive. Detection of diseases and disorders based on the analysis of bodily fluids such as blood ("liquid biopsy") is an attractive alternative. Liquid biopsy is non-invasive and in some cases requires only blood sampling. However, developing accurate and sensitive methods for analyzing liquid biopsy material, including proteins, is challenging, in part due to the low and variable amounts of nucleic acids released into bodily fluids, and recovering nucleic acids from such fluids in an analyzable form is similarly challenging. These sources of variability can obscure the predictive value of mutations (e.g., reassortments such as translocations and indels) between samples. Such mutations can include biomarkers that can be used to assess whether a subject diagnosed with cancer or suspected to have signs of cancer will benefit from a particular type of cancer therapy, such as immuno-oncological (IO) therapy. Isolation and processing of cell-free DNA useful for further analysis in liquid biopsy procedures is an important part of such methods.Therefore, there is a need for improved methods and compositions for analyzing cell-free DNA in, for example, liquid biopsies. Summary of the Invention [Means for solving the problem]

[0006] The purpose of the present disclosure is to meet the need for improving the sensitivity of DNA analysis, such as the analysis of cfDNA derived from tumor cells.Thus, the method herein may include a step that can provide information about DNA mutations and modifications, including but not limited to epigenetic mutations, copy number mutations, and sequence mutations in cfDNA.Such a method that includes DNA analysis can provide even more improved information about the possibility of a particular disease state of a subject.Improved detection of cancer markers in blood allows for more accurate detection (diagnosis) of disorders, and therefore allows for improved treatment.Thus, the following exemplary embodiments are provided:

[0007] Embodiment 1 is a method for analyzing DNA in a blood sample, comprising: a) capturing at least a set of epigenetic target regions of DNA from a blood sample or subsample thereof, comprising contacting the DNA with a plurality of target-specific probes specific for members of the set of epigenetic target regions; the set of epigenetic target regions includes a plurality of type-specific epigenetic target regions that are copy number variants, the type-specific epigenetic target regions being type-specific differentially methylated regions and / or type-specific fragments, thereby providing captured DNA; and Sequencing the captured DNA to determine the levels of type-specific epigenetic target regions The method includes:

[0008] Embodiment 2 is the method of embodiment 1, wherein the type-specific epigenetic target region is a type-specific differentially methylated region.

[0009] Embodiment 3 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises type-specific hypermethylated regions.

[0010] Embodiment 4 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises type-specific hypomethylated regions.

[0011] Embodiment 5 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises target regions that are hypermethylated in immune cells relative to non-immune cell types present in the blood sample.

[0012] Embodiment 6 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises target regions that are differentially methylated in the colon compared to other tissue types.

[0013] Embodiment 7 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises target regions that are differentially methylated in lung compared to other tissue types.

[0014] Embodiment 8 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises target regions that are differentially methylated in breast compared to other tissue types.

[0015] Embodiment 9 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises target regions that are differentially methylated in liver compared to other tissue types.

[0016] Embodiment 10 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises target regions that are differentially methylated in kidney compared to other tissue types.

[0017] Embodiment 11 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises target regions that are differentially methylated in the pancreas relative to other tissue types.

[0018] Embodiment 12 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises target regions that are differentially methylated in prostate compared to other tissue types.

[0019] Embodiment 13 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises target regions that are differentially methylated in skin compared to other tissue types.

[0020] Embodiment 14 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises target regions that are differentially methylated in bladder compared to other tissue types.

[0021] Embodiment 15 is a method according to any one of the preceding embodiments, wherein the hypermethylated target region is methylated to an extent that is at least 10%, 20%, 30%, or at least 40% higher than the average methylation of the target region in the sample.

[0022] Embodiment 16 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises target regions that are hypomethylated in non-immune blood cells compared to the methylation levels of the target regions in a different cell type or tissue type in the sample.

[0023] Embodiment 17 is the method of any one of the preceding embodiments, wherein the type-specific epigenetic target region comprises a type-specific fragment. Embodiment 18 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises fragments specific for immune cells relative to non-immune cell types present in the blood sample.

[0024] Embodiment 19 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises fragments specific for the colon relative to other tissue types.

[0025] Embodiment 20 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises fragments specific for the lung relative to other tissue types.

[0026] Embodiment 21 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises fragments specific for breast relative to other tissue types.

[0027] Embodiment 22 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises fragments specific for liver relative to other tissue types.

[0028] Embodiment 23 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises fragments specific for the kidney relative to other tissue types.

[0029] Embodiment 24 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises fragments specific for the pancreas relative to other tissue types.

[0030] Embodiment 25 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises fragments specific for prostate relative to other tissue types.

[0031] Embodiment 26 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises fragments specific for skin relative to other tissue types.

[0032] Embodiment 27 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises fragments specific for bladder relative to other tissue types.

[0033]

[0046] Embodiment 28 is the method of any one of the preceding embodiments, comprising identifying at least one cell type or tissue type that is the source of the type-specific epigenetic target region.

[0034] Embodiment 29 is the method of the immediately preceding embodiment, wherein the level of a type-specific epigenetic target region originating from a cell type or tissue type is determined.

[0035] Embodiment 30 is a method as in the immediately preceding embodiment, wherein the level of a type-specific epigenetic target region originating from an immune cell, a non-immune blood cell, colon, lung, breast, liver, kidney, prostate, skin, bladder, or pancreas is determined.

[0036] Embodiment 31 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises copy number variants having abnormally high copy numbers.

[0037] Embodiment 32 is the method of any one of the preceding embodiments, wherein the plurality of type-specific epigenetic target regions comprises at least one copy number variant that comprises a duplication.

[0038] Embodiment 33 is the method of any one of the preceding embodiments, wherein the blood sample is a plasma sample.

[0039] Embodiment 34 is the method according to any one of embodiments 1 to 32, wherein the blood sample is a whole blood sample.

[0040] Embodiment 35 is a method according to any one of the preceding embodiments, wherein the blood sample is fractionated prior to capturing at least the set of epigenetic target regions of DNA.

[0041] Embodiment 36 is the method of any one of the preceding embodiments, wherein the DNA is cfDNA.

[0042] Embodiment 36.1 is a method according to any one of embodiments 35 to 36, wherein the analysis of the DNA includes quantifying at least one epigenetic feature of a target region of the DNA, and optionally, the epigenetic feature includes methylation.

[0043] Embodiment 36.2 is the method of any one of embodiments 35-36, wherein analyzing the DNA comprises detecting or quantifying one or more genetic variants in one or more target regions of the DNA.

[0044] Embodiment 37 is the method of any one of the preceding embodiments, comprising partitioning the DNA into a plurality of aliquots by contacting the DNA with an agent that recognizes methylcytosines in the DNA, the plurality comprising a first aliquot and a second aliquot, the first aliquot comprising DNA with a higher proportion of methylcytosines than the second aliquot.

[0045] Embodiment 38 is a method according to the immediately preceding embodiment, wherein dispensing is performed before capture.

[0046] Embodiment 39 is the method according to the immediately preceding embodiment, wherein partitioning is performed after capture and before sequencing.

[0047] Embodiment 40 is the method of any one of embodiments 37 to 39, wherein the agent that recognizes methylcytosine is a methyl-binding agent.

[0048] Embodiment 41 is the method of the immediately preceding embodiment, wherein the methyl-binding reagent is an antibody.

[0049] Embodiment 42 is the method according to embodiments 40-41, wherein the methyl-binding reagent specifically recognizes 5-methylcytosine.

[0050] Embodiment 43 is the method according to embodiments 40 to 42, wherein the methyl-binding reagent is immobilized on a solid support.

[0051] Embodiment 44 is the method of any one of the preceding embodiments, wherein the partitioning comprises immunoprecipitation of methylated DNA.

[0052] Embodiment 45 is the method of any one of embodiments 37 to 44, wherein the partitioning comprises partitioning based on binding to a protein, and optionally the protein is a methylated protein, an acetylated protein, a non-methylated protein, a non-acetylated protein, and / or optionally the protein is a histone.

[0053] Embodiment 46 is the method of the immediately preceding embodiment, wherein the partitioning comprises contacting DNA of the sample with a binding reagent that is specific for the protein and that is immobilized on a solid support.

[0054] Embodiment 47 is the method of any one of the preceding embodiments, comprising contacting the DNA with at least one methylation-sensitive restriction enzyme (MSRE) and / or at least one methylation-dependent restriction enzyme (MDRE).

[0055] Embodiment 48 is a method according to any one of the preceding embodiments, comprising subjecting the sample or one or more subsamples to a procedure that affects a first nucleobase of the DNA differently than a second nucleobase.

[0056] Embodiment 49 is a method according to any one of the preceding embodiments, comprising determining the methylation level of the type-specific differentially methylated target region.

[0057] Embodiment 50 is a method according to any one of the preceding embodiments, wherein the set of epigenetic target regions comprises a CTCF binding site and / or a transcription start site.

[0058] Embodiment 51 is the method of any one of the preceding embodiments, wherein capturing comprises capturing a sequence-variable target region of DNA and contacting the DNA with a plurality of target-specific probes specific for the sequence-variable target region.

[0059] Embodiment 52 is a method according to any one of the preceding embodiments, comprising ligating an adaptor to the DNA, thereby generating an adaptor-ligated DNA.

[0060] Embodiment 53 is the method of the immediately preceding embodiment, wherein the adaptor-ligated DNA is amplified prior to sequencing.

[0061] Embodiment 54 is the method according to any one of embodiments 37 to 53, wherein the aliquots are pooled prior to sequencing.

[0062] Embodiment 55 is the method of any one of the preceding embodiments, wherein the sample is obtained from a subject.

[0063] Embodiment 56 is a method according to the immediately preceding embodiment, comprising determining the likelihood that the subject has cancer or precancer.

[0064] Embodiment 57 is a method according to the immediately preceding embodiment, comprising determining the likelihood that the subject has cancer.

[0065] Embodiment 58 is the method of the immediately preceding embodiment, wherein the cancer is a cancer of the cell or tissue type from which the target region originates.

[0066] Embodiment 59 is a method according to any one of embodiments 56 to 58, wherein the cancer is a cancer of the cell or tissue type that is the origin of the target region at a higher level than the level present in a sample obtained from a healthy subject.

[0067] Embodiment 60 is the method of the immediately preceding embodiment, wherein the cancer is a lymphocytic cancer.

[0068] Embodiment 61 is the method of the immediately preceding embodiment, wherein the cancer is leukemia, lymphoma, or myeloma.

[0069] Embodiment 62 is the method of any one of embodiments 56 to 59, wherein the cancer is a bone marrow cancer.

[0070] Embodiment 63 is a method according to any one of embodiments 56 to 59, wherein the cancer is colorectal cancer, lung cancer, breast cancer, prostate cancer, skin cancer, gastric cancer, pancreatic cancer, bladder cancer, or kidney cancer.

[0071] Embodiment 64 is the method of embodiment 56, comprising determining the likelihood that the subject has precancer.

[0072] Embodiment 65 is the method of the immediately preceding embodiment, wherein the precancer is an adenoma.

[0073] Embodiment 66 is the method of the immediately preceding embodiment, wherein the adenoma is an advanced adenoma.

[0074] Embodiment 67 is the method of any one of embodiments 64-66, wherein the precancer is colorectal precancer, lung precancer, breast precancer, prostate precancer, skin precancer, gastric precancer, pancreatic precancer, bladder precancer, or renal precancer.

[0075]

[0046] Embodiment 68 is the method of any one of the preceding embodiments, wherein the sequencing comprises generating a plurality of sequencing reads, and the method further comprises mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads, and processing the mapped sequence reads to determine the likelihood that the subject has cancer or precancer.

[0076] Embodiment 69 is the method of any one of the preceding embodiments, wherein the sample is obtained from a subject previously diagnosed with cancer and who has undergone one or more prior cancer treatments, and optionally, the sample is obtained at one or more preselected time points after the one or more prior cancer treatments.

[0077] Embodiment 70 is the method of the immediately preceding embodiment, further comprising determining a cancer recurrence score, optionally wherein the subject's cancer recurrence status is determined to be at risk of cancer recurrence if the Cancer Recurrence Score is determined to be at or above a predetermined threshold, or the subject's cancer recurrence status is determined to be at lower risk of cancer recurrence if the Cancer Recurrence Score is below the predetermined threshold.

[0078] Embodiment 71 is the method of the immediately preceding embodiment, further comprising comparing the subject's cancer recurrence score to a predetermined cancer recurrence threshold, and the subject is classified as being a candidate for subsequent cancer treatment if the cancer recurrence score is above the cancer recurrence threshold, or as not being a candidate for subsequent cancer treatment if the cancer recurrence score is below the cancer recurrence threshold.

[0079] Embodiment 72 is a method for screening for cancer, comprising carrying out the method of any one of embodiments 1 to 71 on samples from multiple subjects, wherein the presence or level of at least one epigenetic target region indicates that the corresponding subject may have cancer.

[0080] Embodiment 73 is a method for monitoring residual cancer or detecting the presence or absence of recurrent cancer, comprising carrying out a method according to any one of embodiments 1 to 71, wherein the presence or level of at least one epigenetic target region indicates cancer status or the presence or absence of recurrent cancer.

[0081] Embodiment 74 is a method for identifying a therapy for treating a disease, optionally wherein the disease is cancer, the method comprising carrying out a method according to any one of embodiments 1 to 71, wherein the presence or level of at least one epigenetic target region indicates a suitable therapy for treating the disease.

[0082] Embodiment 75 is a method for partitioning a sample into a plurality of aliquots, including a first aliquot and a second aliquot, the first aliquot having a higher proportion of DNA with cytosine modifications than the second aliquot; contacting the second aliquot with a methylation-dependent nuclease, thereby degrading non-specifically distributed DNA in the second aliquot to generate a treated second aliquot, and optionally contacting the first aliquot with a methylation-sensitive endonuclease, thereby degrading non-specifically distributed DNA in the first aliquot to generate a treated first aliquot; Capturing a first set of target regions comprising epigenetic target regions from the first subsample or at least a portion of the processed first subsample. 13. The method of any one of the preceding embodiments, further comprising:

[0083] Embodiment 76 is a method for analyzing DNA in a sample, comprising the steps of: a) capturing at least a set of epigenetic target regions of DNA from a sample, the capturing comprising contacting the DNA with a plurality of target specific probes specific for members of the set of epigenetic target regions, the set of epigenetic target regions comprising a plurality of type-specific epigenetic target regions that are copy number variants, the type-specific epigenetic target regions being type-specific differentially methylated regions and / or type-specific fragments, thereby providing captured DNA; b) partitioning the sample into a plurality of aliquots, including a first aliquot and a second aliquot, the first aliquot having a higher proportion of DNA with cytosine modifications than the second aliquot; c) contacting the second aliquot with a methylation-dependent nuclease, thereby degrading non-specifically distributed DNA in the second aliquot to generate a treated second aliquot, and optionally contacting the first aliquot with a methylation-sensitive endonuclease, thereby degrading non-specifically distributed DNA in the first aliquot to generate a treated first aliquot; and d) capturing a first set of target regions comprising epigenetic target regions from the first subsample or at least a portion of the processed first subsample. The method includes:

[0084] Embodiment 77 is the method of the immediately preceding embodiment, wherein the cytosine modification is methylation.

[0085] Embodiment 78 is the method according to any one of embodiments 75 to 77, wherein the cytosine modification is methylation at the 5-position of cytosine.

[0086] Embodiment 79 is the method of any one of embodiments 75 to 78, wherein the first aliquot is contacted with a methylation-sensitive endonuclease.

[0087] Embodiment 80 is the method of the immediately preceding embodiment, wherein the methylation-sensitive endonuclease cleaves unmethylated CpG sequences.

[0088] Embodiment 81 is a method according to any one of embodiments 75 to 80, wherein the methylation-sensitive endonuclease is one or more of AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, and SnaBI.

[0089] Embodiment 82 is the method of the immediately preceding embodiment, wherein the methylation-sensitive endonuclease is one or more of BstUI, HpaII, Hin6I, HhaI, or AccII, optionally wherein the methylation-sensitive endonuclease is (i) BstUI and HpaII, (ii) BstUI, HpaII, and Hin6I, or (iii) HhaI and AccII.

[0090] Embodiment 83 is the method of any one of embodiments 75 to 82, wherein the methylation-dependent endonuclease cleaves a methylated CpG sequence.

[0091] Embodiment 84 is the method of any one of embodiments 75 to 83, wherein the methylation-dependent endonuclease is one or more of MspJI, LpnPI, FspEI, or McrBC.

[0092] Embodiment 85 is the method of any one of embodiments 75 to 84, wherein the first partial sample is subjected to a procedure that affects a first nucleobase in the DNA differently than a second nucleobase in the DNA of the first partial sample, the first nucleobase being a modified or unmodified nucleobase, the second nucleobase being a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase having the same base pairing specificity.

[0093] Embodiment 86 is the method according to the immediately preceding embodiment, wherein the procedure in which the first aliquot is provided alters the base-pairing specificity of the first nucleobase without substantially altering the base-pairing specificity of the second nucleobase.

[0094] Embodiment 87 is the method of embodiment 85 or 86, wherein the first nucleobase is a modified or unmodified cytosine and the second nucleobase is a modified or unmodified cytosine.

[0095] Embodiment 88 is the method of any one of embodiments 85 to 87, wherein the first nucleobase comprises an unmodified cytosine (C).

[0096] Embodiment 89 is the method of any one of embodiments 85 to 88, wherein the second nucleobase comprises 5-methylcytosine (mC).

[0097] Embodiment 90 is the method of any one of embodiments 85 to 89, wherein the procedure by which the first aliquot is provided comprises bisulfite conversion.

[0098] Embodiment 91 is the method of any one of embodiments 85 to 87, wherein the first nucleobase comprises mC.

[0099] Embodiment 92 is the method of any one of embodiments 85 to 89, wherein the second nucleobase comprises 5-hydroxymethylcytosine (hmC).

[0100] Embodiment 93 is the method of embodiment 89, wherein the procedure in which the first aliquot is subjected comprises protection of 5hmC.

[0101] Embodiment 94 is the method of embodiment 92, wherein the procedure to which the first aliquot is subjected comprises Tet-assisted bisulfite conversion.

[0102] Embodiment 95 is the method of embodiment 92, wherein the procedure to which the first aliquot is subjected comprises a Tet-assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.

[0103] Embodiment 96 is the method of embodiment 95, wherein the substituted borane reducing agent is 2-picoline borane or borane pyridine.

[0104] Embodiment 97 is the method of any one of embodiments 85-87, 91-93, or 95-96, wherein the second nucleobase comprises C.

[0105] Embodiment 98 is the method of any one of embodiments 91-93 or 97, wherein the procedure to which the first aliquot is subjected comprises protection of hmC followed by Tet-assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.

[0106] Embodiment 99 is the method of embodiment 98, wherein the substituted borane reducing agent is 2-picoline borane or borane pyridine.

[0107] Embodiment 100 is a method according to any one of embodiments 88, 89, 91 to 93, or 97, wherein the procedure to which the first aliquot sample is subjected comprises protection of hmC, followed by deamination of mC and / or C.

[0108] Embodiment 101 is the method of embodiment 100, wherein deamination of mC and / or C comprises treatment with an AID / APOBEC family DNA deaminase enzyme.

[0109] Embodiment 102 is a method according to any one of embodiments 93 or 97 to 101, wherein the protection of hmC comprises glycosylation of hmC.

[0110] Embodiment 103 is the method of any one of embodiments 85 to 87, 89, 91, or 97, wherein the step of subjecting the first aliquot sample includes a chemically assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.

[0111] Embodiment 104 is the method of embodiment 103, wherein the substituted borane reducing agent is 2-picoline borane or borane pyridine.

[0112] Embodiment 105 is the method of any one of embodiments 95-97, 89, 91, 97, or 103-104, wherein the first nucleobase comprises hmC.

[0113] Embodiment 105.1 is a method according to any one of embodiments 85 to 105, wherein the plurality of target-specific probes specific for members of the set of epigenetic target regions comprises at least a portion of the probe specific for a modification state of at least one base in the sequence to which the probe hybridizes, and optionally, the modification state is a modified (e.g., methylated) state, a converted modified (e.g., methylated) state, an unmodified state, or an unconverted state.

[0114] Embodiment 105.2 is a method according to any one of embodiments 85 to 105, wherein a plurality of target-specific probes specific to members of the set of epigenetic target regions comprises at least a portion of the probe capable of hybridizing to both sequences comprising converted bases at positions that may be modified and sequences comprising unconverted bases at positions that may be modified, and optionally at least a portion of the probe comprises bases that hybridize indiscriminately to positions that are complementary to the positions that may be modified.

[0115]

[0041] Embodiment 106 is the method of any one of the preceding embodiments, further comprising partitioning the sample into a plurality of aliquots, including a first aliquot and a second aliquot, wherein the DNA of the first aliquot and the DNA of the second aliquot are differentially tagged; after differential tagging, a portion of the DNA from the second aliquot is added to the first aliquot or the processed first aliquot, or at least a portion thereof, thereby forming a pool, and the sequence variable target regions and the epigenetic target regions are captured from the pool.

[0116] Embodiment 107 is the method of the immediately preceding embodiment, wherein the pool comprises about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less of the DNA of the second aliquot sample.

[0117] Embodiment 108 is the method of the immediately preceding embodiment, wherein the pool comprises about 70-90%, about 75-85%, or about 80% of the DNA of the second aliquot sample.

[0118] Embodiment 109 is the method according to any one of embodiments 106 to 108, wherein the pool comprises substantially all of the DNA of the first subsample.

[0119] Embodiment 110 is the method according to any one of embodiments 106 to 109, wherein the pool comprises substantially all of the DNA of the first aliquot or the processed first aliquot.

[0120] Embodiment 111 is a method according to any one of embodiments 106 to 110, wherein the first set of target regions is captured from the first partial sample or at least a portion of the processed first partial sample after formation of the pool. [Brief description of the drawings]

[0121] [Figure 1] FIG. 1 is a schematic diagram of one example of a system suitable for use in some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0122] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Reference will now be made in detail to certain specific embodiments of the invention. While the invention will be described in conjunction with such embodiments, it will be understood that it is not intended to limit the invention to such embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the invention as defined by the appended claims.

[0123] Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps, which may vary as such.As used herein and in the appended claims, it should be noted that the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.Thus, for example, a reference to "nucleic acid" includes a plurality of nucleic acids, a reference to "cell" includes a plurality of cells, and so on.

[0124] Numeric ranges are inclusive of the numbers defining the range. Measurements and measurable values ​​are understood to be approximations taking into account significant digits and error associated with measurement. Additionally, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" are not intended to be limiting. It is to be understood that both the foregoing general and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0125] Unless otherwise noted in the specification above, embodiments herein that are described as "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the described components, and embodiments herein that are described as "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the described components, and embodiments herein that are described as "consisting essentially of" various components are also contemplated as "consisting of" or "comprising" the described components (this synonymy does not apply to the use of such terms in the claims).

[0126] The section headings used herein are for organizational purposes and should not be construed as limiting the subject matter of this disclosure in any way. In the event that any document or other material incorporated by reference conflicts with any express content of this specification, including definitions, the present specification shall control. I. Definition

[0127] "Solid tissue" or "solid tissue cells" as used herein refer to tissues or cells, respectively, in or derived from solid tissue. Solid tissue cells exclude circulating cell types, such as cells normally found in blood or lymph. Examples of solid tissue cell types include, but are not limited to, colon, lung, breast, skin, prostate, stomach, pancreas, bladder, kidney, and liver.

[0128] "Cell-free DNA", "cfDNA molecules", or simply "cfDNA" includes DNA molecules that are naturally present in a subject in an extracellular form (e.g., in blood, serum, plasma, or other bodily fluids such as lymph, cerebrospinal fluid, urine, or sputum). cfDNA was previously present in one or more cells of a large, complex biological organism, e.g., a mammal, but has been released from the cell(s) into fluids found within the organism, and can be obtained from a sample of the fluid, without the need to perform an in vitro cell lysis step. cfDNA molecules may occur as DNA fragments.

[0129] A "target region" in the context of a nucleic acid refers to a locus or a genetic region comprising multiple loci, for which capture, identification, and / or detection is sought, e.g., by use of a probe (e.g., by sequence complementarity). A "target region set" or "set of target regions" refers to multiple genomic loci that are targeted for identification and / or capture, for example, by use of a set of probes (e.g., by sequence complementarity).

[0130] "Sequence variable target region" refers to a target region that may exhibit sequence changes, such as nucleotide substitutions (i.e., single base mutations), insertions, deletions, or gene fusions or translocations, in neoplastic cells (e.g., tumor cells and cancer cells) compared to normal cells. A sequence variable target region set is a set of sequence variable target regions. In some embodiments, a sequence variable target region is a target region that may exhibit changes affecting less than or equal to 50 contiguous nucleotides, such as less than or equal to 40, 30, 20, 10, 5, 4, 3, or 2 nucleotides, or affecting one nucleotide.

