Compositions and methods for analyzing cell-free DNA in methylation partitioning assays

Abstract

To provide compositions and methods for analyzing cell-free DNA in a methylation partitioning assay.SOLUTION: Provided herein is a DNA analysis method comprising partitioning a sample into at least a first subsample and a second subsample, the first subsample comprising DNA (e.g., cell-free DNA) with a cytosine modification in a greater proportion, the first subsample undergoing a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample, and DNA being sequenced to distinguish the first nucleobase from the second nucleobase.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 908,569, filed September 30, 2019, which is incorporated herein by reference for all purposes. [Background technology]

[0002] FIELD OF THE INVENTION The present disclosure provides compositions and methods related to analyzing DNA, for example, cell-free DNA.In some embodiments, the cell-free DNA is the DNA from a subject who has or is suspected of having cancer, and / or the cell-free DNA comprises the DNA from cancer cells.In some embodiments, the DNA is divided into a first sub-sample and a second sub-sample, the first sub-sample comprises DNA with a higher proportion of nucleotide modification (for example, cytosine modification) than the second sub-sample, the first sub-sample is subjected to a procedure that affects the first nucleobase in the DNA so that it is different from the second nucleobase in the DNA of the first sub-sample, and the DNA is sequenced to distinguish the first nucleobase from the second nucleobase in the DNA of the first sub-sample. Summary of the Invention [Means for solving the problem]

[0003] Introduction and Overview Cancer causes millions of deaths worldwide each year. Early detection of cancer can lead to improved outcomes, as early-stage cancers tend to be more susceptible to treatment.

[0004] Inappropriately controlled cell proliferation is a hallmark of cancer, commonly resulting from the accumulation of genetic and epigenetic alterations such as copy number variations (CNVs), single nucleotide variations (SNVs), gene fusions, insertions and / or deletions (indels), cytosine modifications (e.g., 5-methylcytosine, 5-hydroxymethylcytosine, and other more oxidized forms), and epigenetic variations involving the association of DNA with chromatin proteins and transcription factors.

[0005] Biopsy represents a traditional approach to detecting or diagnosing cancer in which cells or tissue are extracted from a potential cancer site and analyzed for relevant phenotypic and / or genotypic traits. Biopsies have the disadvantage of being invasive.

[0006] Cancer detection based on the analysis of body fluids, such as blood ("liquid biopsy"), is an interesting alternative based on the knowledge that DNA from cancer cells is released into body fluids. Liquid biopsies are non-invasive (sometimes only requiring blood sampling). However, due to the low concentration and heterogeneity of cell-free DNA, it has been challenging to develop accurate and sensitive methods for analyzing liquid biopsy material that provide detailed information about nucleic acid base modifications. Isolating and processing a fraction of cell-free DNA useful for further analysis in liquid biopsy procedures is an important part of these methods. Therefore, improved methods and compositions for analyzing cell-free DNA, for example, in liquid biopsies, are needed.

[0007] The present disclosure is based in part on the following recognition. It can be beneficial to analyze nucleic acid base modifications (including, among others, cytosine methylation and / or hydroxymethylation) along with other process steps, such as distribution based on methylation degree and sequencing. For example, in an exemplary embodiment, a DNA sample (e.g., a cfDNA sample) is divided into multiple sub-samples with different amounts of cytosine methylation (e.g., based on binding to MBD (methyl-binding domain or methyl-binding protein) or an antibody specific for methylated cytosine), and then the sub-sample with high levels of methylation is subjected to a procedure that differentially affects different types of given nucleic acid bases (e.g., unmodified cytosine and methylated cytosine, or hydroxymethylated cytosine and methylated cytosine). Sequencing can then be performed to identify the sequences in the first and second sub-samples, and / or identify the positions in the DNA from the first sub-sample where a particular type of nucleic acid base exists. Such methods according to the present disclosure can provide more information about epigenetic modifications in DNA, e.g., cfDNA, than existing approaches, e.g., MeDIP-seq, MBD-seq, BS-seq, Ox-BS-seq, TAP-seq, ACE-seq, hmC-seal, and TAB-seq. See, e.g., Schutsky, EK et al. Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase. Nature Biotech, 2018; doi.10.1038 / nbt.4204 (ACE-Seq); Yu, Miao et al. Base-resolution analysis of 5-hydroxymethylcytosine in the Mammalian Genome. Cell, 2012; 149(6):1368-80 (TAB-Seq); Han, D. A highly sensitive and robust method for genome-wide 5hmC See, for example, "Profiling of rare cell populations." Mol Cell. 2016; 63(4):711-719 (5hmC-Seal); Shen, SY et al. Sensitive tumor detection and classification using plasma cell-free DNA methylomes. Nature. 2018; 563(7732):579-583 (cfMeDIP); Nair, SS et al. Comparison of methyl-DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain (MBD) protein capture for genome-wide DNA. Epigenetics. 2011; 6(1):34-44. Unlike such existing methods, the method according to the present disclosure can provide a combination of information about a first modification, e.g., methylation level, from a partitioning step and additional information about the specific modification and / or its location from a procedure that differentially affects different forms of a given nucleobase. Examples of such procedures include various conversion or separation steps using bisulfite, substituted boranes, base-modifying enzymes, or modified base-specific antibodies that distinguish between different species of a class of nucleobases. In some embodiments, the methods described herein provide a combination of information regarding (i) the overall modification level (e.g., cytosine modifications) of the molecule (e.g., based on their partitioning), and (ii) high-resolution information regarding the identity and / or location of specific modifications (e.g., based on specific conversion of specific modified or unmodified nucleotides, or on sequencing after further partitioning that distinguishes between specific types of modifications, as discussed in detail herein).