[0131] An "epigenetic target region" refers to a target region that may exhibit sequence-independent differences in different cell or tissue types (e.g., a target region that has a different degree of methylation in solid tissue types different from hematopoietic cells), or differences in neoplastic cells, such as tumor or cancer cells, compared to normal cells. In some embodiments, an epigenetic target region exhibits sequence-independent differences in cfDNA originating from tissue types that do not normally contribute substantially to cfDNA, such as lung, colon, compared to background cfDNA, such as cfDNA originating from hematopoietic cells. In some embodiments, an epigenetic target region exhibits sequence-independent differences in cfDNA from subjects with cancer compared to cfDNA from healthy subjects. Examples of sequence-independent changes include, but are not limited to, changes in methylation (increase or decrease), nucleosome distribution, cfDNA fragmentation patterns, CCCTC-binding factor ("CTCF") binding, transcription start sites, and regulatory protein binding regions. An "epigenetic target region set" is a set of epigenetic target regions. Thus, epigenetic target region sets include, but are not limited to, hypermethylated variable target region sets, hypomethylated variable target region sets, and fragmented variable target region sets, such as CTCF binding sites and transcription start sites.For this purpose, the loci susceptible to local amplification and / or gene fusion associated with neoplasm, tumor or cancer can be analyzed similarly to epigenetic target region sets.Because, the detection of copy number changes by sequencing, or the detection of fusion sequences that map to more than one locus in a reference genome, tends to be more similar to the detection of the exemplary epigenetic changes discussed above than the detection of nucleotide substitutions, insertions or deletions, in that, for example, local amplifications and / or gene fusions can be detected with relatively shallow depth sequencing, since their detection does not depend on the accuracy of base calls at one or a few individual positions.

[0132] As used herein, "epigenetic feature" refers to any feature of DNA or chromatin other than the primary sequence (i.e., the sequence of A, C, G, and T bases). Epigenetic features include covalent modifications of bases, such as methylation, and the modifications and location of histones and other stably associated proteins with DNA.

[0133] As used herein, a "differentially methylated region" refers to a region of DNA that has a detectably different degree of methylation in at least one type of tissue compared to the degree of methylation in another type of tissue, or in a sample from a healthy subject compared to the degree of methylation in a subject with a precancer, cancer, or neoplasm. In some embodiments, the differentially methylated region has a detectably higher degree of methylation in at least one type of tissue compared to the degree of methylation in cell-free DNA from a healthy subject. In some embodiments, the differentially methylated region has a detectably lower degree of methylation in at least one type of tissue compared to the degree of methylation in cell-free DNA from a healthy subject. In some embodiments, the differentially methylated region is hypomethylated in erythroid lineage or immature erythrocytes (e.g., reticulocytes) and hypermethylated in at least one non-erythroid cell type or tissue type (e.g., leukocytes, or solid tissue cell types such as epithelial cells, muscle cells, etc.).

[0134] As used herein, "type-specific" in the context of epigenetic mutations refers to epigenetic mutations that are present at a detectably different degree in one cell or tissue type or a plurality of related cell or tissue types compared to other cell or tissue types. Similarly, a "type-specific epigenetic target region" is an epigenetic target region that has a detectably different epigenetic trait in one cell or tissue type or a plurality of related cell or tissue types compared to other cell or tissue types. Exemplary epigenetic traits are discussed in the definition of epigenetic target region given above. For example, a "type-specific differentially methylated region" is a region of DNA that has a detectably different degree of methylation in one cell or tissue type or a plurality of related cell or tissue types compared to other cell or tissue types. Examples of type-specific differentially methylated regions include tissue-specific differentially methylated regions, including those associated with copy number gain in early cancer. In some embodiments, the capture, identification, and / or detection of type-specific differentially methylated regions facilitates the identification of the cell type or tissue type from which the DNA originates. The cell or tissue from which the type-specific differentially methylated region originates may be a wild-type cell or tissue or a neoplastic cell or tissue. In another example, a "type-specific fragment" of DNA is a DNA fragment resulting from a type-specific fragmentation pattern that is detectably present to a different extent in one cell or tissue type or multiple related cell or tissue types compared to other cell or tissue types. In some embodiments, the type-specific fragment is only present in a particular cell or tissue type(s). In some embodiments, the type-specific fragment is detectably present to a greater extent in a particular cell or tissue type(s).

[0135] As used herein, a "blood sample" refers to a sample containing whole blood or a component thereof (eg, plasma, serum, buffy coat, plasma pellet).

[0136] As used herein, "partitioning" of nucleic acids, such as DNA molecules, refers to separating, fractionating, sorting, or enriching a sample or population of nucleic acids into multiple subsamples or subpopulations of nucleic acids based on one or more modifications or features that are present in different proportions in each of the multiple subsamples or subpopulations. Partitioning may include physically distributing nucleic acid molecules based on the presence or absence of one or more methylated nucleic acid bases. A sample or population can be distributed into one or more distributed subsamples or subpopulations based on a trait that indicates a genetic or epigenetic change or a disease state.

[0137] As used herein, the form of a sample "originally isolated" refers to the composition or chemical structure of the sample at the time the sample is isolated and before it is subjected to any procedure that alters the chemical structure of the isolated sample. Similarly, a feature "originally present" in a molecule refers to a feature that is present in the "original molecule" or in a molecule that "originally comprises" the feature before the molecule is subjected to any procedure that alters the chemical structure of the molecule.

[0138] As used herein, "base pairing specificity" refers to the standard DNA base (A, C, G, or T) with which a given base most preferentially pairs. For example, unmodified cytosine and 5-methylcytosine have the same base pairing specificity (i.e., specificity for G), whereas uracil and cytosine have different base pairing specificities, since uracil has base pairing specificity for A and cytosine has base pairing specificity for G. Nevertheless, uracil's ability to form a wobble pair with G is irrelevant, since uracil most preferentially pairs with A among the four standard DNA bases.

[0139] "Capture" of one or more target molecules, such as one or more nucleic acids comprising at least one target region, refers to preferentially isolating or separating the one or more target molecules from non-target molecules.

[0140] As used herein, a "label" is a capture moiety, fluorophore, oligonucleotide, or other moiety that facilitates detection, separation, or isolation of the one to which it is attached.

[0141] As used herein, a "capture moiety" is a molecule that allows for affinity separation of a molecule linked to the capture moiety from a molecule that lacks the capture moiety. Exemplary capture moieties include biotin, which allows for affinity separation by binding to streptavidin that is linked or linkable to a solid phase, or oligonucleotides, which allow for affinity separation by binding to complementary oligonucleotides that are linked or linkable to a solid phase.

[0142] As used herein, "target-specific probe" refers to a probe that specifically binds to a target region, such as an epigenetic target region or a sequence-variable target region. In some embodiments, a target-specific probe includes a capture moiety that facilitates capture of the target region to which it specifically binds.

[0143] As used herein, a "tag" is a molecule, such as a nucleic acid, label, fluorophore, or peptide, that contains information that indicates a characteristic of the molecule with which it is associated. For example, a molecule may have a sample tag (that distinguishes a molecule in one sample from a molecule in a different sample), a molecular tag / molecular barcode / barcode (that distinguishes different molecules from each other (in both unique and non-unique tagging scenarios)), a purification tag, and / or a detectable tag or label.

[0144] As used herein, a "target molecule" is a molecule, such as a protein, carbohydrate, nucleic acid, or lipid, that is targeted for capture, identification, and / or detection. In some embodiments, the target molecule is a nucleic acid that includes an epigenetic target region and / or a sequence variable target region.

[0145] "Specifically binds," in the context of a primer, probe, or other oligonucleotide, protein, or other binding molecule and a target sequence, means that under appropriate hybridization conditions, the primer, oligonucleotide, or probe hybridizes to its target sequence or a copy thereof to form a stable hybrid, while minimizing the formation of stable non-target hybrids. Thus, the primer or probe hybridizes to the target sequence or a copy thereof to a sufficiently greater extent than to the non-target sequence, ultimately allowing capture or detection of the target sequence. Suitable hybridization conditions are well known in the art and can be predicted based on sequence composition or can be determined using routine testing methods (see, e.g., sections 1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57 of Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), particularly sections 9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57, which are incorporated herein by reference).

[0146] A molecule is said to be "produced by a tumor" if it originates from a tumor cell. cfDNA that originates from a tumor cell is "circulating tumor DNA" ("ctDNA"). Tumor cells are neoplastic cells that originate from a tumor, whether they remain in the tumor or are detached from the tumor (e.g., as in the case of metastatic cancer cells and circulating tumor cells).

[0147] The "capture yield" of a collection of probes for a given set of target regions refers to the amount of nucleic acid corresponding to the set of target regions that the collection of probes captures under typical conditions (e.g., the amount or absolute amount relative to another set of target regions). Exemplary typical capture conditions are incubation of sample nucleic acid and probes at 65°C for 10-18 hours in a small reaction volume (approximately 20 μL) containing stringent hybridization buffer. Capture yields may be expressed as absolute values ​​or, in the case of multiple collections of probes, as relative values. When comparing capture yields of multiple sets of target regions, capture yields are normalized to the footprint size of the set of target regions (e.g., on a per kilobase basis). Thus, for example, if the footprint sizes of the first and second target regions are 50 kb and 500 kb, respectively (with a normalization factor of 0.1), the DNA corresponding to the first set of target regions is captured with a higher yield than the DNA corresponding to the second set of target regions if the mass concentration per volume of the captured DNA corresponding to the first set of target regions is greater than 0.1 times the mass concentration per volume of the captured DNA corresponding to the second set of target regions. As a further example, using the same footprint size, if the captured DNA corresponding to the first set of target regions has a mass concentration per volume that is 0.2 times the mass concentration per volume of the captured DNA corresponding to the second set of target regions, the DNA corresponding to the first set of target regions is captured with a capture yield that is 2 times greater than the DNA corresponding to the second set of target regions.

[0148] The term "methylation" or "DNA methylation" refers to the addition of a methyl group to a nucleobase of a nucleic acid molecule. In some embodiments, methylation refers to the addition of a methyl group to a cytosine at a CpG site (cytosine-phosphate-guanine site (i.e., a cytosine followed by a guanine in the 5'->3' direction of a nucleic acid sequence). In some embodiments, DNA methylation refers to the addition of a methyl group to a cytosine at a CpG site (cytosine-phosphate-guanine site (i.e., a cytosine followed by a guanine in the 5'->3' direction of a nucleic acid sequence). 6-methyladenine, etc. In some embodiments, DNA methylation is 5-methylation (modification of the carbon at the 5th position of the 6-membered carbon ring of cytosine). In some embodiments, 5-methylation refers to the addition of a methyl group to the 5C position of cytosine to create 5-methylcytosine (5mC). In some embodiments, methylation includes derivatives of 5mC. Derivatives of 5mC include, but are not limited to, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-caryboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the carbon at the 3rd position of the 6-membered carbon ring of cytosine). In some embodiments, 3C methylation includes the addition of a methyl group to the 3C position of cytosine to create 3-methylcytosine (3mC). Methylation can also occur at non-CpG sites, for example, methylation can occur at CpA, CpT, or CpC sites. DNA methylation can alter the activity of methylated DNA regions. For example, when DNA in a promoter region is methylated, gene transcription can be repressed. DNA methylation is important for normal development, and aberrant methylation can disrupt epigenetic regulation. Disruption of epigenetic regulation, for example repression, can cause diseases such as cancer. Promoter methylation of DNA can indicate cancer.

[0149] The term "hypermethylation" refers to an increased level or degree of methylation of DNA compared to other DNA molecules in a population (e.g., a sample) of DNA molecules. In some embodiments, hypermethylated DNA may include DNA molecules that contain at least one methylated residue, at least two methylated residues, at least three methylated residues, at least five methylated residues, or at least ten methylated residues. As used herein, "type-specific hypermethylation" refers to an increased level or degree of methylation of DNA in one cell or tissue type or multiple related cell or tissue types compared to other cell or tissue types. In some embodiments, the capture, identification, and / or detection of type-specific hypermethylated regions facilitates the identification of the cell or tissue type from which the DNA originates. The cell or tissue from which the type-specific hypermethylated region originates may be a wild-type cell or tissue or a neoplastic cell or tissue.

[0150] The term "hypomethylation" refers to a decreased level or degree of methylation of a nucleic acid molecule(s) relative to other nucleic acid molecules in a population (e.g., a sample) of nucleic acid molecules. In some embodiments, hypomethylated DNA includes unmethylated DNA molecules. In some embodiments, hypomethylated DNA may include DNA molecules containing zero methylated residues, at most one methylated residue, at most two methylated residues, at most three methylated residues, at most four methylated residues, or at most five methylated residues. As used herein, "type-specific hypomethylation" refers to a decreased level or degree of methylation of DNA in one cell or tissue type or multiple related cell or tissue types relative to other cell or tissue types. In some embodiments, the capture, identification, and / or detection of type-specific hypomethylated regions facilitates the identification of the cell or tissue type from which the DNA originates. The cell or tissue from which the type-specific hypomethylated region originates may be a wild-type cell or tissue or a neoplastic cell or tissue.

[0151] The term "agent that recognizes modified nucleobases of DNA", e.g., "agent that recognizes modified cytosines of DNA", refers to a molecule or reagent that binds to or detects one or more modified nucleobases of DNA, e.g., methylcytosines. A "modified nucleobase" is a nucleobase that includes a difference in chemical structure from an unmodified nucleobase. In the case of DNA, the unmodified nucleobase is adenine, cytosine, guanine, or thymine. In some embodiments, the modified nucleobase is a modified cytosine. In some embodiments, the modified nucleobase is a methylated nucleobase. In some embodiments, the modified cytosine is a methylcytosine, e.g., 5-methylcytosine. In such embodiments, the cytosine modification is methyl. Agents that recognize methylcytosines of DNA include, but are not limited to, "methyl-binding reagents", which herein refer to reagents that bind to methylcytosines. Methyl-binding reagents include, but are not limited to, methyl-binding domains (MBDs), methyl-binding proteins (MBPs), and antibodies specific for methylcytosines. In some embodiments, such antibodies bind to 5-methylcytosines of DNA. In some such embodiments, the DNA may be single-stranded or double-stranded.

[0152] The terms "or combinations thereof" and "or combinations thereof" as used herein refer to any and all permutations and combinations of the terms listed before the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, if order is important in the particular context, BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, combinations including repeats of one or more items or terms are explicitly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, and CABABB. Those skilled in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0153] "Or" is used in its inclusive sense, i.e., equivalent to "and / or," unless the context requires otherwise. II. Exemplary Methods A. Methods for Analyzing DNA with Improved Sensitivity

[0154] The method for analyzing DNA herein includes contacting the DNA with a plurality of target-specific probes specific to members of an epigenetic target region set that includes target regions with type-specific epigenetic mutations and copy number mutations. In some embodiments, the epigenetic target region set consists of target regions with type-specific epigenetic mutations and copy number mutations. In some such embodiments, the plurality of target-specific probes consists of probes specific to members of the epigenetic target region set. In some embodiments, the target region includes type-specific differentially methylated regions and copy number variants. In some embodiments, the target region includes type-specific fragments resulting from type-specific fragmentation patterns and copy number variants. In some embodiments, the type-specific epigenetic mutations are present in a higher proportion in the wild-type genome of one or more cell types or tissue types compared to the wild-type genome of other cell types or tissue types. In some embodiments, the DNA is derived from a blood sample obtained from the subject. In some embodiments, the method herein detects an abnormal level of type-specific DNA, such as cfDNA, in the sample. For example, detection of a higher than normal level of DNA, such as cfDNA, originating from solid tissue in a blood sample can indicate the presence of a disease related to solid tissue. In some embodiments, DNA analysis is used to determine the likelihood that a subject has cancer or precancer.

[0155] In some embodiments, the copy number of the target region is amplified or abnormally high, which facilitates increasing the sensitivity of detection of the target region. In some embodiments, the target region is a type-specific hypermethylated region. In some embodiments, the target region is a type-specific hypomethylated region. In some embodiments, the type-specific differentially methylated region is differentially methylated in one or more relevant cell types. In some such embodiments, the target region is differentially methylated in immune cells compared to non-immune cells. In some embodiments, the type-specific differentially methylated region is differentially methylated in one or more relevant tissue types. In some such embodiments, the target region is differentially methylated in one or more solid tissue types compared to cell types normally found in a sample, such as a blood sample. In some such embodiments, the target region is differentially methylated in one or more solid tissue types other than bladder tissue compared to cell types normally found in a sample, such as a blood sample. In some embodiments, the target region excludes regions that are differentially methylated in bladder. In some embodiments, the target region is a type-specific fragment. In some embodiments, the type-specific fragments result from a fragmentation pattern that is specific to one or more relevant cell types. In some such embodiments, the fragmentation pattern is specific to immune cells compared to non-immune cells. In some embodiments, the type-specific fragmentation pattern is specific to one or more relevant tissue types. In some such embodiments, the fragmentation pattern is specific to one or more solid tissue types compared to cell types typically found in a sample, such as a blood sample.

[0156] In some embodiments, the copy number variation of one or more target regions is local amplification.In some such embodiments, the local amplification is associated with cancer.In some embodiments, the multiple target regions include one or more regions of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAF1.For example, in some embodiments, the multiple target regions include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the above-mentioned genes.Thus, in some embodiments, the multiple target-specific probes include probes that each specifically bind to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the above-mentioned genes.

[0157] In some embodiments, the target region comprises type-specific epigenetic mutations specific to DNA, such as cfDNA, originating from immune cells compared to DNA, such as cfDNA, originating from non-immune cells. In some such embodiments, the plurality of target regions comprises target regions that are differentially methylated in immune cells compared to non-immune cells. In some such embodiments, the plurality of target regions comprises target regions that are hypermethylated in at least some types of immune cells compared to non-immune cells. In some embodiments, the plurality of target regions comprises target regions that are hypomethylated in at least some types of immune cells compared to non-immune cells. In some embodiments, the plurality of target regions comprises a fragmentation pattern that is more prevalent in immune cells compared to non-immune cells. In some embodiments, the target region comprises type-specific epigenetic mutations specific to cfDNA originating from a plurality of immune cell types compared to other immune cell types and non-immune cells present in the sample. In some such embodiments, the plurality of immune cell types comprises naive and activated lymphocytes; monocytes and macrophages; or myelocytes, neutrophils, and eosinophils. In some embodiments, the plurality of immune cell types comprises naive T cells, naive B cells, effector CD4 T cells, effector CD8 T cells, Treg cells, plasma cells, and memory cells. In some embodiments, the plurality of immune cell types comprises metamyelocytes. In some embodiments, the plurality of immune cell types comprises natural killer (NK) cells.

[0158] In some embodiments, the target region comprises type-specific epigenetic mutations specific to DNA, such as cfDNA, originating from solid tissue, compared to DNA, such as cfDNA, originating from other cell types or tissue types, such as other cell types found in the sample. In some such embodiments, the multiple target regions comprise target regions that are differentially methylated in solid tissue types compared to other tissue types or cell types in the sample. In some embodiments, the multiple target regions comprise fragmentation patterns that are more prevalent in solid tissue types compared to other tissue types or cell types in the sample. In some embodiments, the solid tissue type is colon, lung, breast, liver, kidney, prostate, skin, bladder, or pancreas.

[0159] In some embodiments, the plurality of target regions comprises type-specific hypermethylated regions. In some embodiments, the hypermethylated target regions are methylated to an extent that is at least 10%, at least 20%, at least 30%, or at least 40% higher than the average methylation of the target regions in the sample. In some embodiments, the hypermethylated target regions are methylated to an extent that is 5-10%, 10-20%, 10-30%, 20-30%, 30-40%, 40-50%, or 10-50% higher than the average methylation of the target regions in the sample. In some embodiments, the hypermethylated target regions are methylated to an extent that is at least 10%, at least 20%, at least 30%, or at least 40% higher than the average methylation of DNA in the sample. In some embodiments, the hypermethylated target region is methylated to an extent that is 5-10%, 10-20%, 10-30%, 20-30%, 30-40%, 40-50%, or 10-50% higher than the average methylation of the DNA in the sample. In some embodiments, the hypermethylated target region is methylated to an extent that is at least 10%, at least 20%, at least 30%, or at least 40% higher than the average methylation of the corresponding target region in DNA originating from a cell type or tissue type than one or more associated cell types or tissue types of the type-specific hypermethylated target region. In some embodiments, the hypermethylated target region is methylated to an extent that is 5-10%, 10-20%, 10-30%, 20-30%, 30-40%, 40-50%, or 10-50% higher than the average methylation of the corresponding target region in DNA originating from a cell type or tissue type than one or more associated cell types or tissue types of the type-specific hypermethylated target region. In some embodiments, the type-specific hypermethylated target regions are hypermethylated in healthy cells (e.g., healthy cells of one or more solid tissue types) or healthy subjects. In such embodiments, the methylation status of such target regions may not, in itself, be directly indicative of the presence of disease.The methylation status of such target regions indicates the cell type or tissue type from which the DNA originates, and if the cell type or tissue type from which the DNA originates is not expected to be present at significant levels in a given sample, for example, if the DNA is derived from colon tissue in a blood sample, it can indicate the presence of disease in the subject from which the sample was obtained. In some embodiments, the type-specific hypermethylated target regions are hypermethylated in healthy cells and subjects, as well as in diseased cells and subjects with disease. In some such embodiments, the degree of methylation is further increased in diseased cells compared to healthy cells, thereby further increasing the sensitivity of detection of type-specific DNA that may be indicative of disease in the subject from which it was obtained.

[0160] In some embodiments, the plurality of target regions comprises a type-specific fragmentation pattern. In some embodiments, fragments produced by such type-specific fragmentation patterns are present at levels at least 10%, at least 20%, at least 30%, or at least 40% higher than the average level of fragments in samples obtained from healthy subjects. In some embodiments, fragments produced by such type-specific fragmentation patterns are present at levels 5-10%, 10-20%, 10-30%, 20-30%, 30-40%, 40-50%, or 10-50% higher than the average level of fragments in samples obtained from healthy subjects. In some embodiments, the type-specific fragmentation patterns are present in healthy cells or healthy subjects. In such embodiments, the presence of the corresponding fragments may not be directly indicative of the presence of disease. The presence of such fragmentation is indicative of the cell type or tissue type from which the DNA originated, and the presence of DNA originating from a cell type or tissue type not expected to be present in a given sample, e.g., DNA derived from colonic tissue in a blood sample, may indicate the presence of disease in the subject from which the sample was obtained. In some such embodiments, the level of fragments corresponding to type-specific fragmentation patterns is further increased in diseased cells compared to healthy cells, thereby further increasing the sensitivity of detection of type-specific DNA that may indicate disease in the subject from which it is obtained.Exemplary approaches for analyzing DNA fragmentation patterns are provided in, for example, WO2022040163A1, US11352670B2, US20200056245A1, US10297342B2, US10741270B2, US10453556B2, US9892230B2, EP3617324A1, and EP2860266B1, each of which is incorporated herein by reference in its entirety for all purposes.

[0161] In some embodiments, the multiple target regions include copy number variants, such as local amplifications or duplications, with abnormally high copy numbers. In some such embodiments, the increased copy number of the target regions further increases the sensitivity of the method described herein. In some embodiments, the copy number variants are type-specific copy number variants. In some embodiments, the copy number variants are abnormally high and are associated with precancer or cancer. In some embodiments, the copy number variants are copy number amplifications known to occur in early cancer or precancer. In some such embodiments, the copy number variants are present in subjects with disease. In some embodiments, the copy number variants are abnormally high in diseased cells compared to healthy cells, thereby increasing the sensitivity of detection of type-specific DNA that may indicate disease in the subjects from which it is obtained. B. Target

[0162] In some embodiments, the DNA or nucleic acid from a subject is obtained from a subject having cancer or precancer and / or from a sample obtained from a subject having cancer or precancer. In some embodiments, the sample is obtained from a subject suspected of having cancer or precancer. In some embodiments, the sample is obtained from a subject having a tumor. In some embodiments, the sample is obtained from a subject suspected of having a tumor. In some embodiments, the sample is obtained from a subject having a neoplasm. In some embodiments, the sample is obtained from a subject suspected of having a neoplasm. In some embodiments, the sample is obtained from a subject in remission of a tumor, cancer, or neoplasm (e.g., after chemotherapy, surgical resection, radiation therapy, or a combination thereof). In any of the above-mentioned embodiments, the precancer, cancer, tumor, or neoplasm, or suspected precancer, cancer, tumor, or neoplasm, may be of the bladder, head and neck, lung, colon, rectum, kidney, breast, prostate, skin, or liver. In some embodiments, the precancer, cancer, tumor, or neoplasm, or suspected precancer, cancer, tumor, or neoplasm, is of the lung. In some embodiments, the precancer, cancer, tumor, or neoplasm, or suspected precancer, cancer, tumor, or neoplasm, is of the colon or rectum. In some embodiments, the precancer, cancer, tumor, or neoplasm, or suspected precancer, cancer, tumor, or neoplasm, is of the breast. In some embodiments, the precancer, cancer, tumor, or neoplasm, or suspected precancer, cancer, tumor, or neoplasm, is of the prostate. In any of the above embodiments, the subject may be a human subject. In some embodiments, the sample is obtained from a subject having stage I cancer, stage II cancer, stage III cancer, or stage IV cancer. C. Analysis of Nucleic Acids Using Target-Specific Probes

[0163] The method for analyzing DNA or nucleic acid from a sample herein comprises contacting DNA with a plurality of target-specific probes that are specific to the members of a set of epigenetic target regions that comprise or consist of target regions that have both type-specific epigenetic mutations and copy number mutations.In some embodiments, the type-specific epigenetic mutation is differential methylation.In some embodiments, the type-specific mutation is type-specific fragmentation pattern.In some embodiments, the copy number mutation is abnormally high copy number.

[0164] In some embodiments, the methods disclosed herein further comprise contacting DNA from the sample with probes specific for the sequence variable target regions and / or the second set of epigenetic target regions, and capturing DNA that anneals to the probes specific for the sequence variable set of target regions and / or the second epigenetic set. Such embodiments further comprise sequencing the captured DNA using a method such as those disclosed herein.

[0165] The method can be used to diagnose the presence of a condition, particularly cancer or precancer, in a subject, characterize the condition (e.g., stage the cancer or determine the heterogeneity of the cancer), monitor the response to the treatment of the condition, and provide a prognostic risk of the onset of the condition or the subsequent course of the condition. The present disclosure can also be useful for determining the effectiveness of a particular treatment option. If the treatment is successful, fewer cancer cells will shed DNA, and thus the success of the treatment option may reduce the amount of copy number mutations or rare mutations detected in the blood of the subject. In other examples, this may not occur. In another example, perhaps a particular treatment option may correlate with the genetic profile of the cancer over time. This correlation may be useful in selecting a therapy.

[0166] Additionally, if the cancer is observed to be in remission following treatment, the method can be used to monitor for residual disease or recurrence of the disease.

[0167] The types and number of cancers that can be detected include blood cancer, brain cancer, lung cancer, skin cancer, nose cancer, throat cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, intestinal cancer, rectal cancer, colon cancer, prostate cancer, thyroid cancer, bladder cancer, head and neck cancer, kidney cancer, oral cancer, stomach cancer, solid tumors, heterogeneous tumors, and homogeneous tumors, etc. The type and / or stage of cancer can be detected from genetic mutations including mutations, rare mutations, indels, copy number variations, transversions, translocations, recombinations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structural changes, gene fusions, chromosomal fusions, gene truncations, gene amplifications, gene duplications, chromosomal damage, DNA damage, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5-methylcytosine.

[0168] In some embodiments, the methods described herein involve identifying the presence of nucleic acid, such as DNA, produced by a tumor (or neoplastic cell, or cancer cell) or by a pre-cancerous cell.

[0169] Genetic data can also be used to characterize specific forms of cancer. Cancers are often heterogeneous in both composition and stage. Genetic profile data can allow characterization of specific subtypes of cancer, which can be important in diagnosing or treating that specific subtype. This information can also provide clues to the subject or attending physician regarding the prognosis of a particular type of cancer, allowing the subject or attending physician to adapt treatment options according to disease progression. Some cancers can progress to become more aggressive and genetically unstable. Other cancers can remain benign, inactive, or dormant. The disclosed system and method can be useful in determining disease progression.

[0170] Furthermore, the disclosed method can be used to characterize the heterogeneity of abnormal conditions in a subject. Such a method can include, for example, generating a profile of DNA from a subject, the profile including a plurality of data obtained from epigenetic and mutational analysis. In some embodiments, the abnormal condition is cancer or precancer. In some embodiments, the abnormal condition can result in a heterogeneous genomic population. In the example of cancer, it is known that some tumors contain tumor cells of different stages of cancer. In other examples, the heterogeneity can include multiple disease foci. Again, in the example of cancer, there can be multiple tumor foci, perhaps one or more of which are the result of metastasis spreading from the primary site.

[0171] The methods can be used to generate a profile, fingerprint, or dataset that is the sum of information derived from different cells of a heterogeneous disease, which may include structural mutation identity and levels, copy number mutations, epigenetic mutations, or other mutation analyses, either alone or in combination.

[0172] The method can be used to diagnose, prognose, monitor, or observe pre-cancer, cancer, or other disease.In some embodiments, the method herein does not include diagnosing, prognosing, or monitoring fetus, and therefore does not relate to non-invasive prenatal testing.In other embodiments, such methodology can be used in pregnant subjects to diagnose, prognose, monitor, or observe cancer or other disease in fetal subjects where DNA and other polynucleotides may co-circulate with maternal molecules.

[0173] An exemplary method for analyzing DNA includes the following steps: 1. Preparing an extracted DNA sample (e.g., plasma DNA extracted from a human sample) by ligating adapters to the DNA and amplifying the DNA. 2. Partitioning the adaptor-ligated DNA into a plurality of differentially methylated subsamples by contacting the DNA with an agent that recognizes modified cytosines, such as methylcytosines, in the DNA. 3. Capturing DNA containing type-specific hypermethylated target regions having abnormally high copy numbers from the distributed aliquot samples by contacting the aliquot samples with a target-specific probe containing biotin and then contacting the aliquot samples with a solid support containing a binding partner of the capture moiety, such as a solid support containing streptavidin. 4. Enriching and / or eluting the captured DNA. 5. Re-amplifying the captured DNA and assaying it in multiplex on an NGS instrument. 6. Analyzing the NGS data with the molecular tags of the adapters used to identify unique molecules.

[0174] In some embodiments, before capturing DNA, the distributed DNA can be amplified by PCR amplification.In some embodiments, determining the level of the captured sequence facilitates disease diagnosis or identification of appropriate treatment.In some embodiments, the presence of one or more captured sequences or changes in their levels indicate the presence of a disease or disorder, such as cancer or precancer, in a subject.In some embodiments, detection of target molecules in combination with cfDNA analysis of sequence-independent changes in epigenetic target regions, such as cfDNA analysis described herein, indicates the presence of a disease or disorder, such as cancer, precancer, or other disorder that causes changes in nucleic acid, in a subject, compared to healthy subjects. D. DNA Analysis and / or Distribution

[0175] The method disclosed herein comprises analyzing DNA in a sample.In such a method, different forms of DNA (e.g., hypermethylated DNA and hypomethylated DNA) can be physically separated based on one or more characteristics of DNA.This technique can be used, for example, to determine whether a certain sequence is hypermethylated or hypomethylated.

[0176] Methylation profiling may include determining methylation patterns across different regions of a genome. For example, after distributing molecules based on the degree of methylation (e.g., the relative number of methylated nucleobases per molecule) and sequencing, the sequences of molecules in different distribution fractions can be mapped against a reference genome. This can show regions of the genome that are more highly methylated or less highly methylated compared to other regions. In this way, genomic regions can have different degrees of methylation as opposed to individual molecules.

[0177] By partitioning the nucleic acid molecules in a sample, rare signals can be increased, for example, by enriching rare nucleic acid molecules that are more abundant in one partitioned fraction of the sample. For example, genetic mutations that are present in hypermethylated DNA but less abundant (or absent) in hypomethylated DNA can be more easily detected by partitioning the sample into hypermethylated and hypomethylated nucleic acid molecules. By analyzing multiple partitioned fractions of a sample, multidimensional analysis of single molecules can be performed, thus achieving higher sensitivity. Partitioning can include physically partitioning nucleic acid molecules into partitioned fractions or subsamples based on the presence or absence of one or more methylated nucleic acid bases. Samples can be partitioned into partitioned fractions or subsamples based on a characteristic that indicates differential gene expression or disease state. Samples can be partitioned based on a characteristic or combination that results in a difference in signal between normal and disease states during the analysis of nucleic acids, for example, cell-free DNA (cfDNA), non-cfDNA, tumor DNA, circulating tumor DNA (ctDNA), and cell-free nucleic acid (cfNA).

[0178] In some embodiments, hypermethylated and / or hypomethylated variable epigenetic target regions are analyzed to determine whether they exhibit differential methylation signatures in tumor cells, or cell types that do not normally contribute to the DNA sample being analyzed (such as cfDNA), and / or specific immune cell types.

[0179] In some cases, the heterogeneous DNA in the sample is distributed into two or more distribution fractions (e.g., at least 3, 4, 5, 6, or 7 distribution fractions). In some embodiments, each distribution fraction is differentially tagged. The tagged distribution fractions can then be pooled together for collective sample preparation and / or sequencing. The distribution-tagging-pooling step may be performed more than once, with each round of distribution being tagged using a differential tag based on a different characteristic (examples are provided herein) and distinguishing it from other distribution fractions and distribution means. In other examples, the differentially tagged distribution fractions are sequenced separately.

[0180] In some embodiments, sequence reads from the differentially tagged pool DNA are obtained and analyzed in silico. The tags are used to sort the reads from different distribution fractions. Analysis to detect genetic mutations can be performed at the level of each distribution fraction as well as the level of the total nucleic acid population. For example, the analysis can include in silico analysis to determine genetic variants such as CNVs, SNVs, indels, fusions, etc. in the nucleic acids of each distribution fraction. In some cases, the in silico analysis can include determining chromatin structure. For example, the coverage of sequence reads can be used to determine nucleosome positions in chromatin. Higher coverage can be correlated with higher nucleosome occupancy in genomic regions, and lower coverage can be correlated with lower nucleosome occupancy or nucleosome depleted regions (NDRs).

[0181] Examples of characteristics that can be used for partitioning include sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and / or proteins that bind to DNA. The resulting partitioned fractions include one or more of the following nucleic acid forms: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), short DNA fragments, and long DNA fragments. In some embodiments, partitioning is generally performed based on cytosine modification (e.g., cytosine methylation) or methylation, and optionally combined with at least one additional partitioning step that may be based on any of the above-mentioned characteristics or forms of DNA. In some embodiments, a heterogeneous population of nucleic acids is partitioned into nucleic acids that have one or more epigenetic modifications and nucleic acids that do not have one or more epigenetic modifications. Examples of epigenetic modifications include the presence or absence of methylation, the level of methylation, the type of methylation (e.g., 5-methylcytosine versus other types of methylation such as adenine methylation and / or cytosine hydroxymethylation), and the association and level of association with one or more proteins, such as histones. Alternatively or in addition, the heterogeneous population of nucleic acids can be divided into nucleic acid molecules that are associated with nucleosomes and nucleic acid molecules that lack nucleosomes.Alternatively or in addition, the heterogeneous population of nucleic acids can be divided into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).Alternatively or in addition, the heterogeneous population of nucleic acids can be divided based on nucleic acid length (e.g., molecules that are up to 160bp and molecules that have a length greater than 160bp).

[0182] The agents used to partition the population of nucleic acids in a sample may be affinity agents such as antibodies, natural binding partners, or variants thereof with the desired specificity (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68-72 (2011)), or artificial peptides selected, for example, by phage display, to have specificity for a given target. In some embodiments, the agents used for partitioning are agents that recognize modified nucleobases. In some embodiments, the modified nucleobase recognized by the agent is a modified cytosine, such as methylcytosine (e.g., 5-methylcytosine). In some embodiments, the modified nucleobase recognized by the agent is the product of a procedure that affects a first nucleobase in DNA differently than a second nucleobase in the DNA of the sample. In some embodiments, the modified nucleobase may be a "converted nucleobase," meaning that its base-pairing specificity has been altered by the procedure. For example, in certain procedures, unmethylated or unmodified cytosine is converted to dihydrouracil, or more generally, at least one modified or unmodified form of cytosine undergoes deamination, resulting in uracil (considered a modified nucleobase in the context of DNA) or a further modified form of uracil. Examples of partitioning agents include antibodies, such as antibodies that recognize modified nucleobases that may be modified cytosines, such as methylcytosine (e.g., 5-methylcytosine). In some embodiments, the partitioning agent is an antibody that recognizes modified cytosines other than 5-methylcytosine, such as 5-carboxylcytosine (5caC). Alternative partitioning agents include the methyl-binding domains (MBDs) and methyl-binding proteins (MBPs) described herein, including proteins such as MeCP2.

[0183] Additional non-limiting examples of partitioning agents are histone-binding proteins that can separate histone-bound nucleic acids from free or unbound nucleic acids. Examples of histone-binding proteins that can be used in the methods disclosed herein include RBBP4, RbAp48, and SANT domain peptides.

[0184] In some embodiments, partitioning may include both binary partitioning and partitioning based on degree / level of modification. For example, methylated fragments may be partitioned by methylated DNA immunoprecipitation (MeDIP), or all methylated fragments may be partitioned from non-methylated fragments using a methyl-binding domain protein (e.g., MethylMinder Methylated DNA Enrichment Kit (ThermoFisher Scientific)). Additional partitioning may then include eluting fragments with different levels of methylation by adjusting the salt concentration in the solution with the methyl-binding domain and bound fragments. With increasing salt concentration, fragments with higher methylation levels are eluted.

[0185] Analysis of the DNA may include detection or quantification of the DNA of interest. Analysis of the DNA may include detecting genetic variants and / or epigenetic features (e.g., DNA methylation and / or DNA fragmentation).

[0186] In some embodiments, the methylation level can be determined using partitioning, modification-sensitive conversion such as bisulfite conversion, direct detection during sequencing, methylation-sensitive restriction enzyme digestion, methylation-dependent restriction enzyme digestion, or any other suitable technique. For example, different forms of DNA (e.g., hypermethylated DNA and hypomethylated DNA) can be physically partitioned based on one or more characteristics of the DNA. For example, DNA can be partitioned using methylated DNA binding proteins (e.g., MBD such as MBD2, MBD4, or MeCP2) or antibodies specific for 5-methylcytosine (e.g., MeDIP). This technique can be used, for example, to determine whether a particular sequence is hypermethylated or hypomethylated. In some embodiments, DNA fragmentation patterns can be determined based on the end points and / or center points of DNA molecules such as cfDNA molecules.

[0187] In some cases, the final partitioned fraction is enriched for nucleic acids with different degrees of modification (over- or under-representation of the modification). Over- and under-representation can be defined by comparing the number of modifications made to the nucleic acid with the median number of modifications per strand in the population. For example, if the median number of 5-methylcytosine residues of nucleic acids in a sample is two, then nucleic acids containing more than two 5-methylcytosine residues will have this modification over-represented, and nucleic acids with one or zero 5-methylcytosine residues will have this modification under-represented. The effect of the affinity separation is that the bound phase is enriched for nucleic acids with over-represented modifications, and the non-bound phase (i.e., in solution) is enriched for nucleic acids with under-represented modifications. Nucleic acids in the bound phase can be eluted before further processing.

[0188] Using MeDIP or MethylMiner® Methylated DNA Enrichment Kit (ThermoFisher Scientific), sequential elution can be used to partition different levels of methylation. For example, a low methylation partition fraction (no methylation) can be separated from a methylation partition fraction by contacting the nucleic acid population with the MBD of the kit attached to magnetic beads. The beads are used to separate the methylated nucleic acids from the unmethylated nucleic acids. One or more elution steps are then performed sequentially to elute the nucleic acids with different levels of methylation. For example, a first set of methylated nucleic acids may be eluted with a salt concentration of 160 mM or higher, e.g., at least 150 mM, at least 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or 2000 mM. After such methylated nucleic acids are eluted, magnetic separation is once again used to separate the more highly methylated nucleic acids from the less methylated nucleic acids. The elution and magnetic separation steps can be repeated to generate various distribution fractions, such as a low methylation distribution fraction (enriched for nucleic acids with no methylation), a methylation distribution fraction (enriched for nucleic acids with low levels of methylation), and a high methylation distribution fraction (enriched for nucleic acids with high levels of methylation).

[0189] In some methods, nucleic acids bound to agents used for affinity separation-based partitioning are subjected to a wash step, which washes away nucleic acids that are weakly bound to the affinity agent, and such nucleic acids can be enriched for nucleic acids that have a modification level close to the average or median value (i.e., intermediate between those that remain bound to the solid phase and those that do not upon initial contact of the sample with the agent).

[0190] Affinity separation results in at least two, and sometimes three or more, partitioned fractions of nucleic acids with different degrees of modification. Although the partitioned fractions are still separate, the nucleic acids of at least one partitioned fraction, usually two or three (or more), are linked to a nucleic acid tag, usually provided as a component of an adaptor, such that the nucleic acids of the different partitioned fractions receive different tags that distinguish the members of one partitioned fraction from the members of another partitioned fraction. The tags linked to the nucleic acid molecules of the same partitioned fraction may be the same or different from each other. However, when different from each other, the tags may share part of their code in order to identify the molecules to which they are attached as being from a particular partitioned fraction.

[0191] For further details regarding partitioning nucleic acid samples based on characteristics such as methylation, see WO2018 / 119452, which is incorporated herein by reference.

[0192] In some embodiments, partitioning includes contacting the DNA with a methylation-sensitive restriction enzyme (MSRE) and / or a methylation-dependent restriction enzyme (MDRE). After treatment of the DNA with an MSRE or MDRE, the DNA can be partitioned based on size to generate highly methylated (longest DNA molecules after MSRE treatment and shortest DNA fragments after MDRE treatment), intermediate (intermediate length DNA molecules after MSRE or MDRE treatment), and low methylated (shortest DNA molecules after MSRE treatment and longest DNA fragments after MDRE treatment) subsamples.

[0193] In some embodiments, partitioning is performed by contacting the nucleic acid with the methyl-binding domain ("MBD") of a methyl-binding protein ("MBP"). In some such embodiments, the nucleic acid is contacted with the entire MBP. In some embodiments, the MBD binds 5-methylcytosine (5mC), and the MBP comprises an MBD, which is referred to herein interchangeably as a methyl-binding protein or a methyl-binding domain protein. In some embodiments, the MBD is coupled to paramagnetic beads, such as Dynabeads® M-280 streptavidin, via a biotin linker. Partitioning into fractions with different degrees of methylation can be performed by eluting the fractions with increasing NaCl concentrations.

[0194] In some embodiments, the bound DNA is eluted by contacting the antibody or MBD with a protease, such as proteinase K. This may be performed instead of or in addition to the elution step using NaCl as discussed above.

[0195] Examples of modified nucleobase recognizing agents contemplated herein include, but are not limited to, the following: (a) MeCP2 is a protein that preferentially binds 5-methyl-cytosine over unmodified cytosine. (b) RPL26, PRP8, and the DNA mismatch repair protein MHS6 preferentially bind 5-hydroxymethyl-cytosine over unmodified cytosine. (c) FOXK1, FOXK2, FOXP1, FOXP4, and FOXI3 bind preferentially to 5-formyl-cytosine over unmodified cytosine (Iurlaro et al., Genome Biol. 14: R119 (2013)). (d) An antibody specific for one or more methylated or modified nucleobases or their conversion products, such as 5mC, 5caC, or DHU.

[0196] In general, elution is a function of the number of modifications, such as the number of methylation sites per molecule, with increasing salt concentration eluting molecules with more methylation. To elute DNA into different populations based on the degree of methylation, a series of elution buffers with increasing NaCl concentrations can be used. The salt concentration can range from about 100 nM to about 2500 mM NaCl. In one embodiment, the process results in three (3) partitioned fractions. The molecules are contacted with a solution at a first salt concentration that includes molecules that include an agent that recognizes modified nucleobases, and the molecules may be attached to a capture moiety such as streptavidin. At the first salt concentration, some populations of molecules will bind to the agent and some will remain unbound. The unbound population can be separated as a "hypomethylated" population. For example, the first partitioned fraction enriched in hypomethylated forms of DNA is the partitioned fraction that remains unbound at a lower salt concentration, e.g., 100 mM or 160 mM. A second partition fraction enriched for intermediately methylated DNA is eluted using an intermediate salt concentration, for example, between 100 mM and 2000 mM, which is also separated from the sample. A third partition fraction enriched for highly methylated forms of DNA is eluted using a high salt concentration, for example, at least about 2000 mM.

[0197] In some embodiments, methylated DNA is purified using a monoclonal antibody raised against 5-methylcytidine (5mC). To obtain single-stranded DNA fragments, DNA is denatured, for example at 95°C. Protein G coupled to standard or magnetic beads and incubation with anti-5mC antibody followed by washing is used to immunoprecipitate the DNA bound to the antibody. Such DNA can then be eluted. The partitioned fraction may include non-precipitated DNA and one or more partitioned fractions eluted from the beads.

[0198] In some embodiments, the distributed fractions of DNA are desalted and concentrated in preparation for the enzymatic steps of library preparation.

[0199] The sequence that contains abnormally high copy number may tend to be hypermethylated.Therefore, in some embodiments, the DNA that is contacted with the target-specific probe specific to the member of the epigenetic target region set that contains multiple target regions that are both type-specific differentially methylated regions and copy number variants comprises at least a portion of hypermethylated distribution fraction.The DNA that originates from or comprises at least a portion of hypermethylated distribution fraction may or may not be combined with the DNA that originates from or comprises at least a portion of one or more other distribution fractions, such as intermediate distribution fraction or hypomethylated distribution fraction.

[0200] In some embodiments, methylation is detected using modification-sensitive conversion. Modification-sensitive conversion refers to any technique that differentially modifies a first nucleobase but does not modify a second nucleobase in a modification-dependent manner, for example, methylation (or hydroxymethylation, or formylation, or carboxylation, etc.) versus unmodified, and / or modified in one manner versus modified in another manner (e.g., methylation versus hydroxymethylation). Examples of such conversion techniques include bisulfite conversion, which converts unmodified cytosine and certain modified cytosines (e.g., 5-formylcytosine (fC) or 5-carboxylcytosine (caC)) to uracil, but does not convert other modified cytosines (e.g., 5-methylcytosine, 5-hydroxymethylcytosine). Performing bisulfite conversion can facilitate the identification of positions that contain mC or hmC using sequence reads. For an exemplary description of bisulfite conversion, see, e.g., Moss et al., Nat Commun. 2018; 9: 5068.

[0201] Examples of such conversion techniques include oxidative bisulfite (Ox-BS) conversion. Performing Ox-BS conversion can facilitate identifying the position containing mC using sequence reads. For an exemplary description of oxidative bisulfite conversion, see, for example, Booth et al., Science 2012; 336: 934-937.

[0202] Examples of such conversion techniques also include Tet-assisted bisulfite (TAB) conversion. For example, as described in Yu et al., Cell 2012; 149: 1368-80, β-glucosyltransferase can be used to protect hmC (forming 5-glucosylhydroxymethylcytosine (ghmC)), then mC can be converted to caC using a TET protein such as mTet1, and then bisulfite treatment can be used to convert C and caC to U, while ghmC remains unaffected. Thus, when using TAB conversion, the first nucleobase comprises one or more of unmodified cytosine, fC, caC, mC, or other cytosine forms affected by bisulfite, and the second nucleobase comprises hmC. Performing TAB conversion can facilitate identifying positions that contain hmC using sequence reads.

[0203] Examples of such conversion techniques also include Tet-assisted conversions using substituted borane reducing agents, where appropriate, the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. See, for example, Liu et al., Nature Biotechnology 2019; 37:424-429 (e.g., attached Figure 1 and attached Note 7). Performing a TAP conversion can facilitate identifying positions containing unmodified C using sequence reads. This procedure encompasses Tet-assisted pyridine borane sequencing (TAPS), which is described in more detail in Liu et al. 2019, supra.

[0204] Alternatively, protection of hmC (e.g., using βGT) may be combined with Tet-assisted conversion with a substituted borane reducing agent. Such implementation of TAPSβ conversion can facilitate using sequence reads to distinguish positions that contain unmodified C or hmC from positions that contain mC on the other hand. For an exemplary description of this type of conversion, see, for example, Liu et al., Nature Biotechnology 2019; 37:424-429.

[0205] Examples of such conversion techniques also include APOBEC coupling epigenetic (ACE) conversion.Performing ACE conversion can facilitate using sequence reads to distinguish between positions that contain hmC and positions that contain mC or unmodified C.For an exemplary description of ACE conversion, see, for example, Schutsky et al., Nature Biotechnology 2018; 36: 1083-1090.

[0206] Examples of such conversion techniques include enzymatic conversion of nucleobases, as in, for example, EM-Seq. See, e.g., Vaisvila R, et al. (2019) EM-seq: Detection of DNA methylation at single base resolution from picograms of DNA. bioRxiv; DOI: 10.1101 / 2019.12.20.884692v1, available at: www.biorxiv.org / content / 10.1101 / 2019.12.20.884692v1.

[0207] In some embodiments, methylation is detected using methylation-sensitive restriction enzymes (MSREs). For example, a sample, a subsample, or a portion of a sample can be subjected to digestion with one or more MSREs to cleave non-methylated sequences. Exemplary MSREs include AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, and SnaBI. In some embodiments, at least two methylation-sensitive nucleases are used. In some embodiments, at least three methylation-sensitive nucleases are used. In some embodiments, the methylation sensitive nucleases include BstUI and HpaII. In some embodiments, the two methylation sensitive nucleases include HhaI and AccII. In some embodiments, the methylation sensitive nucleases include BstUI, HpaII, and Hin6I. In some embodiments, the portion of the sample contacted with one or more MSREs includes hypermethylated DNA or is or includes a hypermethylated DNA distribution fraction that can be obtained as described elsewhere herein.