[0008] The method may further comprise capturing two sets of target regions from DNA.In some embodiments, the set of target regions comprises a set of sequence variable target regions and a set of epigenetic target regions.Each of these sets can provide useful information for determining the possibility that a sample contains DNA from cancer cells.In some embodiments, the capture yield of the set of sequence variable target regions is higher than the capture yield of the set of epigenetic target regions.Due to the difference in capture yield, for example, during simultaneous sequencing, for example, in the same sequencing cell or the same pool of sequencing material, the set of sequence variable target regions can enable deeper and therefore more accurate sequencing, while the set of epigenetic target regions can enable shallower and broader coverage.

[0009] The set of epigenetic target regions can be analyzed in various ways. For example, if the acceptable confidence level for modifications at specific positions is lower than the acceptable confidence level for accuracy in the set of sequence-variable target regions (e.g., if the purpose is to understand the frequency of different types of modifications at various loci, and it is not necessary to understand the exact positions where modifications occur), the analysis can use a method that does not rely on high accuracy in sequencing specific nucleotides within the target. Examples include determining the degree of modification, such as methylation, and / or the distribution and size of fragments, which can indicate normal or abnormal chromatin structure in the cells from which the fragments were obtained. Such analysis can be performed by sequencing, and requires less data (e.g., the number of sequence reads or the depth of sequencing coverage) than determining the presence or absence of sequence mutations such as base substitutions, insertions, or deletions.

[0010] The present disclosure aims to meet the need for improved analysis of cell-free DNA and / or provide other advantages. Accordingly, the following exemplary embodiments are provided.

[0011] Embodiment 1 is a method for analyzing DNA in a sample, comprising: a) partitioning the sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample contains DNA with a higher proportion of nucleotide modifications (e.g., cytosine modifications) than the second sub-sample; b) subjecting the first sub-sample to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first sub-sample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base-pairing specificity; and c) sequencing the DNA in the first sub-sample and the DNA in the second sub-sample so as to distinguish a first nucleobase from a second nucleobase in the DNA of the first sub-sample.

[0012] Embodiment 2 is the method of embodiment 1, wherein the DNA comprises cell-free DNA (cfDNA) obtained from the test subject.

[0013] Embodiment 3 is a method of analyzing DNA in a sample, including cell-free DNA (cfDNA) obtained from a test subject, comprising: a) partitioning the sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample contains DNA with a higher proportion of nucleotide modifications (e.g., cytosine modifications) than the second sub-sample; b) subjecting the first sub-sample to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first sub-sample, wherein the first nucleobase is a modified or unmodified nucleobase and the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and wherein the first nucleobase and the second nucleobase have the same base-pairing specificity; and c) sequencing the DNA in at least a first sub-sample so as to distinguish a first nucleobase from a second nucleobase in the DNA of the first sub-sample; The method includes:

[0014] Embodiment 4 is the method of embodiment 3, wherein step c) comprises sequencing DNA in at least the first sub-sample.