[0208] In some embodiments, DNA fragmentation is detected by determining the end and / or midpoint of the fragments of sequenced DNA (e.g., cfDNA).For example, fragmentation patterns may differ depending on whether fragments originate from tumor or from healthy cells.To detect tumor cell-derived DNA in cfDNA based on fragmentation, the presence or absence of increased levels of abnormal fragments can be determined in the region with copy number amplification (e.g., proportional to the degree of amplification), for example, the increase and abnormality are compared with control sample or healthy sample.

[0209] In some embodiments, a sample or aliquot (e.g., a first, second, or third aliquot prepared as described herein by partitioning a sample based on the level of cytosine modification, such as cytosine methylation, e.g., 5-methylation) is contacted with a methylation-dependent or methylation-sensitive nuclease. Unless otherwise specified, when partitioning is performed based on cytosine modification, the first aliquot is the aliquot having a higher level of modification, the second aliquot is the aliquot having a lower level of modification, and the third aliquot, if present, has a level of modification intermediate between the first and second aliquots.

[0210] As discussed above, the partitioning procedure may result in incomplete sorting of DNA molecules between the aliquots. A methylation-dependent nuclease or a methylation-sensitive nuclease may be selected to degrade non-specifically partitioned DNA. For example, the second aliquot may be contacted with a methylation-dependent nuclease, such as a methylation-dependent restriction enzyme, which degrades non-specifically partitioned DNA (e.g., methylated DNA) in the second aliquot to generate a processed second aliquot. Alternatively or additionally, the first aliquot may be contacted with a methylation-sensitive endonuclease, such as a methylation-sensitive restriction enzyme, which degrades non-specifically partitioned DNA in the first aliquot to generate a processed first aliquot. Degradation of non-specifically partitioned DNA in either or both the first or second aliquot is proposed as improving the performance of methods that rely on accurate partitioning of DNA based on cytosine modifications, for example, to detect the presence of aberrantly modified DNA in a sample, to determine the tissue of origin of the DNA, and / or to determine whether a subject has cancer. For example, such degradation can improve sensitivity and / or simplify downstream analysis. In general, if the non-specifically partitioned DNA is highly methylated, such as in a low-methylation partitioned fraction, a methylation-dependent nuclease, such as a methylation-dependent restriction enzyme, should be used. Conversely, if the non-specifically partitioned DNA is low-methylated, such as in a high-methylation partitioned fraction, a methylation-sensitive nuclease, such as a methylation-sensitive restriction enzyme, should be used. A methylation-dependent nuclease, such as a methylation-dependent restriction enzyme, preferentially cuts methylated DNA over unmethylated DNA, and a methylation-sensitive nuclease, such as a methylation-sensitive restriction enzyme, preferentially cuts unmethylated DNA over methylated DNA.

[0211] When the partial sample is contacted with the nuclease, one or more nucleases can be used. In some embodiments, the partial sample is contacted with multiple nucleases. The partial sample can be contacted with the nucleases sequentially or simultaneously. If the nucleases are active under similar conditions (such as buffer composition), it may be advantageous to use the nucleases simultaneously to avoid unnecessary sample manipulation. The second partial sample can be contacted with more than one methylation-dependent restriction enzyme to more completely degrade non-specifically distributed hypermethylated DNA. Similarly, the first partial sample can be contacted with more than one methylation-sensitive restriction enzyme to more completely degrade non-specifically distributed hypomethylated and / or unmethylated DNA.

[0212] In some embodiments, the methylation-dependent nuclease comprises one or more of MspJI, LpnPI, FspEI, or McrBC. In some embodiments, at least two methylation-dependent nucleases are used. In some embodiments, at least three methylation-dependent nucleases are used. In some embodiments, the methylation-dependent nuclease comprises FspEI. In some embodiments, the methylation-dependent nuclease comprises FspEI and MspJI, for example, used sequentially.

[0213] In some embodiments, the methylation-sensitive nuclease comprises one or more of AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, and SnaBI. In some embodiments, at least two methylation-sensitive nucleases are used. In some embodiments, at least three methylation-sensitive nucleases are used. In some embodiments, the methylation-sensitive nuclease comprises BstUI and HpaII. In some embodiments, the two methylation-sensitive nucleases comprise HhaI and AccII. In some embodiments, the methylation-sensitive nucleases include BstUI, HpaII, and Hin6I.

[0214] In some embodiments, FspEI is used to digest nucleic acid molecules in at least one subsample (e.g., low methylation distribution fraction). In some embodiments, BstUI, HpaII, and Hin6I are used to digest nucleic acid molecules in at least one subsample (e.g., high methylation distribution fraction), and FspEI is used to digest nucleic acid molecules in at least one other subsample (e.g., low methylation distribution fraction). In embodiments that include intermediately methylated distribution fractions, the nucleic acid molecules therein can be digested with methylation-sensitive or methylation-dependent nucleases. In some embodiments, the nucleic acid molecules in the intermediately methylated distribution fractions are digested with the same nuclease(s) as the high methylation distribution fraction. For example, the intermediately methylated distribution fractions may be pooled with the high methylation distribution fractions, and then the pooled distribution fractions may be subjected to digestion. In some embodiments, the nucleic acid molecules in the intermediately methylated distribution fractions are digested with the same nuclease(s) as the low methylation distribution fraction. For example, intermediately methylated distribution fractions may be pooled with less methylated distribution fractions and the pooled distribution fractions may then be subjected to digestion.

[0215] In some embodiments, after the step of tagging or attaching adapters to both ends of DNA, the aliquot sample is contacted with nuclease as described above.The tag or adapter may be resistant to cleavage by nuclease using any of the methods described above.In this method, the cleavage product lacks tags or adapters at both ends, so that cleavage can prevent non-specifically distributed molecules from being carried over into the analysis.

[0216] Alternatively, the step of tagging or attaching the adapter can be performed after cleavage by the nuclease described above. The cleaved molecules in the sequence reads can then be identified based on having an end (attachment point with the tag or adapter) that corresponds to the nuclease recognition site. Processing the molecules in this manner may allow information to be obtained from the cleaved molecules, for example, observing somatic mutations. When tagging or attaching adapters after contacting a partial sample with a nuclease to analyze low molecular weight DNA such as cfDNA, it may be desirable to remove high molecular weight DNA (such as contaminating genomic DNA) from the sample before the contacting step. It may also be desirable to use a nuclease that can be heat-inactivated at a relatively low temperature (e.g., 65°C or lower, or 60°C or lower) to avoid denaturation of the DNA, as denaturation may interfere with the subsequent ligation step.

[0217] When the sample is divided into three aliquots, including a third aliquot containing intermediately methylated molecules, in some embodiments, the third aliquot is contacted with a methylation-sensitive nuclease. Such a step may have any of the characteristics described elsewhere herein for the contacting step, and may be performed before or after the tagging or adapter attachment step as discussed above. In some embodiments, the first and third aliquots are combined before contacting with a methylation-sensitive nuclease. Such a step may have any of the characteristics described elsewhere herein for the contacting step, and may be performed before or after the tagging or adapter attachment step as discussed above. In some embodiments, the first and third aliquots are differentially tagged before being combined.

[0218] Alternatively, when the sample is divided into three aliquots, including a third aliquot containing intermediately methylated molecules, in some embodiments, the third aliquot is contacted with a methylation-dependent nuclease. Such a step may have any of the characteristics described elsewhere herein for the contacting step, and may be performed before or after the tagging or adapter attachment step as discussed above. In some embodiments, the second and third aliquots are combined before contacting with a methylation-dependent nuclease. Such a step may have any of the characteristics described elsewhere herein for the contacting step, and may be performed before or after the tagging or adapter attachment step as discussed above. In some embodiments, the second and third aliquots are differentially tagged before being combined.

[0219] In some embodiments, the DNA is purified after contacting with the nuclease, for example using SPRI beads. Such purification may occur after heat inactivation of the nuclease. Alternatively, purification may be omitted, and subsequent steps, such as, for example, amplification, may be performed on the aliquot containing the heat inactivated nuclease. In another embodiment, the contacting step may occur in the presence of a purification reagent, such as SPRI beads, for example to minimize losses associated with tube transfer. After cleavage and heat inactivation, the SPRI beads can be cleaned up and reused by adding a molecular crowding reagent (e.g., PEG) and salt.

[0220] In some embodiments, when modification-sensitive conversion is performed on a sample or subsample, subsequent capture of one or more sets of target regions (e.g., at least a set of epigenetic target regions) from the sample or subsample is performed using target-specific probes that are specific for the modification state (e.g., of at least one base of the sequence to which the probe hybridizes), e.g., probes that are complementary to target sequences that have undergone conversion (e.g., conversion of modified or unmodified cytosine to uracil or their analogs, such as DHU, which preferentially pairs with adenine) or, if desired, have not undergone conversion. Thus, the probes may be specific for sequences in which a modification of interest, such as methylation, was or was not present. In some embodiments, when modification-sensitive conversion is performed on a sample or sub-sample, subsequent capture of one or more sets of target regions (e.g., at least a set of epigenetic target regions) from the sample or sub-sample uses target-specific probes that include probes that can hybridize to target sequences regardless of modification state (e.g., containing nucleobases that indiscriminately pair at positions that may or may not have undergone conversion of modified or unmodified cytosines to uracil or their analogs, such as DHU, which preferentially pairs with adenine; e.g., inosine can pair with C or U).

[0221] In some embodiments, such methods include preparing a pool comprising at least a portion of the DNA of the second subsample (also referred to as the low methylation distribution fraction) and at least a portion of the DNA of the first subsample (also referred to as the high methylation distribution fraction). For example, target regions comprising epigenetic target regions and / or sequence variable target regions can be captured from the pool. The step of capturing a set of target regions from at least a portion of the subsamples described elsewhere herein includes a capture step performed on a pool comprising DNA from the first and second subsamples. The step of amplifying the DNA in the pool may be performed prior to capturing the target regions from the pool. The capture step may have any of the features described elsewhere herein.

[0222] Epigenetic target regions may exhibit differences in methylation levels and / or fragmentation patterns depending on whether they originate from a tumor or a healthy cell, or from what type of tissue, as discussed elsewhere herein. Sequence-variable target regions may exhibit differences in sequence depending on whether they originate from a tumor or a healthy cell.

[0223] Analysis of epigenetic target regions from low methylation distribution fractions may not be as informative in some applications as analysis of sequence variable target regions from high methylation distribution fractions and low methylation distribution fractions, and epigenetic target regions from high methylation distribution fractions. Thus, in methods where sequence variable target regions and epigenetic target regions are captured, the latter may be captured to a lesser extent than one or more of sequence variable target regions from high methylation distribution fractions and low methylation distribution fractions, and epigenetic target regions from high methylation distribution fractions. For example, sequence variable target regions can be captured from a portion of high methylation distribution fractions and low methylation distribution fractions that are not pooled, and pools can be prepared with a portion (e.g., most, substantially all, or all) of DNA from high methylation distribution fractions and zero or a portion (e.g., a small amount) of DNA from low methylation distribution fractions. In such approaches, sequencing of epigenetic target regions from low methylation distribution fractions can be reduced or eliminated, thereby reducing the amount of sequencing data that is sufficient for further analysis.

[0224] In some embodiments, including small amounts of DNA from the low methylation distribution fraction in the pool facilitates quantification, e.g., on a relative basis, of one or more epigenetic features (e.g., methylation or other epigenetic feature(s) discussed in detail elsewhere herein).

[0225] In some embodiments, the pool may contain a small amount of low methylation distribution fraction DNA, e.g., less than about 50% of the low methylation distribution fraction DNA, such as about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less of the low methylation distribution fraction DNA. In some embodiments, the pool contains about 5%-25% of the low methylation distribution fraction DNA. In some embodiments, the pool contains about 10%-20% of the low methylation distribution fraction DNA. In some embodiments, the pool contains about 10% of the low methylation distribution fraction DNA. In some embodiments, the pool contains about 15% of the low methylation distribution fraction DNA. In some embodiments, the pool contains about 20% of the low methylation distribution fraction DNA.

[0226] In some embodiments, the pool comprises a portion of the highly methylated distribution fraction, which may be at least about 50% of the DNA of the highly methylated distribution fraction. For example, the pool may comprise at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the DNA of the highly methylated distribution fraction. In some embodiments, the pool comprises 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100% of the DNA of the highly methylated distribution fraction. In some embodiments, the second pool comprises all or substantially all of the highly methylated distribution fraction.

[0227] In some embodiments, such methods include preparing a first pool comprising at least a portion of DNA from a low methylated distribution fraction. In some embodiments, such methods include preparing a second pool comprising at least a portion of DNA from a high methylated distribution fraction. In some embodiments, the first pool further comprises a portion of DNA from a high methylated distribution fraction. In some embodiments, the second pool further comprises a portion of DNA from a low methylated distribution fraction. In some embodiments, the first pool comprises a majority of DNA from a low methylated distribution fraction, and optionally a minority of DNA from a high methylated distribution fraction. In some embodiments, the second pool comprises a majority of DNA from a high methylated distribution fraction, and a minority of DNA from a low methylated distribution fraction. In some embodiments comprising an intermediately methylated distribution fraction, the second pool comprises at least a portion of DNA from an intermediately methylated distribution fraction, e.g., a majority of DNA from an intermediately methylated distribution fraction. In some embodiments, the first pool comprises a majority of DNA from a low methylated distribution fraction, and the second pool comprises a majority of DNA from a high methylated distribution fraction and a majority of DNA from an intermediately methylated distribution fraction.

[0228] In some embodiments, such methods include capturing at least a first set of target regions from a first pool, e.g., the first pool is as set forth in any of the above embodiments. In some embodiments, the first set includes sequence variable target regions. In some embodiments, the first set includes hypomethylated variable target regions and / or fragmented variable target regions. In some embodiments, the first set includes sequence variable target regions and fragmented variable target regions. In some embodiments, the first set includes sequence variable target regions, hypomethylated variable target regions, and fragmented variable target regions. A step of amplifying the DNA in the first pool may be performed prior to this capturing step. In some embodiments, capturing the first set of target regions from the first pool includes contacting the DNA of the first pool with a first set of target specific probes. In some embodiments, the first set of target specific probes includes target binding probes specific for sequence variable target regions. In some embodiments, the first set of target specific probes includes target binding probes specific for sequence variable target regions, hypomethylated variable target regions, and / or fragmented variable target regions.

[0229] In some embodiments, such methods include capturing a second set of target regions or a plurality of sets of target regions from a second pool, e.g., the first pool is as shown in any of the above embodiments. In some embodiments, the second plurality includes epigenetic target regions, such as hypermethylated variable target regions and / or fragmented variable target regions. In some embodiments, the second plurality includes sequence variable target regions and epigenetic target regions, such as hypermethylated variable target regions and / or fragmented variable target regions. A step of amplifying the DNA in the second pool may be performed prior to this capturing step. In some embodiments, capturing a second plurality of sets of target regions from the second pool includes contacting the DNA of the first pool with a second set of target specific probes, the second set of target specific probes including target binding probes specific for sequence variable target regions and target binding probes specific for epigenetic target regions. In some embodiments, the first set of target regions and the second set of target regions are not identical. For example, the first set of target regions may include one or more target regions that are not present in the second set of target regions. Alternatively or in addition, the second set of target regions may include one or more target regions that are not present in the first set of target regions. In some embodiments, at least one hypermethylated variable target region is captured from the second pool but not from the first pool. In some embodiments, a plurality of hypermethylated variable target regions is captured from the second pool but not from the first pool. In some embodiments, the first set of target regions includes sequence variable target regions and / or the second set of target regions includes epigenetic target regions. In some embodiments, the first set of target regions includes sequence variable target regions and fragmentation variable target regions, and the second set of target regions includes epigenetic target regions, such as hypermethylated variable target regions and fragmentation variable target regions.In some embodiments, the first set of target regions includes sequence variable target regions, fragmented variable target regions, and includes hypomethylated variable target regions, and the second set of target regions includes epigenetic target regions, such as hypermethylated variable target regions and fragmented variable target regions.

[0230] In some embodiments, the first pool comprises a majority of the DNA from the low methylation distribution fraction and a portion (e.g., about half) of the DNA from the high methylation distribution fraction, and the second pool comprises a portion (e.g., about half) of the DNA from the high methylation distribution fraction. In some such embodiments, the first set of target regions comprises sequence variable target regions and / or the second set of target regions comprises epigenetic target regions. The sequence variable target regions and / or epigenetic target regions may be as set forth in any of the embodiments described elsewhere herein. E. Adapter ligation or addition; tagging

[0231] In some embodiments, the disclosed method includes analyzing DNA in a sample. In such methods, adapters may be added to the DNA. This can be done simultaneously with the amplification procedure, for example, by providing the adapters at the 5' portion of the primer before or after the amplification step (when PCR is used, this may be called library preparation PCR or LP-PCR). In some embodiments, the adapters are added by other techniques, such as ligation. In some such methods, a first adapter is added to the nucleic acid by ligation to its 3' end, which may include ligation to single-stranded DNA, before any partitioning or capture step. The adapter can be used as a priming site for second strand synthesis, for example, using a universal primer and a DNA polymerase. A second adapter can then be ligated to at least the 3' end of the second strand of the now double-stranded molecule. In some embodiments, the first adapter includes an affinity tag, such as biotin, and the nucleic acid ligated to the first adapter is bound to a solid support (e.g., beads), which may include a binding partner for the affinity tag, such as streptavidin. For further discussion of related procedures, see Gansauge et al., Nature Protocols 8:737-748 (2013). Commercially available kits for sequencing library preparation that are compatible with single-stranded nucleic acids are available, such as the Accel-NGS® Methyl-Seq DNA Library Kit from Swift Biosciences. In some embodiments, after adaptor ligation, the nucleic acid is amplified.

[0232] Preferably, the adaptors contain a sufficient number of different tags such that the number of tag combinations results in a low probability, e.g., a 95, 99 or 99.9% probability, that two nucleic acids with the same start and stop points will receive the same tag combination. Adaptors, whether they have the same or different tags, may contain the same or different primer binding sites, although preferably the adaptors contain the same primer binding sites.

[0233] In some embodiments, after attachment of the adaptors, the nucleic acid is subjected to amplification, which can, for example, use a universal primer that recognizes the primer binding site of the adaptor.

[0234] In some embodiments, the method includes partitioning the DNA or a subsample or portion of the DNA after the attachment of the adaptor, and contacting the DNA with an agent that preferentially binds to nucleic acids with epigenetic modifications. The nucleic acid is partitioned into at least two partitioned subsamples that differ in the extent to which the nucleic acid has the modification by binding with the agent. For example, if the agent has an affinity for nucleic acids with modifications, nucleic acids with overabundance of the modification (compared to the median abundance in the population) will preferentially bind to the agent, while nucleic acids with underabundance of the modification will not bind or will be more easily eluted from the agent. The nucleic acid can then be amplified from a primer that binds to the primer binding site in the adaptor. Alternatively, partitioning may be performed before the attachment of the adaptor, in which case the adaptor may include a differential tag that includes a component that identifies which partitioned fraction the molecule was in.

[0235] In some embodiments, the nucleic acid is ligated at both ends to a Y-shaped adaptor that comprises a primer binding site and a tag. The molecule is amplified.

[0236] Tagging of DNA molecules is the procedure of attaching or associating a tag to a DNA molecule. Such a tag may be a molecule, such as a nucleic acid, that contains information indicative of a characteristic of the molecule with which the tag is associated. For example, the molecule may have a sample tag (that distinguishes a molecule in one sample from a molecule in another sample), or a molecular tag / molecular barcode / barcode (that distinguishes different molecules from each other (in both unique and non-unique tagging scenarios). In the case of methods that include a partitioning step, it may also include a partitioning tag (that distinguishes a molecule in one partitioned fraction from a molecule in another partitioned fraction). In some embodiments, the adapter that is added to the DNA molecule includes a tag. In certain embodiments, the tag may include a barcode or a combination of barcodes. As used herein, the term "barcode" refers to a nucleic acid molecule having a specific nucleotide sequence, or to the nucleotide sequence itself, depending on the context. A barcode may have, for example, 10-100 nucleotides. A collection of barcodes may have degenerate sequences or sequences with a certain Hamming distance depending on a particular purpose. Thus, for example, a molecular barcode may consist of one barcode or a combination of two barcodes, each attached to a different end of the molecule. Additionally or alternatively, for different distribution fractions and / or samples, different sets of molecular barcodes or molecular tags may be used, such that the barcodes serve as molecular tags due to their individual sequences, and also serve to identify the corresponding distribution fraction and / or sample based on the set of which the barcode is a member.

[0237] In some embodiments, two or more distribution fractions, for example each distribution fraction, are differentially tagged. Tags can be used to label individual polynucleotide population distribution fractions, such that the tag (or tags) are correlated with a particular distribution fraction. Alternatively, tags can be used in embodiments that do not use a distribution step. In some embodiments, a single tag can be used to label a particular distribution fraction. In some embodiments, multiple different tags can be used to label a particular distribution fraction. In embodiments where multiple different tags are used to label a particular distribution fraction, the set of tags used to label one distribution fraction can be easily distinguished from the set of tags used to label other distribution fractions. In some embodiments, the tag may have additional functionality, e.g., the tag may be used to index the sample source or may be used as a unique molecular identifier (which may be used to improve the quality of sequencing data by distinguishing sequencing errors from mutations, e.g., as in Kinde et al., Proc Nat'l Acad Sci USA 108: 9530-9535 (2011); Kou et al., PLoS ONE,11: e0146638 (2016)), or may be used as a non-unique molecular identifier, e.g., as described in U.S. Pat. No. 9,598,731. Similarly, in some embodiments, the tag may have additional functionality, e.g., the tag may be used to index the sample source or may be used as a non-unique molecular identifier (which may be used to improve the quality of sequencing data by distinguishing sequencing errors from mutations).

[0238] In some embodiments, the distribution fraction tagging comprises tagging the molecules in each distribution fraction with a distribution tag. After remixing the distribution fractions (e.g., to reduce the number of required sequencing runs and avoid unnecessary costs) and sequencing the molecules, the source distribution fraction is identified by the distribution tag. In another embodiment, different distribution fractions are tagged with different sets of molecular tags, e.g., composed of a pair of barcodes. In this way, each molecular barcode indicates the source distribution fraction, as well as being useful for distinguishing molecules within the distribution fraction. For example, a first set of 35 barcodes can be used to tag the molecules in the first distribution fraction, and a second set of 35 barcodes can be used to tag the molecules in the second distribution fraction.

[0239] In some embodiments, after partitioning and tagging with partitioning tags, molecules may be pooled for sequencing in a single run. In some embodiments, sample tags are added to molecules, for example, in a step following partitioning tag addition and pooling. Sample tags can facilitate pooling of material generated from multiple samples for sequencing in a single sequencing run.

[0240] Alternatively, in some embodiments, the distribution tag can be correlated with the sample and the distribution fraction.As a simple example, the first tag can indicate the first distribution fraction of the first sample, the second tag can indicate the second distribution fraction of the first sample, the third tag can indicate the first distribution fraction of the second sample, and the fourth tag can indicate the second distribution fraction of the second sample.

[0241] Tags may be attached to molecules that have already been distributed based on one or more characteristics, but the final tagged molecules in the library may no longer have those characteristics. For example, single-stranded DNA molecules may be distributed and tagged, but the final tagged molecules in the library are likely to be double-stranded. Similarly, DNA may be subjected to distribution based on different levels of methylation, but the tagged molecules derived from such molecules in the final library may not be methylated. Thus, the tags attached to molecules in the library typically represent the characteristics of the "parent molecule" from which the final tagged molecule is derived, and not necessarily the characteristics of the tagged molecule itself.

[0242] As an example, the molecules in the first distribution fraction are tagged and labeled using barcodes 1, 2, 3, 4, etc. The molecules in the second distribution fraction are tagged and labeled using barcodes A, B, C, D, etc. The molecules in the third distribution fraction are tagged and labeled using barcodes a, b, c, d, etc. The differentially tagged distribution fractions may be pooled before sequencing. The differentially tagged distribution fractions may be sequenced separately or may be sequenced together at the same time, for example, in the same flow cell of an Illumina sequencer.

[0243] After sequencing, analysis of reads can be performed at the level of each distribution fraction as well as at the level of the total DNA population. Tags are used to sort the reads of different distribution fractions. Analysis can include in silico analysis to determine genetic and epigenetic mutations (one or more of methylation, chromatin structure, etc.) using sequence information, genome coordinate length, coverage, and / or copy number. In some embodiments, higher coverage can be correlated with higher nucleosome occupancy in genomic regions, and lower coverage can be correlated with lower nucleosome occupancy or nucleosome depleted regions (NDRs).

[0244] Molecular tagging refers to a tagging operation that allows the DNA molecule from which a sequence read originates to be differentiated. Tagging strategies can be divided into unique and non-unique tagging strategies. In unique tagging, all or substantially all molecules in a sample have different tags, so that the reads can be assigned to the original molecule based on tag information alone. The tags used in such methods are sometimes called "unique tags". In non-unique tagging, different molecules in the same sample may have the same tag, so that other information in addition to the tag information is used to assign the sequence read to the original molecule. Such information can include start and end coordinates, coordinates to which the molecule is mapped, start coordinates alone or end coordinates alone, etc. The tags used in such methods are sometimes called "non-unique tags". Thus, it is not necessary to uniquely tag every molecule in a sample. It is sufficient to uniquely tag molecules that fall into a distinguishable class in a sample. Thus, molecules of different distinguishable families can have the same tag without losing information about the identity of the tagged molecule.

[0245] In certain embodiments of non-unique tagging, the number of different tags used may be sufficient to make it highly likely (e.g., at least 99%, at least 99.9%, at least 99.99%, or at least 99.999%) that all DNA molecules in a particular group have different tags. Note that when barcodes are used as tags, and when barcodes are attached, for example, randomly, to both ends of the molecules, a combination of barcodes together may constitute a tag. And this number is a function of the number of molecules that are classified into that call. For example, a class may be all molecules that map to the same start-end position of the reference genome. A class may be all molecules that map to a particular locus, for example, a particular base or across a particular region (e.g., up to 100 bases or a gene or exon of a gene). In certain embodiments, the number of different tags used to uniquely identify a number z of molecules in a class can be from any of 2*z, 3*z, 4*z, 5*z, 6*z, 7*z, 8*z, ​​9*z, 10*z, 11*z, 12*z, 13*z, 14*z, 15*z, 16*z, 17*z, 18*z, 19*z, 20*z, or 100*z (e.g., a lower limit) to any of 100,000*z, 10,000*z, 1000*z, or 100*z (e.g., an upper limit).

[0246] For example, in a sample of about 5 ng-30 ng of cell-free DNA, roughly 3000 molecules are expected to map to a particular nucleotide coordinate, with about 3-10 molecules with any given start coordinate expected to share the same end coordinate. Thus, about 50 to about 50,000 different tags (e.g., about 6-220 barcode combinations) may be sufficient to uniquely tag all such molecules. About 1 million to about 20 million different tags would be required to uniquely tag all 3,000 molecules that map across nucleotide coordinates.

[0247] In general, assignment of unique or non-unique tag barcodes in the reactions follows the methods and systems described in U.S. Patent Application Publication Nos. 20010053519, 20030152490, 20110160078, and U.S. Patent Nos. 6,582,908, 7,537,898, and 9,598,731. Tags may be randomly or non-randomly linked to the sample nucleic acids.

[0248] In some embodiments, the tagged nucleic acid is sequenced after loading into microwell plate.Microwell plate may have 96,384 or 1536 microwells.In some cases, they are introduced into microwell with expected ratio of unique tags.For example, unique tags can be loaded so that more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags are loaded per genome sample. In some cases, unique tags can be loaded such that less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags are loaded per genomic sample. In some cases, the average number of unique tags loaded per sample genome is less than or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags per genome sample.

[0249] In a preferred format, 20-50 different tags (e.g., barcodes) ligated to both ends of a target nucleic acid are used. For example, 35 different tags (e.g., barcodes) are ligated to both ends of a target molecule to create 35x35 permutations, which is equivalent to 1225 for 35 tags. Such a number of tags is sufficient to ensure a high probability (e.g., at least 94%, 99.5%, 99.99%, 99.999%) that different molecules with the same start and end points will receive different combinations of tags. Other barcode combinations include any number between 10 and 500, such as about 15x15, about 35x35, about 75x75, about 100x100, about 250x250, about 500x500.

[0250] In some cases, the unique tag may be an oligonucleotide of predetermined or random or semi-random sequence. In other cases, multiple barcodes may be used, and the multiple barcodes are not necessarily unique to each other. In this example, the barcode can be ligated to each molecule, such that the combination of the barcode and the sequence to which it can be ligated creates a unique sequence that can be tracked individually. As described herein, detection of sequence data non-unique barcodes in combination with the beginning (start) and end (end) parts of sequence reads can allow for the assignment of a unique identity to a particular molecule. The length or number of base pairs of each sequence read can also be used to assign a unique identity to such a molecule. As described herein, fragments derived from a single strand of nucleic acid that are assigned a unique identity can thereby allow subsequent identification of fragments derived from the parent strand. F. Enrichment / capture step; Amplification

[0251] The method disclosed herein may comprise enriching or capturing DNA, such as type-specific cfDNA target region that is also copy number variant.In some embodiments, capturing comprises contacting DNA with a probe that is specific to the target region.Enrichment or capture can be carried out on any sample or subsample described herein by using any suitable method known in the art.

[0252] In some embodiments, the probe specific for the target region (i.e., the target-specific probe) comprises a capture moiety that facilitates enrichment or capture of DNA hybridized to the probe. In some embodiments, the capture moiety is biotin. In some such embodiments, streptavidin attached to a solid support, such as a magnetic bead, is used to bind to the biotin. Non-specifically bound DNA that does not contain the target region is washed away from the captured DNA. In some embodiments, the DNA is then dissociated from the probe and eluted from the solid support using a buffer containing a salt wash or another DNA denaturing agent. In some embodiments, the probe is also eluted from the solid support, for example, by disrupting the biotin-streptavidin interaction. In some embodiments, the captured DNA is amplified after elution from the solid support. In some such embodiments, the DNA containing the adapter is amplified using a PCR primer that anneals to the adapter. In some embodiments, the captured DNA is amplified while still attached to the solid support. In some such embodiments, the amplification comprises the use of a PCR primer that anneals to a sequence in the adapter and a PCR primer that anneals to a sequence in the probe that anneals to the target region of the DNA.

[0253] In some embodiments, the methods herein include enriching or capturing DNA comprising epigenetic and / or sequence variable target regions. Such regions may be captured from an aliquot, portion, or subsample of a sample (e.g., a sample that has undergone adapter attachment and amplification), and a step of distributing the DNA with an agent that recognizes methylcytosine is performed on a separate aliquot, portion, or subsample of the sample. Enriching or capturing DNA comprising epigenetic and / or sequence variable target regions may include contacting the DNA with a first or second set of target specific probes. Such target specific probes may have any of the characteristics described herein for a set of target specific probes, including but not limited to those in the embodiments set forth above and in the section on probes below. The capture may be performed on one or more subsamples prepared by the methods disclosed herein. In some embodiments, DNA is captured from a first subsample or a second subsample. In some embodiments, the subsamples are differentially tagged (e.g., as described herein) and then pooled before undergoing capture. Exemplary methods for capturing DNA containing epigenetic and / or sequence variable target regions can be found, for example, in WO2020 / 160414, which is hereby incorporated by reference herein.

[0254] The capture step or steps can be carried out using conditions suitable for specific nucleic acid hybridization, which generally depend in part on the characteristics of the probe, such as length, base composition, etc. Those skilled in the art will be familiar with suitable conditions, given their general knowledge in the art of nucleic acid hybridization.

[0255] In some embodiments, the method described herein comprises capturing a plurality of sets of target regions of cfDNA obtained from a subject. The target regions may contain differences depending on whether they originate from a tumor or from a healthy cell or from a certain cell type. In the capture step, a capture set of cfDNA molecules is generated. In some embodiments, cfDNA molecules corresponding to the set of sequence-variable target regions are captured with a higher capture yield in the capture set of cfDNA molecules than cfDNA molecules corresponding to the set of epigenetic target regions. In some embodiments, the method described herein comprises contacting cfDNA obtained from a subject with a set of target-specific probes, the set of target-specific probes being configured to capture cfDNA corresponding to the set of sequence-variable target regions with a higher capture yield than cfDNA corresponding to the set of epigenetic target regions. For additional discussion of the capture step, capture yield, and related aspects, see WO2020 / 160414, which is incorporated herein by reference for all purposes.

[0256] It may be beneficial to capture cfDNA corresponding to a set of sequence-variable target regions at a higher capture yield than cfDNA corresponding to a set of epigenetic target regions, since a deeper depth of sequencing may be required to analyze the sequence-variable target regions with sufficient reliability or accuracy than may be required to analyze epigenetic target regions. The amount of data required to determine fragmentation patterns (e.g., to investigate breakage of transcription start sites or CTCF binding sites) or fragment abundances (e.g., in high- and low-methylation distribution fractions) is generally less than the amount of data required to determine the presence or absence of cancer-associated sequence mutations. Capturing a set of target regions at different yields may facilitate sequencing target regions to different sequencing depths in the same sequencing run (e.g., using pooled mixtures and / or within the same sequencing cell). Copy number variations such as local amplifications are somatic mutations, but can be detected by sequencing based on read frequency, similar to the techniques for detecting certain epigenetic changes, such as methylation changes. Therefore, they can be considered epigenetic target regions for functional reasons. In addition, regions that exhibit copy number variation that are also hypermethylated or fragmented variable target regions may exhibit epigenetic variation and are therefore considered epigenetic target regions.

[0257] In some embodiments, the DNA is amplified. In some embodiments, the amplification is performed before the capture step. In some embodiments, the amplification is performed after the capture step. In some embodiments, the amplification is performed before and after the capture step. In various embodiments, such methods further include sequencing the captured DNA, for example, to different degrees of sequencing depth for the set of epigenetic target regions and the set of sequence variable target regions, consistent with the discussion herein. In some embodiments, an RNA probe is used. In some embodiments, a DNA probe is used. In some embodiments, a single-stranded probe is used. In some embodiments, a double-stranded probe is used. In some embodiments, a single-stranded RNA probe is used. In some embodiments, a double-stranded DNA probe is used.

[0258] In some embodiments, the capture step is performed simultaneously in the same vessel using probes for a set of sequence variable target regions and probes for a set of epigenetic target regions, e.g., the probes for the set of sequence variable target regions and the set of epigenetic target regions and the capture probes are in the same composition. This approach provides a relatively simple workflow.

[0259] In some embodiments, the adapter is included in the DNA, as described herein. In some embodiments, the tag may be or include a barcode and is included in the DNA. In some embodiments, such a tag is included in the adapter. The tag can facilitate identification of the origin of the nucleic acid. For example, the barcode can allow identification of the source (e.g., subject) from which the DNA originated after pooling multiple samples for parallel sequencing. This can be done simultaneously with the amplification procedure, for example by providing a barcode in the 5' portion of the primer, as described herein. In some embodiments, the adapter and the tag / barcode are provided by the same primer or primer set. For example, the barcode may be located 3' of the adapter and 5' of the target hybridization portion of the primer. Alternatively, the barcode can be added by other techniques, such as ligation, if desired, with the adapter of the same ligation substrate.

[0260] Additional details regarding amplification, tags, and barcodes are discussed herein, which may be combined to the extent practicable with any of these embodiments. G. Captured DNA; target region

[0261] In some embodiments, the nucleic acid captured or enriched using the methods described herein comprises captured DNA. In some embodiments, the captured DNA comprises a region that contains both type-specific epigenetic mutations and copy number mutations. In some embodiments, the mutations are present in healthy cells but are not normally present in sample types such as blood samples. In some embodiments, the mutations are present in abnormal cells (e.g., hyperplastic, metaplastic, or neoplastic cells).

[0262] In some embodiments, the first captured epigenetic target region set captured from the sample or the first subsample comprises a hypermethylated variable target region. In some embodiments, the hypermethylated variable target region shows type-specific hypermethylation in healthy cfDNA derived from one or more related cell types or tissue types. Without wishing to be bound by any particular theory, the presence of cancer cells may increase the discharge of DNA (e.g., from cancer and / or surrounding tissues) into the bloodstream. Therefore, the distribution of tissues from which cfDNA originates may change upon carcinogenesis. Thus, an increase in the level of hypermethylated variable target region in the first subsample may be an indication of the presence of cancer (or recurrence depending on the subject's medical history).

[0263] In some embodiments, the method herein comprises capturing a second set of captured epigenetic target regions from the sample or the second subsample. In some embodiments, the second set of epigenetic target regions comprises hypomethylated variable target regions. Without wishing to be bound by any particular theory, cancer cells may shed more DNA into the bloodstream than healthy cells of the same tissue type. Therefore, the distribution of tissues from which cfDNA originates may change during carcinogenesis. Thus, an increase in the level of hypomethylated variable target regions in the second subsample may be an indication of the presence (or recurrence depending on the subject's medical history) of cancer.

[0264] In addition, the set of captured target regions may contain DNA corresponding to a set of sequence variable target regions. The capture sets can be combined to provide a combined capture set.

[0265] In some embodiments, where the capture set includes DNA corresponding to the set of sequence variable target regions and the set of epigenetic target regions, including the combined capture set discussed above, the DNA corresponding to the set of sequence variable target regions is at a higher concentration than the DNA corresponding to the set of epigenetic target regions, e.g., at 1.1-1.2 fold concentration, at 1.2-1.4 fold concentration, at 1.4-1.6 fold concentration, at 1.6-1.8 fold concentration, at 1.8-2.0 fold concentration, at 2.0-2.2 fold concentration, at 2.2-2.4 fold concentration, at 2.4-2.6 fold concentration, at 2.6-2.8 fold concentration, at 2.8-3.0 fold concentration, at 3.0-3.5 fold concentration, at 3.5-4.0, at 4.0-4.5 fold concentration, at 4.5-5.0 fold concentration, at 5.0-5.5 fold concentration. , 5.5-6.0x concentration, 6.0-6.5x concentration, 6.5-7.0x concentration, 7.0-7.5x concentration, 7.5-8.0x concentration, 8.0-8.5x concentration, 8.5-9.0x concentration, 9.0-9.5x concentration, 9.5-10.0x concentration, 10-11x concentration, 11-12x concentration, 12-13x concentration, 13-14x concentration, 1 It may be present at a concentration of 4-15x, 15-16x, 16-17x, 17-18x, 18-19x, 19-20x, 20-30x, 30-40x, 40-50x, 50-60x, 60-70x, 70-80x, 80-90x, or 90-100x. As discussed in the definitions section, the degree of concentration difference corresponds to a normalization of the footprint size of the target region. 1.Target area

[0266] In some embodiments, the captured DNA comprises the region with type-specific epigenetic mutation and copy number mutation.In some embodiments, the epigenetic target region set consists of the target region with type-specific epigenetic mutation and copy number mutation.In some embodiments, type-specific epigenetic mutation, for example, differential methylation or type-specific fragmentation pattern, is likely to distinguish the DNA from one or more related cell types or tissue types from the DNA from other cell types or tissue types present in the sample or subject.

[0267] In some embodiments, the captured epigenetic target region set captured from the sample or first subsample comprises a hypermethylated variable target region. In some embodiments, the hypermethylated variable target region is differentially or exclusively hypermethylated in one or more related cell types or tissue types. Such hypermethylated variable target regions may be hypermethylated in other cell types or tissue types, but not to the extent observed in one or more related cell types or tissue types. In some embodiments, the hypermethylated variable target region shows even higher methylation in cfDNA derived from diseased cells of one or more related cell types or tissue types. In some embodiments, the target region comprises a hypermethylated region with abnormally high copy number. In some such embodiments, the target region is hypermethylated in healthy and diseased colon tissues and has abnormally high copy number in precancerous or cancerous colon tissues. Examples of such target regions are shown in Table 1 below. [Table 1-1] [Table 1-2]

[0268] In some embodiments, the captured epigenetic target region set captured from the sample or subsample comprises hypomethylated variable target regions.In some embodiments, the hypomethylated variable target regions are exclusively hypomethylated in one or more related cell types or tissue types.Such hypomethylated variable target regions may be hypomethylated in other cell types or tissue types, but not to the extent observed in one or more related cell types or tissue types.

[0269] Without wishing to be bound by any particular theory, in individuals with cancer, proliferating or dying cancer cells may release more DNA into the bloodstream than cells of healthy individuals and / or healthy cells of the same tissue type.Therefore, the distribution of cell types and / or tissues from which cfDNA originates may change during carcinogenesis.Therefore, the presence and / or level of cfDNA originating from a certain cell type or tissue type may be an indicator of disease.

[0270] Further exemplary hypermethylated and hypomethylated variable target regions useful for distinguishing between various cell types have been identified by analyzing DNA from various cell types by whole genome bisulfite sequencing, as described, for example, in Scott, CA, Duryea, JD, MacKay, H. et al., "Identification of cell type-specific methylation signals in bulk whole genome bisulfite sequencing data," Genome Biol 21, 156 (2020) (doi.org / 10.1186 / s13059-020-02065-5). Whole genome bisulfite sequence data is available from the Blueprint Consortium and available on the Internet at dcc.blueprint-epigenome.eu.

[0271] In some embodiments, the first and second capture target region sets comprise DNA corresponding to a sequence variable target region set and DNA corresponding to an epigenetic target region set, respectively, for example as described in WO2020 / 160414. The first and second capture sets may be combined to provide a combined capture set. In some embodiments, the sequence variable target region set and the epigenetic target region set may have any of the features described for such sets in WO2020 / 160414, which is incorporated herein by reference in its entirety. In some embodiments, the epigenetic target region set comprises a hypermethylated variable target region set. In some embodiments, the epigenetic target region set comprises a hypomethylated variable target region set. In some embodiments, the epigenetic target region set comprises a CTCF binding region. In some embodiments, the epigenetic target region set comprises a fragmented variable target region. In some embodiments, the epigenetic target region set comprises a transcription start site.

[0272] In some embodiments, the sequence variable target region set includes a plurality of regions known to undergo somatic mutations in cancer. In some aspects, the sequence variable target region set targets a plurality of different genes or genomic regions ("panels") selected such that a determined percentage of subjects with cancer exhibits genetic variants or tumor markers in one or more different genes or genomic regions in the panel. The panel can be selected to restrict the region for sequencing to a certain number of base pairs. The panel may be selected to sequence a desired amount of DNA, for example, by adjusting the affinity and / or amount of probes as described elsewhere herein. The panel may further be selected to achieve a desired sequence read depth. The panel may be selected to achieve a desired sequence read depth or sequence read coverage relative to the amount of base pairs sequenced. The panel may be selected to achieve a theoretical sensitivity, theoretical specificity, and / or theoretical accuracy for detecting one or more genetic variants in a sample.

[0273] The probe for detecting the panel of regions can include the probe for detecting the genomic region of interest (hotspot region).The design of the probe can take into account information about chromatin structure, and / or the probe can be designed to maximize the possibility of capturing a specific site (for example, KRAS codons 12 and 13), and can be designed to optimize capture based on the analysis of cfDNA coverage and fragment size variation that is influenced by nucleosome binding pattern and GC sequence composition.The region used herein can include the non-hotspot region that is optimized based on nucleosome position and GC model.

[0274] The probe for detecting a panel of regions can include the probe for detecting genomic regions of interest (hotspot regions).The design of the probe can take into account information about chromatin structure, and / or the probe can be designed to maximize the possibility that a particular site (e.g., KRAS codons 12 and 13) can be captured, and can be designed to optimize capture based on the analysis of cfDNA coverage and fragment size variation that is influenced by nucleosome binding patterns and GC sequence composition.In addition, the region used herein can include non-hotspot regions that are optimized based on nucleosome position and GC model.

[0275] Examples of lists of genomic locations of interest can be found in WO2020 / 160414, for example, in Table 4. In some embodiments, the sequence variable target region set used in the method of the present disclosure comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at least 70 portions of the genes in Table 3 of WO2020 / 160414. In some embodiments, the sequence variable target region set used in the method of the present disclosure comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or at least 73 portions of the genes in Table 4 of WO2020 / 160414. Additionally or alternatively, suitable target region sets are available from the literature. For example, Gale et al., PLoS One 13: e0194630 (2018), incorporated herein by reference, describes a panel of 35 cancer-related gene targets that can be used as part or all of a sequence variable target region set.These 35 targets are AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MED12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, and U2AF1.

[0276] In some embodiments, the set of sequence variable target regions includes target regions from at least 10, 20, 30, or 35 cancer-associated genes, such as those listed above and in WO2020 / 160414. H. Sequencing

[0277] Generally, the sample nucleic acid, including the nucleic acid flanked by adaptor, can be subjected to sequencing, whether or not it has been previously amplified.Sequencing methods include, for example, Sanger sequencing, high-throughput sequencing, pyrosequencing, sequencing by synthesis, single molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing by ligation, sequencing by hybridization, digital gene expression (Helicos), next-generation sequencing (NGS), single molecule sequencing by synthesis (SMSS) (Helicos), enzymatic methyl sequencing (EM-Seq), Tet-assisted pyridine borane sequencing (TAPS), massively parallel sequencing, clonal single molecule array (Solexa), shotgun sequencing, Ion Torrent, Oxford Nanopore, Roche Genia, Maxim-Gilbert sequencing, primer walking, and sequencing using PacBio, SOLiD, Ion Torrent or Nanopore platform.

[0278] In some embodiments, sequencing includes detecting and / or distinguishing between unmodified and modified nucleobases. For example, single molecule real-time (SMRT) sequencing and nanopore sequencing can facilitate direct detection of, for example, 5-methylcytosine and 5-hydroxymethylcytosine as well as unmodified cytosine. See, for example, Schatz., Nature Methods. 14(4): 347-348 (2017); US9,150,918; and Simpson et al., Nature Methods. 14:407-410, doi:10.1038 / nmeth.4184 (2017). Sequencing reactions can be performed in a variety of sample processing units, which may be multiple lanes, multiple channels, multiple wells, or other means of processing multiple sample sets substantially simultaneously. Sample processing units may also include multiple sample chambers so that multiple runs can be processed simultaneously.

[0279] The sequencing reaction can be performed on one or more forms of nucleic acid, such as nucleic acids known to contain markers for cancer or other diseases. The sequencing reaction can also be performed on any nucleic acid fragments present in the sample. In some embodiments, the sequence coverage of the genome can be less than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100%. In some embodiments, the sequencing reaction can provide sequence coverage of at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the genome. Sequence coverage can be performed for at least 5, 10, 20, 70, 100, 200, or 500 different genes, or up to 5000, 2500, 1000, 500, or 100 different genes.

[0280] Simultaneous sequencing reaction can be carried out using multiplex sequencing.In some cases, cell-free nucleic acid can be sequenced using at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions.In other cases, cell-free nucleic acid can be sequenced using less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions.Sequencing reactions can be carried out sequentially or simultaneously.Subsequent data analysis can be carried out on all or part of sequencing reactions. In some cases, data analysis may be performed on at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions. In other cases, data analysis may be performed on less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions. Exemplary read depths are 1000-50000 reads per locus (base). III. ADDITIONAL FEATURES OF CERTAIN DISCLOSED METHODS A. Sample

[0281] The sample may be any biological sample isolated from a subject. The sample may be a body sample. The sample may include body tissues or fluids such as known or suspected solid tumors, whole blood, platelets, serum, plasma, stool, red blood cells, white blood cells or leukocytes, endothelial cells, tissue biopsy, cerebrospinal fluid, synovial fluid, lymphatic fluid, ascites, interstitial or extracellular fluid, fluid in the space between cells, gingival crevicular fluid, bone marrow, pleural fluid, pleural fluid, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, and urine. The sample is preferably a body fluid, in particular blood and its fractions, cerebrospinal fluid, pleural fluid, saliva, sputum, or urine. The sample may be in the original form isolated from the subject, or may have been subjected to further processing to remove or add components such as cells, or to enrich one component relative to another. Thus, the preferred body fluid for analysis is plasma or serum, which contains cell-free nucleic acid.

[0282] In some embodiments, the population of nucleic acids is obtained from serum, plasma or blood samples from subjects suspected of having neoplasm, tumor, precancer or cancer, or from subjects previously diagnosed with neoplasm, tumor, precancer or cancer.The population comprises nucleic acids with various levels of sequence mutation, epigenetic mutation, and / or post-replicative or post-transcriptional modification.Post-replicative modifications include, in particular, modifications of cytosine at the 5th position of nucleic acid base, such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine.

[0283] The sample can be isolated or obtained from the subject and transported to the site of sample analysis. The sample can be stored and shipped at a desired temperature, for example, room temperature, 4°C, -20°C, and / or -80°C. The sample can be isolated or obtained from the subject at the site of sample analysis. The subject can be a human, a mammal, an animal, a companion animal, a service animal, or a pet. The subject can have cancer, precancer, an infection, transplant rejection, or other disease or disorder related to alterations in the immune system. The subject can have no cancer or detectable symptoms of cancer. The subject can be treated with one or more cancer therapies, for example, any one or more of chemotherapy, antibodies, vaccines, or biologics. The subject can be in remission. The subject may or may not have been diagnosed with cancer or susceptible to any cancer-related genetic mutation / disorder.

[0284] In some embodiments, the sample comprises plasma. The volume of plasma obtained may depend on the desired read depth of the sequenced region. Exemplary volumes are 0.4-40 ml, 5-20 ml, 10-20 ml. For example, the volume may be 0.5 mL, 1 mL, 5 mL 10 mL, 20 mL, 30 mL, or 40 mL. The volume of plasma collected may be 5-20 mL.

[0285] A sample may contain various amounts of nucleic acid, including genomic equivalents. For example, a sample of about 30 ng of DNA may contain approximately 10,000 (10 4 ) haploid human genome equivalents, and for cfDNA, approximately 200 billion (2 × 10 11 Similarly, a sample of about 100 ng of DNA may contain about 30,000 haploid human genome equivalents, and in the case of cfDNA, about 600 billion individual molecules.