[0015] Embodiment 5 is a method for analyzing a sample containing cell-free DNA (cfDNA), comprising: a) partitioning the sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample comprises cfDNA having a higher proportion of nucleotide modifications (e.g., cytosine modifications) than the second sub-sample; b) subjecting the first sub-sample to a procedure that affects a first nucleobase in the cfDNA differently from a second nucleobase in the cfDNA of the first sub-sample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base-pairing specificity; c) capturing at least a set of epigenetic target regions of cfDNA from the first and second sub-samples, thereby providing captured cfDNA; and d) sequencing the captured cfDNA to distinguish the first nucleobase from the second nucleobase in the cfDNA from the first sub-sample. The method includes:

[0016] Embodiment 6 is a method for analyzing a sample containing cell-free DNA (cfDNA), comprising: a) partitioning the sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample comprises cfDNA having a higher proportion of nucleotide modifications (e.g., cytosine modifications) than the second sub-sample; b) subjecting the first sub-sample to a procedure that affects a first nucleobase in the cfDNA differently from a second nucleobase in the cfDNA of the first sub-sample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base-pairing specificity; c) capturing a set of multiple target regions of cfDNA from the first and second sub-samples, thereby providing captured cfDNA, wherein the set of multiple target regions comprises a set of sequence variable target regions and a set of epigenetic target regions; and d) sequencing the captured cfDNA to distinguish the first nucleobase from the second nucleobase in the captured cfDNA from the first sub-sample. The method includes:

[0017] Embodiment 7 is the method of embodiment 6, wherein cfDNA molecules corresponding to the set of sequence variable target regions are captured in the sample with a higher capture yield than cfDNA molecules corresponding to the set of epigenetic target regions.

[0018] Embodiment 8 is a method of isolating cell-free DNA (cfDNA) from a sample, comprising: a) partitioning the sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample comprises cfDNA having a higher proportion of nucleotide modifications (e.g., cytosine modifications) than the second sub-sample; b) subjecting the first sub-sample to a procedure that affects a first nucleobase in the cfDNA differently from a second nucleobase in the cfDNA of the first sub-sample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base-pairing specificity; c) contacting the cfDNA of the first and second sub-samples with target-specific probe sets, the target-specific probe sets comprising target-binding probes specific for a sequence-variable target set and target-binding probes specific for an epigenetic target set, whereby complexes between the target-specific probes and the cfDNA are formed; separating the complexes from cfDNA that is not bound to the target-specific probes, thereby providing captured cfDNA corresponding to the sequence-variable target set and cfDNA corresponding to the epigenetic target set; and d) sequencing the captured cfDNA to distinguish the first nucleobase from the second nucleobase in the cfDNA from the first sub-sample. The method includes:

[0019] Embodiment 9 is the method of embodiment 8, wherein the target-specific probe sets are configured to capture cfDNA corresponding to the sequence-variable target set with a higher capture yield than cfDNA corresponding to the epigenetic target set.

[0020] Embodiment 10 is a method for identifying the presence of DNA produced by a tumor, comprising: a) collecting a cfDNA sample from a test subject; b) partitioning the cfDNA sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample comprises captured cfDNA having a higher proportion of nucleotide modifications (e.g., cytosine modifications) than the second sub-sample; c) subjecting the first sub-sample to a procedure that affects a first nucleobase in the cfDNA differently from a second nucleobase in the cfDNA of the first sub-sample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base-pairing specificity; d) capturing a set of multiple target regions from the cfDNA in the first and second sub-samples, thereby providing a sample containing the captured cfDNA, wherein the set of multiple target regions comprises a set of sequence variable target regions and a set of epigenetic target regions; and e) sequencing the captured cfDNA in the first sub-sample and the captured cfDNA in the second sub-sample to distinguish the first nucleobase from the second nucleobase in the cfDNA of the first sub-sample. The method includes:

[0021] Embodiment 11 is the method of embodiment 10, wherein cfDNA molecules corresponding to the set of sequence variable target regions are captured in the sample with a higher capture yield than cfDNA molecules corresponding to the set of epigenetic target regions.

[0022] Embodiment 12 is the method of any one of embodiments 6 to 11, comprising sequencing cfDNA molecules corresponding to the set of sequence-variable target regions to a higher sequencing depth than cfDNA molecules corresponding to the set of epigenetic target regions.