[0286] The sample may include nucleic acids from different sources, e.g., from cells and acellular samples of the same subject, from cells and acellular samples of different subjects. The sample may include nucleic acids having mutations. For example, the sample may include DNA having germline mutations and / or somatic mutations. A germline mutation refers to a mutation present in the germline DNA of a subject. A somatic mutation refers to a mutation originating from a somatic cell of a subject, e.g., a cancer cell. The sample may include DNA having a cancer-associated mutation (e.g., a cancer-associated somatic mutation). The sample may include epigenetic variants (i.e., chemical or protein modifications), where the epigenetic variants are associated with the presence of a genetic variant, such as a cancer-associated mutation. In some embodiments, the sample includes epigenetic variants associated with the presence of a genetic variant, and the sample does not include a genetic variant.

[0287] Exemplary amounts of cell-free nucleic acid in a sample prior to amplification range from about 1 fg to about 1 μg, e.g., 1 pg to 200 ng, 1 ng to 100 ng, 10 ng to 1000 ng. For example, the amount may be up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acid molecules. The amount may be at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of cell-free nucleic acid molecules. The amount may be up to 1 femtogram (fg), 10 fg, 100 fg, 1 picogram (pg), 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng, or 200 ng of cell-free nucleic acid molecules. The method may include obtaining between 1 femtogram (fg) and 200 ng.

[0288] Cell-free DNA refers to DNA that is not contained within cells when isolated from a subject. For example, cfDNA can be isolated from a sample as DNA remaining in the sample after removing intact cells without lysing cells or extracting intracellular DNA. Cell-free nucleic acids include DNA, RNA, and hybrids thereof, including genomic DNA, mitochondrial DNA, siRNA, miRNA, circular RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi-binding RNA (piRNA), long non-coding RNA (long ncRNA), or fragments of any of these. Cell-free nucleic acids may be double-stranded, single-stranded, or hybrids thereof. Cell-free nucleic acids may be released into bodily fluids by secretion or cell death processes, such as cell necrosis and apoptosis. Some cell-free nucleic acids, such as circulating tumor DNA (ctDNA), are released into bodily fluids from cancer cells. Others are released from healthy cells. In some embodiments, cfDNA is cell-free fetal DNA (cffDNA). In some embodiments, cell-free nucleic acids are produced by tumor cells. In some embodiments, the cell-free nucleic acid is produced by a mixture of tumor cells and non-tumor cells.

[0289] Cell-free nucleic acids have an exemplary size distribution of about 100-500 nucleotides, with molecules of 110 to about 230 nucleotides accounting for about 90% of the molecules, with the mode at about 168 nucleotides, and a second minor peak in the range of 240-440 nucleotides.

[0290] Cell-free nucleic acids can be isolated from bodily fluids by a fractionation or partitioning step in which the cell-free nucleic acids found in solution are separated from intact cells and other insoluble components of the bodily fluid. Partitioning may include techniques such as centrifugation or filtration. Alternatively, cells in the bodily fluid may be lysed and the cell-free and cellular nucleic acids processed together. Generally, after addition of buffer and washing steps, the nucleic acids can be precipitated with alcohol. Further clean-up steps such as silica-based columns may be used to remove contaminants or salts. Non-specific bulk carrier nucleic acids such as C1 DNA, DNA, or proteins for bisulfite sequencing, hybridization, and / or ligation can be added throughout the reaction to optimize certain aspects of the procedure, such as yield.

[0291] After such processing, the sample may contain various forms of nucleic acid, including double-stranded DNA, single-stranded DNA, and single-stranded RNA. In some embodiments, single-stranded DNA and RNA can be converted to double-stranded forms, which are included in subsequent processing and analysis steps.

[0292] The double-stranded DNA molecules in the sample and the single-stranded nucleic acid molecules converted to double-stranded DNA molecules may be ligated to an adaptor at either one or both ends. Typically, the double-stranded molecules are blunt-ended by treatment with a polymerase having a 5'-3' polymerase and a 3'-5' exonuclease (or proofreading function) in the presence of all four standard nucleotides. Examples of suitable polymerases are Klenow large fragment and T4 polymerase. The blunt-ended DNA molecules may be ligated to at least partially double-stranded adaptors (e.g., Y-shaped or bell-shaped adaptors). Alternatively, complementary nucleotides may be added to the blunt ends of the sample nucleic acid and adaptor to facilitate ligation. Both blunt-end ligation and sticky-end ligation are contemplated herein. In blunt-end ligation, both the nucleic acid molecule and the adaptor tag have blunt ends. In sticky-end ligation, typically, the nucleic acid molecule has an "A" overhang and the adaptor has a "T" overhang. B. Amplification

[0293] The sample nucleic acid flanked by adaptors can be amplified by PCR and other amplification methods. Amplification is typically primed by a primer that binds to the primer binding site of the adaptor that flanks the DNA molecule to be amplified. The amplification method may include cycles of denaturation, annealing, and extension that occur by thermal cycling, or may be isothermal, as in the case of transcription-mediated amplification. Other amplification methods include ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustained sequence-based replication.

[0294] In some embodiments, the method performs dsDNA ligation with T-tailed and C-tailed adapters, resulting in at least 50, 60, 70, or 80% amplification of the double-stranded nucleic acid before it is ligated to the adapter. Preferably, the method increases the amount or number of amplified molecules by at least 10, 15, or 20% compared to a control method performed with only T-tailed adapters. C. Capture part

[0295] As discussed above, the nucleic acid in the sample may be subjected to a capture step in which molecules with certain characteristics are captured and analyzed. Target capture may include the use of a bait set that includes oligonucleotide baits labeled with a capture moiety, such as biotin or other examples described below. The probes may have sequences selected to be arranged side-by-side across a panel of regions, such as genes. In some embodiments, the bait set may have higher and lower capture yields for a set of target regions, such as the capture yields of a sequence-variable target region set and an epigenetic target region set, respectively, as discussed elsewhere herein. Such a bait set is combined with the sample under conditions that allow hybridization of the target molecule with the bait. The captured molecules are then isolated using a capture moiety. DNA capture may include the use of a capture moiety, such as a target-specific probe labeled with biotin, and an oligonucleotide labeled with a second moiety or binding partner that binds to the capture moiety, such as streptavidin. In some embodiments, the capture moiety and binding partner may have higher and lower capture yields for different sets of probes, such as those used to capture sequence variable target region sets and epigenetic target region sets, respectively, as discussed elsewhere herein.Methods that include capture moieties are further described, for example, in US9,850,523, issued December 26, 2017.This document is incorporated herein by reference.

[0296] Capture moieties include, but are not limited to, biotin, avidin, streptavidin, nucleic acids containing specific nucleotide sequences, haptens recognized by antibodies, and magnetically attractive particles. Extraction moieties may be members of binding pairs, such as biotin / streptavidin or hapten / antibody. In some embodiments, the capture moiety attached to the analyte is captured by its binding partner attached to a separable moiety, such as a magnetically attractive particle or a large particle that can be sedimented by centrifugation. The capture moiety may be any type of molecule that allows affinity separation of nucleic acids that have a capture moiety and nucleic acids that lack a capture moiety. Exemplary capture moieties are biotin, which allows affinity separation by binding to streptavidin that is linked or linkable to a solid phase, or oligonucleotides that allow affinity separation by binding to complementary oligonucleotides that are linked or linkable to a solid phase. D. Collection of target-specific probes

[0297] In some embodiments, a collection of target-specific probes is used in the methods described herein. In some embodiments, the collection of target-specific probes includes target binding probes specific to a set of sequence-variable target regions and target binding probes specific to a set of epigenetic target regions. In some embodiments, the capture yield of the target binding probes specific to a set of sequence-variable target regions is higher (e.g., at least 2-fold higher) than the capture yield of the target binding probes specific to a set of epigenetic target regions. In some embodiments, the collection of target-specific probes is configured to have a capture yield specific to a set of sequence-variable target regions that is higher (e.g., at least 2-fold higher) than its capture yield specific to a set of epigenetic target regions.

[0298] In some embodiments, the capture yield of target binding probes specific for the set of sequence variable target regions is at least 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times higher than the capture yield of target binding probes specific for the set of epigenetic target regions. In some embodiments, the capture yield of target binding probes specific for the set of sequence variable target regions is 1.25-1.5, 1.5-1.75, 1.75-2, 2-2.25, 2.25-2.5, 2.5-2.75, 2.75-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 13-14, or 14-15 times higher than the capture yield of target binding probes specific for the set of epigenetic target regions.

[0299] In some embodiments, the collection of target-specific probes is configured to have a capture yield specific for a set of sequence variable target regions that is at least 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times higher than its capture yield specific for a set of epigenetic target regions. In some embodiments, the collection of target-specific probes is configured to have a capture yield specific for a set of sequence variable target regions that is 1.25-1.5, 1.5-1.75, 1.75-2, 2-2.25, 2.25-2.5, 2.5-2.75, 2.75-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 13-14, or 14-15 times higher than its capture yield specific for the set of epigenetic target regions.

[0300] A collection of probes can be configured in a variety of ways to provide higher capture yields for a set of sequence-variable target regions, including concentration, different lengths and / or chemistries (e.g., affecting affinity), and combinations thereof. Affinity can be modulated by adjusting the length of the probe and / or by including nucleotide modifications as discussed below.

[0301] In some embodiments, the target-specific probes specific to the set of sequence variable target regions are present in a higher concentration than the target-specific probes specific to the set of epigenetic target regions. In some embodiments, the concentration of the target-binding probes specific to the set of sequence variable target regions is at least 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times higher than the concentration of the target-binding probes specific to the set of epigenetic target regions. In some embodiments, the concentration of target binding probes specific to the set of sequence variable target regions is 1.25-1.5, 1.5-1.75, 1.75-2, 2-2.25, 2.25-2.5, 2.5-2.75, 2.75-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 13-14, or 14-15 times higher than the concentration of target binding probes specific to the set of epigenetic target regions. In such embodiments, concentration may refer to the average mass per volume concentration of the individual probes in each set.

[0302] In some embodiments, target-specific probes specific to sequence-variable target regions set have higher affinity to their targets than target-specific probes specific to epigenetic target regions set. Affinity can be modulated in any manner known to those skilled in the art, such as by using different probe chemistries. For example, certain nucleotide modifications, such as cytosine 5-methylation (in certain sequence contexts), modifications that introduce heteroatoms at the 2' sugar position, and LNA nucleotides, can increase the stability of double-stranded nucleic acids, indicating that oligonucleotides with such modifications have relatively higher affinity to their complementary sequences. See, for example, Severin et al., Nucleic Acids Res. 39: 8740-8751 (2011); Freier et al., Nucleic Acids Res. 25: 4429-4443 (1997); U.S. Patent No. 9,738,894. Also, longer sequence lengths will generally result in increased affinity. Other nucleotide modifications, such as substituting guanine with the nucleobase hypoxanthine, reduce the amount of hydrogen bonding between the oligonucleotide and its complementary sequence, thereby reducing affinity. In some embodiments, the target-specific probes specific to the set of sequence-variable target regions have modifications that increase affinity for their targets. In some embodiments, alternatively or in addition, the target-specific probes specific to the set of epigenetic target regions have modifications that decrease affinity for their targets. In some embodiments, the target-specific probes specific to the set of sequence-variable target regions have a longer average length and / or a higher average melting temperature than the target-specific probes specific to the set of epigenetic target regions. These embodiments can be combined with each other and / or with the concentration differences discussed above to achieve a desired fold difference in capture yield, such as any fold difference or range discussed above.

[0303] In some embodiments, the target-specific probe comprises a capture moiety. The capture moiety may be any of the capture moieties described herein, such as biotin. In some embodiments, the target-specific probe is covalently or non-covalently linked to a solid support, such as by the interaction of the binding pair of the capture moiety. In some embodiments, the solid support is a bead, such as a magnetic bead.

[0304] In some embodiments, the target specific probes specific to a set of sequence variable target regions and / or the target specific probes specific to a set of epigenetic target regions are sequences selected to be juxtaposed across a panel of regions, such as the bait sets discussed above, e.g., probes comprising capture moieties, and genes.

[0305] In some embodiments, the target-specific probes are provided in a single composition. The single composition may be in solution (liquid or frozen). Alternatively, it may be lyophilized.

[0306] Alternatively, target-specific probes may be provided as multiple compositions, including, for example, a first composition comprising a probe specific to a set of epigenetic target regions, and a second composition comprising a probe specific to a set of sequence-variable target regions.These probes can be mixed in appropriate proportions to provide a combination probe composition with any of the above-mentioned fold differences in concentration and / or capture yield.Alternatively, they may be used in separate capture procedures (e.g., with aliquots of sample, or sequentially with the same sample) to provide a first and second composition comprising capture epigenetic target regions and sequence-variable target regions, respectively. 1. Probes specific to epigenetic target regions

[0307] The probe for the epigenetic target region set may include probes specific to one or more types of target regions that are likely to distinguish the DNA of neoplastic (e.g., tumor or cancer) cells from healthy cells, e.g., non-neoplastic circulating cells.Exemplary types of such regions are discussed in detail herein, e.g., in the above section on capture sets.The probe for the epigenetic target region set may also include probes for one or more control regions, e.g., as described herein.

[0308] In some embodiments, the probes for the set of epigenetic target regions have a footprint of at least 100 kbp, e.g., at least 200 kbp, at least 300 kbp, or at least 400 kbp. In some embodiments, the set of epigenetic target regions has a footprint in the range of 100-20 Mbp, e.g., 100-200 kbp, 200-300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900-1,000 kbp, 1-1.5 Mbp, 1.5-2 Mbp, 2-3 Mbp, 3-4 Mbp, 4-5 Mbp, 5-6 Mbp, 6-7 Mbp, 7-8 Mbp, 8-9 Mbp, 9-10 Mbp, or 10-20 Mbp. In some embodiments, the set of epigenetic target regions has a footprint of at least 20 Mbp. A. Hypermethylated variable target region

[0309] In some embodiments, the probes for the set of epigenetic target regions include probes specific for one or more hypermethylated variable target regions. The hypermethylated variable target regions may also be referred to herein as hypermethylated DMRs (differentially methylated regions). The hypermethylated variable target regions may be any of those listed above. For example, in some embodiments, the probes specific for the hypermethylated variable target regions include probes specific for a plurality of loci listed in Table 1, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 1. In some embodiments, the probes specific for the hypermethylated variable target regions include probes specific for a plurality of loci listed in Table 2, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 2. In some embodiments, the probes specific for the hypermethylated variable target region include probes specific for multiple loci listed in Table 1 or Table 2, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 1 or Table 2. In some embodiments, for each locus included as a target region, there may be one or more probes with hybridization sites that bind between the transcription start site of the gene and the stop codon (the last stop codon of an alternatively spliced ​​gene). In some embodiments, the one or more probes bind within 300 bp, for example within 200 or 100 bp, of the listed position. In some embodiments, the probes have hybridization sites that overlap with the positions listed above. In some embodiments, the probes specific to hypermethylated target regions include probes specific to a subset of 1, 2, 3, 4, or 5 of hypermethylated target regions that collectively exhibit hypermethylation in one, 2, 3, 4, or 5 of breast cancer, colon cancer, kidney cancer, liver cancer, and lung cancer. b. Hypomethylated variable target regions

[0310] In some embodiments, the probe for epigenetic target region set comprises a probe specific for one or more hypomethylated variable target regions. Hypomethylated variable target regions may also be referred to herein as hypomethylated DMR (differentially methylated region). Hypomethylated variable target regions may be any of those listed above. For example, the probe specific for one or more hypomethylated variable target regions may include probes for repetitive elements, such as LINE1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and satellite DNA, as well as intergenic regions that are normally methylated in healthy cells and may show reduced methylation in tumor cells.

[0311] In some embodiments, the probes specific for hypomethylated variable target regions include probes specific for repetitive elements and / or intergenic regions. In some embodiments, the probes specific for repetitive elements include probes specific for one, two, three, four, or five of the following: LINE1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and / or satellite DNA.

[0312] Exemplary probes specific for genomic regions exhibiting cancer-associated hypomethylation include probes specific for nucleotides 8403565-8953708 and / or 151104701-151106035 of human chromosome 1. In some embodiments, probes specific for hypomethylated variable target regions include probes specific for regions overlapping with or including nucleotides 8403565-8953708 and / or 151104701-151106035 of human chromosome 1. c.CTCF binding region

[0313] In some embodiments, the probes for the set of epigenetic target regions include probes specific for CTCF binding regions. In some embodiments, the probes specific for CTCF binding regions include probes specific for at least 10, 20, 50, 100, 200, or 500 CTCF binding regions, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 CTCF binding regions, such as those described above or in the CTCFBSDB or one or more of the Cuddapah et al., Martin et al., or Rhee et al. articles cited above. In some embodiments, the probes for the set of epigenetic target regions include at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, or at least 1000 bp of the upstream and downstream regions of the CTCF binding site. D transcription start site

[0314] In some embodiments, the probes for the set of epigenetic target regions include probes specific to transcription start sites. In some embodiments, the probes specific to the transcription start sites include probes specific to at least 10, 20, 50, 100, 200, or 500 transcription start sites, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 transcription start sites, such as those listed in the DBTSS. In some embodiments, the probes for the set of epigenetic target regions include probes for at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, or at least 1000 bp of sequences upstream and downstream of the transcription start sites. e. Local amplification

[0315] As mentioned above, local amplification is a somatic mutation, but can be detected by read frequency-based sequencing in a similar manner to the method for detecting certain epigenetic changes, such as methylation changes.Therefore, as discussed above, the epigenetic target region set may include regions that may show local amplification in cancer.In some embodiments, the epigenetic target region set specific probes include local amplification specific probes.In some embodiments, the local amplification specific probes include one or more of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAF1 specific probes. For example, in some embodiments, probes specific for local amplification include probes specific for one or more of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the above targets. f. Control region

[0316] It may be useful to include a control region to facilitate data validation. In some embodiments, the probes specific to the set of epigenetic target regions include probes specific to control methylation regions that are expected to be methylated in essentially all samples. In some embodiments, the probes specific to the set of epigenetic target regions include probes specific to control hypomethylation regions that are expected to be hypomethylated in essentially all samples. 2. Probes specific to sequence-variable target regions

[0317] The probes for the set of sequence variable target regions may include probes specific to multiple regions known to undergo somatic mutations in cancer. The probes may be specific to any set of sequence variable target regions described herein. Exemplary sets of sequence variable target regions are discussed in detail herein, for example, in the section above regarding capture sets.

[0318] In some embodiments, the sequence variable target region probe set has a footprint of at least 0.5 kb, e.g., at least 1 kb, at least 2 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, or at least 40 kb. In some embodiments, the epigenetic target region probe set has a footprint in the range of 0.5-100 kb, e.g., 0.5-2 kb, 2-10 kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50 kb, 50-60 kb, 60-70 kb, 70-80 kb, 80-90 kb, and 90-100 kb. In some embodiments, the sequence variable target region probe set has a footprint of at least 50 kbp, e.g., at least 100 kbp, at least 200 kbp, at least 300 kbp, or at least 400 kbp. In some embodiments, the sequence variable target region probe set has a footprint in the range of 100-2000 kbp, e.g., 100-200 kbp, 200-300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900-1,000 kbp, 1-1.5 Mbp, or 1.5-2 Mbp. In some embodiments, the sequence variable target region set has a footprint of at least 2 Mbp.

[0319] In some embodiments, the probes specific for the set of sequence variable target regions include probes specific for at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 of the genes of Table 3. In some embodiments, the probes specific for the set of sequence variable target regions include probes specific for at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 of the SNVs of Table 3. In some embodiments, the probes specific for the set of sequence variable target regions include probes specific for at least 1, at least 2, at least 3, at least 4, at least 5, or 6 of the fusions of Table 3. In some embodiments, the probes specific for the set of sequence variable target regions include probes specific for at least a portion of at least one, at least two, or three indels of Table 3. In some embodiments, the probes specific for the set of sequence variable target regions include probes specific for at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the genes of Table 4. In some embodiments, the probes specific for the set of sequence variable target regions include probes specific for at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the SNVs of Table 4. In some embodiments, the probes specific for the set of sequence variable target regions include probes specific for at least one, at least two, at least three, at least four, at least five, or six of the fusions in Table 4.In some embodiments, the probes specific for the set of sequence variable target regions include probes specific for at least a portion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or 18 of the indels of Table 4. In some embodiments, the probes specific for the set of sequence variable target regions include probes specific for at least a portion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 of the genes of Table 5.

[0320] In some embodiments, the probes specific for the set of sequence variable target regions include probes specific for target regions from at least 10, 20, 30, or 35 cancer-associated genes, such as AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MED12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, and U2AF1. E. Computer Systems

[0321] The methods of the present disclosure can be implemented using or with the assistance of a computer system. Figure 1 shows a computer system 101 programmed or otherwise configured to implement the methods of the present disclosure. The computer system 101 can regulate various aspects of sample preparation, sequencing, and / or analysis. In some examples, the computer system 101 is configured to perform sample analysis, including sample preparation and nucleic acid sequencing (if applicable), for example, according to any of the methods disclosed herein.

[0322] The computer system 101 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 105, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 101 also includes memory or memory locations 110 (e.g., random access memory, read-only memory, flash memory), electronic storage 115 (e.g., hard disk), communication interface 120 (e.g., network adapter) for communication with one or more other systems, and peripheral devices 125, such as cache, other memory, data storage devices, and / or electronic display adapters. The memory 110, storage 115, interface 120, and peripheral devices 125 communicate with the CPU 105 via a communication network or bus (solid lines), such as a motherboard. The storage 115 may be a data storage device (or data repository) for storing data. The computer system 101 may be operatively connected to a computer network 130 with the aid of the communication interface 120. The computer network 130 may be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The computer network 130 may in some cases be a telecommunications network and / or a data network. The computer network 130 may include one or more computer servers that may enable distributed computing, such as cloud computing. The computer network 130 may in some cases implement a peer-to-peer network that may enable devices connected to the computer system 101 to act as clients or servers, with the assistance of the computer system 101.

[0323] CPU 105 may execute sequences of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 110. Examples of operations performed by CPU 105 may include fetch, decode, execute, and write-back.

[0324] The storage device 115 can store files such as drivers, libraries, and saved programs. The storage device 115 can store user generated programs and recorded sessions, as well as output(s) associated with the programs. The storage device 115 can store user data, such as user selections and user programs. The computer system 101 may, in some cases, include one or more additional data storage devices that are external to the computer system 101, such as located on a remote server that communicates with the computer system 101 via an intranet or the Internet. Data can be transferred from one location to another, for example, using a communications network or physical data transfer (e.g., using a hard drive, thumb drive, or other data storage mechanism).

[0325] Computer system 101 can communicate with one or more remote computer systems via network 130. In an embodiment, computer system 101 can communicate with a remote computer system of a user (e.g., an operator). Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., Apple® iPad®, Samsung® Galaxy Tab), a phone, a smartphone (e.g., Apple® iPhone®, Android®-enabled device, Blackberry®), or a personal digital assistant. A user can access computer system 101 via network 130.

[0326] The methods described herein may be implemented by machine (e.g., computer processor) executable code stored in an electronic storage location of computer system 101, such as, for example, memory 110 or electronic storage 115. The machine executable or machine readable code may be provided in the form of software. During use, processor 105 may execute the code. In some cases, the code may be retrieved from storage 115 and stored in memory 110 for immediate access by processor 105. In some cases, electronic storage 115 may be omitted and machine executable instructions are stored in memory 110.

[0327] In one aspect, the present disclosure provides a non-transitory computer-readable medium comprising computer executable instructions that, when executed by at least one electronic processor, carry out at least a part of the method described herein.For example, this method may include: collecting sample from subject, and optionally dividing the sample; contacting the subsample or sample with target-specific probe; capturing the DNA associated with the probe; detecting, sequencing, and / or identifying the level of captured DNA molecules; determining the possibility that the subject has cancer or another disease and / or the appropriate treatment for cancer or other disease.

[0328] The code may be pre-compiled and configured for use on a machine having a processor adapted to execute the code, or may be compiled at run-time. The code may be provided in a programming language that may be selected to enable the code to be executed in a pre-compiled or co-compiled manner.

[0329] Aspects of the systems and methods provided herein, such as the computer system 101, can be embodied in programming. Various aspects of the technology can be considered to be "products" or "articles" in the form of machine (or processor) executable code and / or associated data, typically carried or embodied in some type of machine-readable medium. The machine-executable code can be stored in electronic storage, such as memory (e.g., read-only memory, random access memory, flash memory) or hard disk. A "storage" type medium can include any or all of the tangible memory, such as a computer or processor, or associated modules thereof, such as various semiconductor memories, tape drives, and disk drives, that can provide non-transitory storage at any time for software programming.

[0330] All or parts of the software may be communicated from time to time via the Internet or various other telecommunications networks. Such communication may, for example, allow the software to be loaded from one computer or processor to another, for example, from a management server or host computer to a computer platform of an application server. Thus, other types of media that may carry software elements include optical, electrical, and electromagnetic waves, such as those used between physical interfaces between local devices over wired and optical landline networks, and through various wireless links. Physical elements that transmit such waves, such as wired or wireless or optical links, may also be considered to be media that carry the software. As used herein, unless limited to non-transitory tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions for execution to a processor.

[0331] Thus, a machine-readable medium such as a computer executable code can take many forms, including but not limited to a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices of any computer(s), such as those shown in the drawings, that can be used to implement databases, etc. Volatile storage media include dynamic memories, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that make up a bus in a computer system. Carrier wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punch cards, paper tape, any other physical storage medium having a pattern of holes, a RAM, a ROM, a PROM and EPROM, a Flash-EPROM, any other memory chip or cartridge, a carrier wave carrying data or instructions, a cable or link carrying such a carrier wave, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media can be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0332] The computer system 101 can include or communicate with, for example, an electronic display with a user interface (UI) for providing one or more results of a sample analysis. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0333] Additional details regarding computer systems and networks, databases, and computer program products are found in, for example, Peterson, Computer Networks: A Systems Approach, Morgan Kaufmann, 5th Ed. (2011); Kurose, Computer Networking: A Top-Down Approach, Pearson, 7 th Ed. (2016), Elmasri, Fundamentals of Database Systems, Addison Wesley, 6th Ed. (2010), Coronel, Database Systems: Design, Implementation, & Management, Cengage Learning, 11 th Ed. (2014), Tucker, Programming Languages, McGraw-Hill Science / Engineering / Math, 2nd Ed. (2006), and Rhoton, Cloud Computing Architected: Solution Design Handbook, Recursive Press (2011), each of which is incorporated herein by reference in its entirety. F. Application 1. Cancer and other diseases

[0334] The method can be used to diagnose the presence of a condition, particularly cancer or precancer, in a subject, characterize the condition (e.g., stage the cancer or determine the heterogeneity of the cancer), monitor the response to the treatment of the condition, and provide a prognostic risk of the onset of the condition or the subsequent course of the condition. The present disclosure can also be useful in determining the effectiveness of a particular treatment option. As more cancers die and may excrete nucleic acids upon successful treatment, the success of the treatment option may increase the amount of copy number mutations or any other somatic mutations detected in the blood of the subject. In other examples, this may not occur. In another example, a particular treatment option may correlate with the profile (e.g., genetic profile) of the cancer over time. This correlation may be useful in selecting a therapy. In some embodiments, the hypermethylated variable epigenetic target regions are analyzed to determine whether they exhibit the hypermethylated characteristics of tumor cells or cells that do not normally contribute significantly to cfDNA, and / or the hypomethylated variable epigenetic target regions are analyzed to determine whether they exhibit the hypomethylated characteristics of tumor cells or cells that do not normally contribute significantly to cfDNA.

[0335] In some embodiments, the method is used in cancer screening or for cancer screening.For example, the sample may be from a subject who has not been diagnosed with cancer before.In some embodiments, the subject may or may not have cancer.In some embodiments, the subject may or may not have early stage cancer.In some embodiments, the subject has one or more risk factors for cancer, such as tobacco use (e.g., smoking), overweight or obesity, having high body mass index (BMI), being elderly, malnutrition, high alcohol consumption, or family history of cancer.

[0336] In some embodiments, the subject has been using tobacco for at least 1, 5, 10, or 15 years, for example.In some embodiments, the subject has a high BMI, for example, a BMI of 25 or higher, 26 or higher, 27 or higher, 28 or higher, 29 or higher, or 30 or higher.In some embodiments, the subject is at least 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old.In some embodiments, the subject is malnourished, for example, has a high consumption of one or more of red meat and / or processed meat, trans fat, saturated fat, and refined sugar, and / or has a low consumption of fruits and vegetables, complex carbohydrates, and / or unsaturated fat. High and low consumption can be defined, for example, as being above or below the recommendations of the Dietary Guidelines for Americans 2020-2025 available at www.dietaryguidelines.gov / sites / default / files / 2021-03 / Dietary_Guidelines_for_Americans-2020-2025.pdf, respectively. In some embodiments, the subject has a high alcohol consumption, e.g., consuming an average of at least 3, 4, or 5 drinks per day (a drink is about 1 ounce or 30 mL of 80 proof distilled spirits or equivalent). In some embodiments, the subject has a family history of cancer, e.g., at least one, two, or three blood relatives have been previously diagnosed with cancer. In some embodiments, relatives are at least the third degree of kinship (e.g., great-grandparents, great aunts or uncles, cousins), at least the second degree of kinship (e.g., grandparents, aunts or uncles, or half-siblings), or first degree of kinship (e.g., parents or siblings).

[0337] In addition, if a cancer is observed to be in remission following treatment, the methods of the invention can be used to monitor for residual disease or recurrence of the disease.

[0338] In some embodiments, the methods and systems disclosed herein can be used to identify personalized or targeted therapy for treating a given disease or condition in a patient based on the presence of one or more proteins of interest and / or the classification of nucleic acid variants as somatic or germline in origin.Typically, the disease under consideration is a type of cancer. Non-limiting examples of such cancers include: biliary tract cancer, bladder cancer, head and neck cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, glioma, astrocytoma, breast cancer, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal cancer, colon cancer, hereditary nonpolyposis colorectal cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrial stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, ocular melanoma, uveal melanoma, gallbladder cancer, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinoma, Wilms' tumor, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML). , chronic myelomonocytic leukemia (CMML), liver cancer, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B cell lymphoma, non-Hodgkin's lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, T cell lymphoma, non-Hodgkin's lymphoma, precursor T lymphoblastic lymphoma / leukemia, peripheral T cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal carcinoma, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary neoplasm, acinar cell carcinoma, prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, gastric cancer, gastrointestinal stromal tumor (GIST), uterine cancer, or uterine sarcoma. The type and / or stage of cancer can be detected from genetic alterations including mutations, rare mutations, indels, rearrangements, copy number alterations, transversions, translocations, recombinations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structural alterations, gene fusions, chromosomal fusions, gene truncations, gene amplification, gene duplications, chromosomal damage, DNA damage, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5-methylcytosine.

[0339] In some embodiments, the methods described herein include identifying the presence of a target region and / or DNA produced by a tumor (or neoplastic cell, or cancer cell) or by a precancerous cell. In some embodiments, the methods described herein include determining the level of a target region and / or identifying the presence of DNA produced by a tumor (or neoplastic cell, or cancer cell) or by a precancerous cell. In some embodiments, determining the level of a target region includes determining either an increased level or a decreased level of the target region, where an increased or decreased level of the target region is determined by comparing the level of the target region to a threshold level / value.

[0340] Genetic data can also be used to characterize specific forms of cancer. Cancers are often heterogeneous in both composition and stage. Genetic profile data can allow characterization of specific subtypes of cancer, which can be important in the diagnosis or treatment of that specific subtype. This information can also provide clues to the subject or attending physician regarding the prognosis of a particular type of cancer, allowing either the subject or attending physician to adapt treatment options according to the progression of the disease. Some cancers can progress and become more aggressive and genetically unstable. Other cancers can remain benign, inactive, or dormant. The systems and methods of the present disclosure can be useful in determining disease progression.

[0341] Furthermore, the disclosed method can be used to characterize the heterogeneity of abnormal conditions in a subject. Such a method can include, for example, generating a genetic profile of nucleic acid from a subject, the genetic profile including a plurality of data obtained from sequence variation and epigenetic analysis. In some embodiments, the abnormal condition is cancer. In some embodiments, the abnormal condition can result in a heterogeneous genomic population. In the example of cancer, it is known that some tumors contain tumor cells of cancer at different stages. In other examples, the heterogeneity can include multiple disease foci. Again, in the example of cancer, there can be multiple tumor foci, and perhaps one or more foci are the result of metastasis spreading from the primary site.

[0342] The method can be used to diagnose, prognose, monitor, or observe cancer, precancer, or other disease.In some embodiments, the method herein does not include diagnosing, prognosing, or monitoring fetus, and therefore does not relate to non-invasive prenatal testing.In other embodiments, such methodology can be used in pregnant subjects to diagnose, prognose, monitor, or observe cancer or other disease in fetal subjects where DNA and other polynucleotides may co-circulate with maternal molecules.

[0343] Non-limiting examples of other genetically-based diseases, disorders, or conditions that may be optionally evaluated using the methods and systems disclosed herein include: achondroplasia, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Charcot-Marie-Tooth (CMT) disease, cricketing syndrome, Crohn's disease, cystic fibrosis, Dercum's disease, Down's syndrome, Duane's syndrome, Duchenne muscular dystrophy, factor V Leiden thrombocytosis, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher's disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter's syndrome, Marfan's syndrome, myotonic dystrophy, neurofibromatosis, Noonan's syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly ... anomaly), porphyria, progeria, retinitis pigmentosa, severe combined immunodeficiency (SCID), sickle cell disease, spinal muscular atrophy, Tay-Sachs disease, thalassemia, trimethylaminuria, Turner syndrome, palatocardiofacial syndrome, WAGR syndrome, or Wilson's disease.

[0344] In some embodiments, the method described herein comprises detecting the presence or absence of nucleic acid (e.g., DNA, such as cfDNA) originating from or derived from tumor cells at a preselected time point after a previous cancer treatment of a subject previously diagnosed with cancer.The method may further comprise determining a cancer recurrence score that indicates the presence or level of DNA originating from or derived from tumor cells of the subject.

[0345] When the cancer recurrence score is determined, it can be further used to determine the cancer recurrence status. The cancer recurrence status can be, for example, at risk of cancer recurrence when the cancer recurrence score is above a predetermined threshold. The cancer recurrence status can be, for example, at low or lower risk of cancer recurrence when the cancer recurrence score is above a predetermined threshold. In certain embodiments, a cancer recurrence score equal to a predetermined threshold can result in a cancer recurrence status of either at risk of cancer recurrence or at low or lower risk of cancer recurrence.

[0346] In some embodiments, the cancer recurrence score is compared to a predetermined cancer recurrence threshold, and if the cancer recurrence score is above the cancer recurrence threshold, the subject is classified as a candidate for subsequent cancer treatment, or if the cancer recurrence score is below the cancer recurrence threshold, the subject is classified as not being a candidate for therapy. In certain embodiments, a cancer recurrence score equal to the cancer recurrence threshold may result in the subject being classified as either a candidate for subsequent cancer treatment or not being a candidate for therapy.

[0347] The methods discussed above may further include one or more of any compatible features set forth elsewhere in this specification, including the sections relating to methods for determining a subject's risk of cancer recurrence and / or for classifying a subject as a candidate for subsequent cancer treatment. 2. Methods for determining the risk of cancer recurrence in a subject and / or for classifying a subject as a candidate for subsequent cancer treatment

[0348] In some embodiments, the methods provided herein are methods for determining a subject's risk of cancer recurrence. In some embodiments, the methods provided herein are methods for classifying a subject as a candidate for a subsequent cancer treatment.

[0349] Any of such methods may include collecting a sample from a subject diagnosed with cancer at one or more preselected time points after one or more previous cancer treatments for the subject.The subject may be any of the subjects described herein.The sample may include DNA, for example cfDNA.The DNA may be obtained from a tissue sample or a liquid sample.

[0350] Any of such methods may include contacting the sample or a subsample thereof with a plurality of target-specific probes specific for members of the epigenetic target region set according to any of the embodiments described herein. Such methods may further include capturing a plurality of sets of target regions from DNA derived from the subject, the plurality of sets of target regions including a sequence-variable target region set, thereby generating a combined capture set of DNA molecules. One or more capture steps may be performed according to any of the embodiments described elsewhere herein. Any of such methods may include sequencing the captured DNA molecules, thereby generating a set of sequence information. The captured DNA molecules of the sequence-variable target region set may be sequenced to a deeper sequencing depth than the captured DNA molecules of the epigenetic target region set. Any of such methods may include using the set of sequence information to detect the presence or absence of DNA originating or derived from tumor cells at a preselected time point. The detection of the presence or absence of DNA originating or derived from tumor cells may be performed according to any of those embodiments described elsewhere herein.

[0351] In any of such methods, the previous cancer treatment may include surgery, administration of a therapeutic composition, and / or chemotherapy.

[0352] The method for determining the risk of cancer recurrence of a subject may include determining a cancer recurrence score that indicates the presence or absence or amount of type-specific target regions originating or derived from the subject's tumor cells. The cancer recurrence score can further be used to determine a cancer recurrence status. The cancer recurrence status can be, for example, at risk of cancer recurrence when the cancer recurrence score is above a predetermined threshold. The cancer recurrence status can be, for example, at low or lower risk of cancer recurrence when the cancer recurrence score is above a predetermined threshold. In certain embodiments, a cancer recurrence score equal to a predetermined threshold can result in a cancer recurrence status of either at risk of cancer recurrence or at low or lower risk of cancer recurrence.

[0353] The method for classifying a subject as a candidate for a subsequent cancer treatment may include comparing the subject's cancer recurrence score with a predetermined cancer recurrence threshold, thereby classifying the subject as a candidate for a subsequent cancer treatment if the cancer recurrence score is above the cancer recurrence threshold, or as not a candidate for therapy if the cancer recurrence score is below the cancer recurrence threshold.In certain embodiments, a cancer recurrence score equal to the cancer recurrence threshold may result in the subject being classified as either a candidate for a subsequent cancer treatment or as not a candidate for therapy.In some embodiments, the subsequent cancer treatment comprises the administration of chemotherapy or a therapeutic composition.

[0354] Any such method may include determining a disease-free survival (DFS) period for the subject based on the cancer recurrence score, for example, the DFS period may be 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years, etc.

[0355] In some embodiments, the set of sequence information comprises a sequence variable target region sequence, and determining the cancer recurrence score may comprise determining at least a first subscore indicative of a particular immune cell type, a level of SNVs, insertions / deletions, CNVs, and / or fusions present in the sequence variable target region sequence.

[0356] In some embodiments, the number of mutations in the sequence variable target region selected from 1, 2, 3, 4, or 5 is sufficient to result in a cancer recurrence score in which the first subscore is classified as positive for cancer recurrence. In some embodiments, the number of mutations is selected from 1, 2, or 3.

[0357] In some embodiments, the set of sequence information includes epigenetic target region sequences, and determining the cancer recurrence score includes determining a second subscore indicative of the amount of molecules (obtained from the epigenetic target region sequences) that represent an epigenetic state different from that found in a corresponding sample from a healthy subject (e.g., cfDNA found in a blood sample from a healthy subject, or DNA found in a tissue sample from a healthy subject, the tissue sample being the same tissue type as that obtained from the test subject). Such abnormal molecules (i.e., molecules that have an epigenetic state different from that found in the corresponding sample from the healthy subject) may be consistent with epigenetic changes associated with cancer, such as methylation of a hypermethylated variable target region and / or breakage of a fragmented variable target region, where "breakage" means different from that found in the corresponding sample from the healthy subject.

[0358] In some embodiments, a percentage of molecules corresponding to the hypermethylated variable target region set and / or the fragmented variable target region set that is greater than or equal to a value in the range of 0.001% to 10%, indicating hypermethylation in the hypermethylated variable target region set and / or aberrant fragmentation in the fragmented variable target region set, is sufficient to classify the second subscore as positive for cancer recurrence. The range may be 0.001% to 1%, 0.005% to 1%, 0.01% to 5%, 0.01% to 2%, or 0.01% to 1%.

[0359] In some embodiments, any of such methods may include determining the percentage of tumor DNA from the percentage of molecules in the set of sequence information that exhibit one or more characteristics indicative of originating from a tumor cell. This can be done for molecules corresponding to some or all of the epigenetic target regions, including, for example, one or both of the hypermethylated variable target regions and the fragmented variable target regions (hypermethylation of the hypermethylated variable target regions and / or aberrant fragmentation of the fragmented variable target regions can be considered indicative of originating from a tumor cell). This can be done for molecules corresponding to sequence variable target regions, for example, molecules that include alterations consistent with cancer, such as SNVs, indels, CNVs, and / or fusions. The percentage of tumor DNA can be determined based on a combination of molecules corresponding to epigenetic target regions and molecules corresponding to sequence variable target regions.

[0360] The determination of the cancer recurrence score may be based, at least in part, on the proportion of tumor DNA, 10 -11 ~1 or 10 -10 A percentage of tumor DNA greater than a threshold in the range of 1 to 1 is sufficient to classify the Cancer Recurrence Score as positive for cancer recurrence. -10 ~10 -9 , 10 -9 ~10 -8 , 10 -8 ~10 -7 , 10 -7 ~10 -6 , 10 -6 ~10 -5 , 10 -5 ~10 -4 , 10 -4 ~10 -3 , 10 -3 ~10 -2 , or 10 -2 ~10 -1 A percentage of tumor DNA greater than or equal to a threshold value in the range of at least 10 is sufficient to classify the Cancer Recurrence Score as positive for cancer recurrence. -7The proportion of tumor DNA greater than the threshold is sufficient to classify the cancer recurrence score as positive for cancer recurrence. The determination that the proportion of tumor DNA is greater than a threshold, such as the threshold corresponding to any of the above-mentioned embodiments, can be based on cumulative probability. For example, if the cumulative probability that the proportion of tumor is greater than any of the thresholds in the above-mentioned ranges exceeds a probability threshold of at least 0.5, 0.75, 0.9, 0.95, 0.98, 0.99, 0.995 or 0.999, the sample is considered positive. In some embodiments, the probability threshold is at least 0.95, such as 0.99.

[0361] In some embodiments, the set of sequence information includes sequence variable target region sequences and epigenetic target region sequences, and determining the cancer recurrence score includes determining a first subscore indicative of the amount of SNVs, insertions / deletions, CNVs, and / or fusions present in the sequence variable target region sequences, and a second subscore indicative of the amount of abnormal molecules in the epigenetic target region sequences, and combining the first and second subscores to provide a cancer recurrence score. When combining the first and second subscores, they can be combined by applying a threshold value to each subscore independently (e.g., greater than a predetermined number of mutations (e.g., >1) in the sequence variable target region and greater than a predetermined percentage of abnormal molecules (i.e., molecules with an epigenetic state different from that found in the corresponding sample from a healthy subject, e.g., tumor) in the epigenetic target region), or by training a machine learning classifier to determine the status based on multiple positive and negative training samples.

[0362] In some embodiments, a combined score value in the range of -4 to 2 or -3 to 1 is sufficient to classify the Cancer Recurrence Score as positive for cancer recurrence.

[0363] In any embodiment in which the Cancer Recurrence Score is classified as positive for cancer recurrence, the subject's Cancer Recurrence Status may be classified as being at risk for cancer recurrence and / or the subject may be classified as a candidate for subsequent cancer treatment.

[0364] In some embodiments, the cancer is any one of the types of cancer described elsewhere herein, for example, colorectal cancer. 3. Therapy and Related Administration

[0365] In certain embodiments, the methods disclosed herein relate to identifying and administering personalized therapy to patients. In some embodiments, determining the level of a particular nucleic acid facilitates the selection of appropriate treatment. In some embodiments, the patient or subject has a given disease, disorder, or condition. Essentially any cancer therapy (e.g., surgery, radiation, and / or chemotherapy, etc.) can be included as part of such methods. In certain embodiments, the therapy administered to the subject includes at least one chemotherapeutic agent. In some embodiments, the chemotherapeutic agent may include alkylating agents (e.g., but not limited to, chlorambucil, cyclophosphamide, cisplatin, and carboplatin), nitrosoureas (e.g., but not limited to, carmustine and lomustine), antimetabolites (e.g., but not limited to, Fluorauracil, methotrexate, and fludarabine), plant alkaloids and natural products (e.g., but not limited to, vincristine, paclitaxel, and topotecan), antitumor antibiotics (e.g., but not limited to, bleomycin, doxorubicin, and mitoxantrone), hormonal agents (e.g., but not limited to, prednisone, dexamethasone, tamoxifen, and leuprolide), and biological response modifiers (e.g., but not limited to, herceptin and avastin, erbitux and rituxan). In some embodiments, the chemotherapy administered to the subject may include FOLFOX or FOLFIRI. In certain embodiments, the subject can be administered a therapy that includes at least one PARP inhibitor. In certain embodiments, the PARP inhibitor can include, among others, olaparib, talazoparib, rucaparib, niraparib (brand name ZEJULA). Typically, the therapy includes at least one immunotherapy (or immunotherapeutic agent). Immunotherapy generally refers to a method for enhancing immune response to a given cancer type. In certain embodiments, immunotherapy refers to a method for enhancing T cell response to tumor or cancer.

[0366] In some embodiments, therapy is individualized based on the status of nucleic acid variants, whether somatic or germline in origin. In some embodiments, essentially any cancer therapy (such as surgery, radiation, and / or chemotherapy) can be included as part of such methods. Typically, individualized therapy includes at least one immunotherapy (or immunotherapeutic agent). Immunotherapy generally refers to a method for enhancing immune response to a given cancer type. In certain embodiments, immunotherapy refers to a method for enhancing T cell response to tumor or cancer.

[0367] In some embodiments, the immunotherapy or immunotherapeutic agent targets immune checkpoint molecules.Certain tumors can evade the immune system by hijacking immune checkpoint pathways.Therefore, targeting immune checkpoints has emerged as an effective approach to combat tumors' ability to evade the immune system and activate antitumor immunity against certain cancers.Pardoll, Nature Reviews Cancer, 2012, 12:252-264.

[0368] In certain embodiments, the immune checkpoint molecule is an inhibitory molecule that reduces signals involved in T cell responses to antigens. For example, CTLA4 is expressed on T cells and plays a role in downregulating T cell activation by binding to CD80 (also known as B7.1) or CD86 (also known as B7.2) on antigen presenting cells. PD-1 is another inhibitory checkpoint molecule expressed on T cells. PD-1 limits the activity of T cells in peripheral tissues during inflammatory responses. In addition, the ligands of PD-1 (PD-L1 or PD-L2) are commonly upregulated on the surface of many different tumors, resulting in downregulation of anti-tumor immune responses in the tumor microenvironment. In certain embodiments, the inhibitory immune checkpoint molecule is CTLA4 or PD-1. In other embodiments, the inhibitory immune checkpoint molecule is a ligand of PD-1, such as PD-L1 or PD-L2. In other embodiments, the inhibitory immune checkpoint molecule is a ligand of CTLA4, such as CD80 or CD86. In other embodiments, the inhibitory immune checkpoint molecule is lymphocyte activation gene 3 (LAG3), killer cell immunoglobulin-like receptor (KIR), T cell membrane protein 3 (TIM3), galectin 9 (GAL9), or adenosine A2a receptor (A2aR).

[0369] Antagonists targeting such immune checkpoint molecules can be used to enhance antigen-specific T cell responses against certain cancers. Thus, in certain embodiments, the immunotherapy or immunotherapeutic agent is an inhibitory immune checkpoint molecule antagonist. In certain embodiments, the inhibitory immune checkpoint molecule is PD-1. In certain embodiments, the inhibitory immune checkpoint molecule is PD-L1. In certain embodiments, the inhibitory immune checkpoint molecule antagonist is an antibody (e.g., a monoclonal antibody). In certain embodiments, the antibody or monoclonal antibody is an anti-CTLA4, anti-PD-1, anti-PD-L1, or anti-PD-L2 antibody. In certain embodiments, the antibody is a monoclonal anti-PD-1 antibody. In some embodiments, the antibody is a monoclonal anti-PD-L1 antibody. In certain embodiments, the monoclonal antibody is a combination of an anti-CTLA4 antibody and an anti-PD-1 antibody, an anti-CTLA4 antibody and an anti-PD-L1 antibody, or an anti-PD-L1 antibody and an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is one or more of pembrolizumab (Keytruda®) or nivolumab (Opdivo®). In certain embodiments, the anti-CTLA4 antibody is ipilimumab (Yervoy®). In certain embodiments, the anti-PD-L1 antibody is one or more of atezolizumab (Tecentriq®), avelumab (Bavencio®), or durvalumab (Imfinzi®).

[0370] In certain embodiments, the immunotherapy or immunotherapeutic agent is an antagonist (e.g., an antibody) against CD80, CD86, LAG3, KIR, TIM3, GAL9, or A2aR. In other embodiments, the antagonist is a soluble form of an inhibitory immune checkpoint molecule, such as a soluble fusion protein comprising the extracellular domain of an inhibitory immune checkpoint molecule and the Fc domain of an antibody. In certain embodiments, the soluble fusion protein comprises the extracellular domain of CTLA4, PD-1, PD-L1, or PD-L2. In some embodiments, the soluble fusion protein comprises the extracellular domain of CD80, CD86, LAG3, KIR, TIM3, GAL9, or A2aR. In one embodiment, the soluble fusion protein comprises the extracellular domain of PD-L2 or LAG3.

[0371] In certain embodiments, the immune checkpoint molecule is a costimulatory molecule that amplifies the signal involved in T cell response to antigen. For example, CD28 is a costimulatory receptor expressed on T cells. When T cells bind to antigen via T cell receptor, CD28 binds to CD80 (also known as B7.1) or CD86 (also known as B7.2) on antigen-presenting cells, amplifying T cell receptor signaling and promoting T cell activation. Because CD28 binds to the same ligands (CD80 and CD86) as CTLA4, CTLA4 can counter or regulate the costimulatory signaling mediated by CD28. In certain embodiments, the immune checkpoint molecule is a costimulatory molecule selected from CD28, inducible T cell costimulator (ICOS), CD137, OX40, or CD27. In other embodiments, the immune checkpoint molecule is a ligand for a costimulatory molecule, including, for example, CD80, CD86, B7RP1, B7-H3, B7-H4, CD137L, OX40L, or CD70.

[0372] Agonists that target these costimulatory checkpoint molecules can be used to enhance antigen-specific T cell responses against certain cancers. Thus, in certain embodiments, the immunotherapy or immunotherapeutic agent is an agonist of a costimulatory checkpoint molecule. In certain embodiments, the agonist of a costimulatory checkpoint molecule is an agonist antibody, preferably a monoclonal antibody. In certain embodiments, the agonist antibody or monoclonal antibody is an anti-CD28 antibody. In other embodiments, the agonist antibody or monoclonal antibody is an anti-ICOS, anti-CD137, anti-OX40, or anti-CD27 antibody. In other embodiments, the agonist antibody or monoclonal antibody is an anti-CD80, anti-CD86, anti-B7RP1, anti-B7-H3, anti-B7-H4, anti-CD137L, anti-OX40L, or anti-CD70 antibody.

[0373] In certain embodiments, the status of nucleic acid variants of a sample from a subject, as being of somatic or germline origin, can be compared with a database of comparator results of a reference population to identify personalized or targeted therapy for the subject.Typically, the reference population includes patients with the same cancer or disease type as the subject, and / or patients who are undergoing or have undergone the same therapy as the subject.Personalized or targeted therapy(s) can be identified when the nucleic acid variants and comparator results meet certain classification criteria (e.g., substantially or nearly identical).

[0374] In certain embodiments, the personalized therapy described herein is typically administered parenterally (e.g., intravenously or subcutaneously). Pharmaceutical compositions that include immunotherapeutic agents are typically administered intravenously. Certain therapeutic agents are administered orally. However, personalized therapy (e.g., immunotherapeutic agents, etc.) can also be administered by any method known in the art, for example, bucally, sublingually, rectally, vaginally, intraurethrally, topically, intraocularly, intranasally, and / or intraauricularly, including by tablet, capsule, granule, aqueous suspension, gel, spray, suppository, salve, or ointment, etc.

[0375] Therapeutic options for treating particular genetically-based diseases, disorders, or conditions other than cancer are generally well known to those of skill in the art and will be apparent given the particular disease, disorder, or condition being considered.

[0376] In some embodiments, for example, when a genetic variant is detected, therapy is personalized based on the status of the nucleic acid variant as being of somatic or germline origin. In some embodiments, essentially any cancer therapy (e.g., surgery, radiation, and / or chemotherapy, etc.) can be included as part of such methods. Typically, personalized therapy includes at least one immunotherapy (or immunotherapeutic agent). Immunotherapy generally refers to a method for enhancing immune response to a given cancer type. In certain embodiments, immunotherapy refers to a method for enhancing T cell response to tumor or cancer.

[0377] In certain embodiments, the status of nucleic acid variants of a sample from a subject as somatic or germline origin can be compared with a database of comparator results of a reference population to identify individualized or targeted therapy for the subject.Typically, the reference population includes patients with the same cancer or disease type as the subject, and / or patients who are undergoing or have undergone the same therapy as the subject.Individualized or targeted therapy(s) can be identified when the nucleic acid variants and comparator results meet certain classification criteria (e.g., substantially or nearly match).

[0378] In certain embodiments, the personalized therapy described herein is typically administered parenterally (e.g., intravenously or subcutaneously). Pharmaceutical compositions that include immunotherapeutic agents are typically administered intravenously. Certain therapeutic agents are administered orally. However, personalized therapy (e.g., immunotherapeutic agents, etc.) can also be administered by methods such as buccal, sublingual, rectal, vaginal, intraurethral, ​​topical, intraocular, intranasal, and / or intraaural, and administration can include tablets, capsules, granules, aqueous suspensions, gels, sprays, suppositories, salves, or ointments, etc. IV. Kit

[0379] Also provided are kits comprising the compositions described herein. The kits can be used in carrying out the methods described herein. In some embodiments, the kits comprise a plurality of target-specific probes. In some embodiments, the plurality of target-specific probes comprises or consists of probes comprising a capture moiety that hybridizes to a target region having type-specific epigenetic mutations and copy number mutations. In some embodiments, the kits comprise a solid support linked to a binding partner of the capture moiety. In some embodiments, the kits comprise an adaptor. In some embodiments, the kits comprise a PCR primer that anneals to the adaptor. In some embodiments, the kits comprise additional elements elsewhere herein. In some embodiments, the kits comprise instructions for carrying out the methods described herein.

[0380] In some embodiments, the kit further comprises an agent that recognizes methylcytosine in DNA. In some such embodiments, the agent is an antibody or a methyl-binding protein or domain. In some embodiments, the kit comprises a target-specific probe that specifically binds to the set of sequence variable target regions. In some such embodiments, the target-specific probe comprises a capture moiety.

[0381] Kit includes ALK, APC, BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN, RBI, TP53, MET, AR, ABLl, AKTl, ATM, CDHl, CSFIR, CTNNBl, ERBB 4, EZH2, FGFRl, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, PTPN11, RET, SMAD4, It may further comprise a plurality of oligonucleotide probes selectively hybridizing to at least 5, 6, 7, 8, 9, 10, 20, 30, 40 or all genes selected from the group consisting of SMARCB1, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAF1, BRCA1, CCND2, CDK6, NF1, TP53, ARID1A, BRCA2, CCNE1, ESR1, RIT1, GATA3, MAP2K1, RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA and NTRK1. The number of genes that the oligonucleotide probes can selectively hybridize to can vary. For example, the number of genes may include 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, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54. The kit may include a container comprising a plurality of oligonucleotide probes and instructions for performing any of the methods described herein.

[0382] The kit may include at least 4, 5, 6, 7, or 8 different library adapters with different molecular barcodes and the same sample barcode. The library adapters may not be sequencing adapters. For example, the library adapters do not include flow cell sequences or sequences that allow for the formation of hairpin loops for sequencing. Various variations and combinations of molecular barcodes and sample barcodes are described throughout and are applicable to the kit. Furthermore, in some cases, the adapters are not sequence adapters. In addition, the adapters provided with the kit may further include sequencing adapters. The sequencing adapters may include sequences that hybridize to one or more sequencing primers. The sequencing adapters may further include sequences that hybridize to a solid support, such as flow cell sequences. For example, the sequencing adapters may be flow cell adapters. The sequencing adapters can be attached to one or both ends of the polynucleotide fragments. In some cases, the kit may include at least 8 different library adapters with different molecular barcodes and the same sample barcode. The library adapters may not be sequencing adapters. The kit may further include a sequencing adaptor having a first sequence that selectively hybridizes to the library adaptor and a second sequence that selectively hybridizes to the flow cell sequence. In another example, the sequencing adaptor may be hairpin shaped. For example, the hairpin shaped adaptor may include a complementary double-stranded portion and a loop portion, and the double-stranded portion may be attached (e.g., ligated) to a double-stranded polynucleotide. The hairpin shaped sequencing adaptor may be attached to both ends of a polynucleotide fragment to generate a circular molecule that is sequenced multiple times. The sequencing adaptor may include one or more barcodes. For example, the sequencing adaptor may include a sample barcode. The sample barcode may include a predetermined sequence. The sample barcode may be used to identify the source of the polynucleotide.The sample barcode may be at least 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, or more (or any length described throughout) nucleobases, e.g., at least 8 bases. As described above, the barcode may be a continuous or discontinuous sequence.

[0383] The library adaptors may be blunt ended and Y-shaped and may be less than or equal to 40 nucleobases in length. Other variations of library adaptors can be found throughout and are applicable to the kit.

[0384] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the above specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific illustrations, configurations, or relative proportions shown herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the disclosed embodiments described herein can be used in the practice of the present invention. It is therefore contemplated that the present disclosure will cover any such alternatives, modifications, variations, or equivalents. It is intended that the scope of the present invention is defined by the following claims, and that methods and structures within the scope of such claims and their equivalents are covered thereby. Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made therein without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, the methods, systems, computer readable media, and / or component features, steps, elements, or other aspects thereof may all be used in various combinations.

[0385] All patents, patent applications, websites, other publications or documents, and accession numbers, etc. cited herein are incorporated by reference in their entirety for all purposes as if each individual item was specifically and individually indicated to be incorporated by reference. Where different versions of sequences are associated with accession numbers at different times, the version associated with the accession number at the effective filing date of this application is intended. By effective filing date, we mean the earlier of the actual filing date or, if applicable, the filing date of the priority application referencing the accession number. Similarly, where different versions of a publication or website, etc., were published at different times, the latest published version at the effective filing date of this application is intended unless otherwise indicated. EXAMPLES

[0386] Example 1 Analysis of cfDNA to detect type-specific DMRs that are also CNVs associated with early stage colorectal cancer in subjects Samples from healthy subjects and subjects with early stage colorectal cancer are analyzed in a blood-based assay to detect hypermethylated regions associated with colon tissues that may have abnormally high copy numbers, and test whether such signals are predictive of colorectal cancer or colorectal precancer. cfDNA is extracted from the plasma of these subjects and then partitioned based on binding to MBD attached to beads. The beads are washed with increasing salt concentrations. Unbound DNA and DNA from these washes result in three partitioned fractions of increasingly methylated cfDNA (low methylation, residual methylation, and high methylation partitioned fractions). The cfDNA molecules in the partitioned fractions are purified to remove salt and concentrated in preparation for the enzymatic steps of library preparation.

[0387] After enrichment of the cfDNA in the partitioned fractions, adapters containing molecular barcodes are added to the cfDNA. These molecular barcodes are non-unique, and each partitioned fraction is ligated to an adapter with a non-unique molecular barcode that is distinguishable from the barcodes of the adapters used in the other partitioned fractions. After ligation, if necessary, the highly methylated partitioned fraction is treated with a methylation-sensitive restriction enzyme to degrade the mispartitioned unmethylated DNA. The partitioned fractions are pooled together and amplified by PCR.

[0388] After PCR, the amplified DNA is washed and concentrated before enrichment. Once concentrated, the DNA is combined with a salt buffer and a biotinylated RNA target-specific probe that specifically binds to DMRs that are hypermethylated in colon tissue compared to blood cells (i.e., the other major cell type present in the sample) and have abnormally high copy numbers compared to the wild-type copy numbers expected for hypermethylated regions. This mixture is incubated overnight.

[0389] The biotinylated RNA target-specific probes hybridized to the DNA are captured with streptavidin-conjugated magnetic beads and separated from the uncaptured amplified DNA by a series of salt-based washes, thereby enriching the sample. After enrichment, further amplification is performed using primers that add sample indexes to the amplicons. An aliquot of the enriched sample is sequenced using an Illumina NovaSeq sequencer. The sequence reads generated by the sequencer are then analyzed using bioinformatics tools / algorithms. Molecular barcodes are used to identify unique molecules as well as deconvolute the sample into differentially distributed molecules. The hypermethylated target region sequences are analyzed to detect methylated cfDNA molecules in regions associated with colonic tissue. Relative methylation frequency is determined as the total number of methylation percentages (high + residual) normalized by input cfDNA. Colon-specific hypermethylated DMRs with abnormally high CNVs can provide highly sensitive detection of information associated with a subject's cancer status (e.g., more sensitive detection than would be obtained from analysis of colon-specific hypermethylated DMRs present at wild-type copy number). Example 2 Analysis of cfDNA to detect type-specific DMRs that are also CNVs associated with early stage colorectal cancer in subjects

[0390] Samples from healthy subjects and subjects with early stage colorectal cancer are analyzed by blood-based assay to detect hypermethylated regions associated with colonic tissues that may have abnormally high copy numbers, and test whether such signals predict colorectal cancer or colorectal precancer. cfDNA is extracted from the plasma of these subjects and then combined with antibodies specific to methylcytosine. Magnetic beads conjugated with protein G are used to immunoprecipitate the antibodies and the DNA bound to them, thus dividing hypermethylated DNA and hypomethylated DNA. First, any DNA that is unmethylated or lowly methylated is eluted from the beads using buffers containing increasing concentrations of salt. Finally, highly methylated DNA is washed away from the antibodies specific to methylcytosine using high salt buffers. Unbound DNA and DNA from these washes result in three distribution fractions of cfDNA that are increasingly methylated (hypomethylated, residually methylated, and hypermethylated distribution fractions). The cfDNA molecules in the partitioned fractions are purified to remove salts and concentrated in preparation for the enzymatic steps of library preparation.

[0391] After the cfDNA in the distribution fraction is concentrated, a first adaptor is added to the cfDNA by ligation to its 3' end. The adaptor is used as a priming site for second strand synthesis using a universal primer and DNA polymerase. The first adaptor contains biotin, and the nucleic acid ligated to the first adaptor is bound to beads containing streptavidin. A second adaptor is then ligated to the 3' end of the second strand of the now double-stranded molecule. These adaptors contain non-unique molecular barcodes, and each distribution fraction is ligated to an adaptor that has a non-unique molecular barcode that is distinguishable from the barcodes of the adaptors used in other distribution fractions. After ligation, the highly methylated distribution fraction is treated with a methylation-sensitive restriction enzyme to degrade the mis-distributed non-methylated DNA. The distribution fractions are pooled together and amplified by PCR.

[0392] After PCR, the amplified DNA is washed and concentrated before enrichment. Once concentrated, the amplified DNA is combined with a salt buffer and a biotinylated RNA target-specific probe that specifically binds to DMRs that are hypermethylated in colon tissue compared to blood cells (i.e., the other major cell type present in the sample) and have abnormally high copy numbers compared to the wild-type copy numbers expected for hypermethylated regions. This mixture is incubated overnight.

[0393] The biotinylated RNA target-specific probes hybridized to DNA are captured with streptavidin-conjugated magnetic beads and separated from the non-captured amplified DNA by a series of salt-based washes, thereby enriching the sample. After enrichment, an aliquot of the enriched sample is sequenced using an Illumina NovaSeq sequencer. The sequence reads generated by the sequencer are then analyzed using bioinformatics tools / algorithms. Molecular barcodes are used to identify unique molecules, as well as deconvolute the sample into differentially distributed molecules. The hypermethylated target region sequences are analyzed to detect methylated cfDNA molecules in regions associated with colon tissue. Relative methylation frequency is determined as the total number of methylation fractions (high + residual) normalized by input cfDNA. Colon-specific hypermethylated DMRs with abnormally high CNVs can provide highly sensitive detection of information associated with a subject's cancer status (e.g., more sensitive than would be obtained from the analysis of colon-specific hypermethylated DMRs present at wild-type copy number). Example 2 Analyzing the cfDNA to detect type-specific fragments that are also CNVs associated with early stage colorectal cancer in the subject.

[0394] Obtain samples of cfDNA from healthy subjects and subjects with early stage colorectal cancer, and analyze as described in Example 1, except that the partitioning step may be omitted and different biotinylated RNA target-specific probes are used. The target-specific probes are known to show different fragmentation patterns in colon tissues compared to blood cells (i.e., the main cell type contributing to cfDNA samples), and specifically bind to DNA regions that may show abnormally high copy numbers in colorectal cancer compared to wild-type copy numbers. The probes and the DNA hybridized to the probes are captured and sequenced as described in Example 1. The target region fragments are analyzed to detect cfDNA molecules associated with colon tissue. Colon-specific fragments with abnormally high CNV can provide highly sensitive detection of information associated with the cancer status of the subject. Example 3 Epigenetic and sequence variability analysis of cfDNA to detect the presence or absence of cancer in a subject

[0395] cfDNA samples from healthy subjects and subjects with early stage colorectal cancer are obtained, prepared, captured, sequenced, and analyzed as described in Example 1 or 2, except that in addition to epigenetic target region set or target specific probe (i.e., probe that binds to colon specific DMR or colon specific fragmentation pattern fragment), sequence variable target region set of target specific probe is also used. In particular, biotinylated RNA target specific probe comprises a probe for sequence variable target region set that includes a sequence known to be mutated in colon cancer. The probe for sequence variable region set has a footprint of about 50kb.

[0396] The sequence-variable target region sequences are analyzed to detect genomic alterations such as SNVs, insertions, deletions, and fusions that can be called with sufficient certainty to distinguish actual tumor variants from technical errors (e.g., PCR errors, sequencing errors). The epigenetic target region sequences are analyzed independently to detect hypermethylated cfDNA molecules derived from colon tissue. Finally, the results of both analyses are combined to generate a final determination of the likelihood of cancer or precancer in the subject from whom the sample was obtained.

Claims

1. A method for analyzing DNA in a blood sample, comprising: a) capturing at least a set of epigenetic target regions of DNA from said blood sample or a sub-sample thereof, said capturing comprising contacting said DNA with a plurality of target-specific probes specific to members of said set of epigenetic target regions, wherein said set of epigenetic target regions comprises a plurality of type-specific epigenetic target regions that are copy number variants, said type-specific epigenetic target regions being type-specific differentially methylated regions and / or type-specific fragments, whereby captured DNA is provided, and sequencing said captured DNA and determining the levels of said type-specific epigenetic target regions A method comprising the steps of:

2. (a) said type-specific epigenetic target regions are type-specific differentially methylated regions, and / or (b) said type-specific epigenetic target regions comprise type-specific fragments, The method according to claim 1.

3. Said plurality of type-specific epigenetic target regions are: (a) type-specific hypermethylated regions, (b) type-specific hypomethylated regions, (c) target regions that are hypermethylated in immune cells compared to non-immune cell types present in said blood sample, (d) (i) in the colon compared to other tissue types, (ii) in the lung compared to other tissue types, (iii) in the breast compared to other tissue types, (iv) in the liver compared to other tissue types, (v) in the kidney compared to other tissue types, (vi) in the pancreas compared to other tissue types, (vii) in the prostate compared to other tissue types, (viii) in the skin compared to other tissue types, and / or (ix) in the bladder compared to other tissue types, target regions that are differentially methylated, (e) target regions that are hypomethylated in non-immune blood cells compared to the methylation levels of target regions of different cell types or tissue types in said sample, (f) fragments specific to immune cells compared to non-immune cell types present in said blood sample, (g) (i) in the colon compared to other tissue types, (ii) in the lung compared to other tissue types, (iii) in the breast compared to other tissue types, (iv) in the liver compared to other tissue types, (v) in the kidney compared to other tissue types, (vi) in the pancreas as compared to other tissue types, (vii) in the prostate as compared to other tissue types, (viii) in the skin as compared to other tissue types, and / or (ix) in the bladder as compared to other tissue types specific fragments, (h) copy number variants having an abnormally high copy number, and / or (i) at least one copy number variant containing duplications The method according to claim 1 or claim 2, comprising.

4. (a) The hypermethylated target region is methylated to an extent that is at least 10%, 20%, 30%, or at least 40% higher than the average methylation of the target region in the sample, and / or (b) identifying at least one cell type or tissue type that is the origin of the type-specific epigenetic target region, for example, determining the level of the type-specific epigenetic target region originating from the cell type or tissue type, and optionally determining the level of the type-specific epigenetic target region originating from immune cells, non-immune blood cells, colon, lung, breast, liver, kidney, prostate, skin, bladder, or pancreas The method according to claim 1.

5. (a) The blood sample is a plasma sample, or the blood sample is a whole blood sample, (b) The blood sample is fractionated prior to capturing at least the set of epigenetic target regions of DNA, and / or (c) The DNA is cfDNA, The method according to claim 1.

6. distributing the DNA into a plurality of sub-samples by contacting the DNA with an agent that recognizes methylcytosine in the DNA, the plurality including a first sub-sample and a second sub-sample, the first sub-sample including DNA having a higher proportion of methylcytosine than the second sub-sample, and optionally, (a) the distributing is performed before the capturing, or after the capturing and before the sequencing, (b) the agent that recognizes methylcytosine is a methyl-binding reagent, for example, the methyl-binding reagent (i) is an antibody, (ii) specifically recognizes 5-methylcytosine, and / or (iii) is immobilized on a solid support, (c) the distributing includes immunoprecipitation of methylated DNA, and / or (d) The said distributing includes distributing based on binding to a protein, and optionally, the said protein is a methylated protein, an acetylated protein, a non-methylated protein, a non-acetylated protein, and / or optionally, the said protein is a histone. For example, the said distributing includes contacting the DNA of the said sample with a binding reagent that is specific to the said protein and immobilized on a solid support. The method according to claim 1. **Claim 7** (a) including contacting the said DNA with at least one methylation-sensitive restriction enzyme (MSRE) and / or at least one methylation-dependent restriction enzyme (MDRE); For example, the said at least one MSRE is one or more of AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, and SnaBI. For example, the said at least one MSRE is (i) BstUI and HpaII, (ii) HhaI and AccII, or (iii) BstUI, HpaII, and Hin6I; For example, the said at least one MDRE is one or more of MspJI, LpnPI, FspEI, or McrBC; For example, the said first partial sample is contacted with the said at least one MSRE and / or the said second partial sample is contacted with the said at least one MDRE; (b) including subjecting the said sample or one or more partial samples to a procedure that has a different effect on the first nucleobase of the said DNA than on the second nucleobase, and / or (c) including determining the methylation level of the type-specific differential methylation target region; The method according to claim 1. **Claim 8** (a) The said set of epigenetic target regions includes CTCF binding sites and / or transcription start sites. (b) said capturing comprises capturing the sequence-variable target region of said DNA, said capturing comprising contacting said DNA with a plurality of target-specific probes specific for said sequence-variable target region, and / or (c) said method comprises ligating an adapter to said DNA, thereby generating adapter-ligated DNA, and optionally said adapter-ligated DNA is amplified prior to said sequencing, The method according to claim 1.

9. The method according to claim 6, wherein said partial samples are pooled prior to said sequencing.

10. said sample is obtained from a subject, and optionally said level of said type-specific epigenetic target region indicates that said subject has a likelihood of having cancer or pre-cancer, for example (a) said cancer is a cancer of the cell type or tissue type that is the origin of the target region, (b) said cancer is a cancer of the cell type or tissue type that is the origin of said target region at a level higher than the level present in a sample obtained from a healthy subject, (c) said cancer is a lymphocyte cancer, for example said cancer is leukemia, lymphoma, or myeloma, or said cancer is a bone marrow cancer, or said cancer is colorectal cancer, lung cancer, breast cancer, prostate cancer, skin cancer, stomach cancer, pancreatic cancer, bladder cancer, or kidney cancer, (d) said pre-cancer is an adenoma, for example said adenoma is an advanced adenoma, and / or (e) said pre-cancer is colorectal pre-cancer, lung pre-cancer, breast pre-cancer, prostate pre-cancer, skin pre-cancer, stomach pre-cancer, pancreatic pre-cancer, bladder pre-cancer, or kidney pre-cancer, The method according to claim 1.

11. said sequencing comprises generating a plurality of sequencing reads, said method further comprising mapping said plurality of sequence reads to one or more reference sequences to generate mapped sequence reads, and processing said mapped sequence reads to determine said likelihood that said subject has cancer or pre-cancer, The method according to claim 1.

12. said sample is obtained from a subject previously diagnosed with cancer and having received one or more previous cancer treatments, and optionally said sample is obtained at one or more preselected time points after said one or more previous cancer treatments, (a) The method further comprises, optionally, determining a cancer recurrence score, and optionally, the cancer recurrence status of the subject is determined to be at risk of cancer recurrence if the cancer recurrence score is at or above a predetermined threshold, or the cancer recurrence status of the subject is determined to be at lower risk of cancer recurrence if the cancer recurrence score is below the predetermined threshold, and optionally, (b) The method further comprises comparing the cancer recurrence score of the subject to a predetermined cancer recurrence threshold, and the subject is classified as a candidate for subsequent cancer treatment if the cancer recurrence score exceeds the cancer recurrence threshold, or is classified as not a candidate for subsequent cancer treatment if the cancer recurrence score is below the cancer recurrence threshold, The method according to claim 1.

13. Detecting methylation using modified sensitivity conversion, for example, the conversion technique is (a) Bisulfite conversion, (b) Oxidative bisulfite conversion, (c) Tet-assisted bisulfite conversion, (d) Tet-assisted conversion using a substituted borane reducing agent, optionally, the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane, Tet-assisted conversion using a substituted borane reducing agent, (e) Protection of hmC combined with Tet-assisted conversion by a substituted borane reducing agent, (f) APOBEC-coupled epigenetic conversion, or (g) Enzymatic conversion of nucleobases The method according to claim 1, comprising.

14. The method according to claim 6, wherein the DNA of the first partial sample and the DNA of the second partial sample are differentially tagged.

15. Use of the method according to claim 1 for generating a profile, fingerprint or dataset that is the sum of information from different cells of the heterogeneous disease of the subject from which the sample is derived, for example, the dataset includes identity and level of structural variation, copy number variation, epigenetic variation, or other mutation analysis, alone or in combination.

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