[0023] Embodiment 13 is the method of embodiment 12, wherein the captured cfDNA molecules of the sequence-variable target set are sequenced to a sequencing depth that is at least two-fold higher than the captured cfDNA molecules of the epigenetic target region set.

[0024] Embodiment 14 is the method of embodiment 12, wherein the captured cfDNA molecules of the sequence-variable target set are sequenced to a sequencing depth that is at least three times higher than the captured cfDNA molecules of the epigenetic target region set.

[0025] Embodiment 15 is the method of embodiment 12, wherein the captured cfDNA molecules of the sequence-variable target set are sequenced to a sequencing depth that is 4 to 10 times higher than the captured cfDNA molecules of the epigenetic target region set.

[0026] Embodiment 16 is the method of embodiment 12, wherein the captured cfDNA molecules of the sequence-variable target set are sequenced to a sequencing depth that is 4 to 100 times higher than the captured cfDNA molecules of the epigenetic target region set.

[0027] Embodiment 17 is the method of any one of embodiments 6 to 16, wherein the captured cfDNA molecules of the sequence-variable target set and the captured cfDNA molecules of the epigenetic target region set are sequenced in the same sequencing cell.

[0028] Embodiment 18 is the method according to any one of the preceding embodiments, wherein the DNA is amplified before sequencing, or wherein the method comprises a capture step and the DNA is amplified before the capture step.

[0029] Embodiment 19 is the method of embodiments 5 to 18, further comprising the step of ligating a barcode-containing adapter to the DNA before capture, where the ligation step is optionally performed before or simultaneously with amplification.

[0030] Embodiment 20 is the method of any one of embodiments 5 to 19, wherein the set of epigenetic target regions comprises a set of hypermethylated variable target regions.

[0031] Embodiment 21 is the method of any one of embodiments 5 to 20, wherein the set of epigenetic target regions comprises a set of hypomethylated variable target regions.

[0032] Embodiment 22 is the method of embodiment 20 or 21, wherein the set of epigenetic target regions comprises a set of methylation control target regions.

[0033] Embodiment 23 is the method of any one of embodiments 5 to 22, wherein the set of epigenetic target regions comprises a set of fragmented variable target regions.

[0034] Embodiment 24 is the method of embodiment 23, wherein the set of fragmented variable target regions comprises a transcription start site region.

[0035] Embodiment 25 is the method of embodiment 23 or 24, wherein the set of fragmented variable target regions comprises a CTCF binding region.

[0036] Embodiment 26 is a method according to any one of embodiments 5 to 25, wherein capturing the set of multiple target regions of cfDNA comprises contacting the cfDNA with target binding probes specific for the set of sequence-variable target regions and target binding probes specific for the set of epigenetic target regions.

[0037] Embodiment 27 is the method of embodiment 26, wherein the target binding probes specific for the set of sequence variable target regions are present at a higher concentration than the target binding probes specific for the set of epigenetic target regions.

[0038] Embodiment 28 is the method of embodiment 26, wherein the target binding probes specific for the set of sequence variable target regions are present at a concentration that is at least two-fold greater than the target binding probes specific for the set of epigenetic target regions.

[0039] Embodiment 29 is the method of embodiment 26, wherein the target binding probes specific for the set of sequence variable target regions are present at a concentration at least four or five times greater than the target binding probes specific for the set of epigenetic target regions.

[0040] Embodiment 30 is the method of embodiment 26, wherein the target binding probes specific for the set of sequence variable target regions are present at a concentration that is at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than the target binding probes specific for the set of epigenetic target regions, or wherein the target binding probes specific for the set of sequence variable target regions are present at a concentration in the range of 2 to 3, 3 to 4, 4 to 5, 5 to 7, 7 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 times greater than the concentration of the target binding probes specific for the set of epigenetic target regions.

[0041] Embodiment 31 is a method according to any one of embodiments 26 to 30, wherein the target binding probes specific for the set of sequence-variable target regions have a higher target binding affinity than the target binding probes specific for the set of epigenetic target regions.

[0042] Embodiment 32 is the method of any one of embodiments 6 to 32, wherein the footprint of the set of epigenetic target regions is at least two times larger than the size of the set of sequence variable target regions.

[0043] Embodiment 33 is the method of embodiment 32, wherein the footprint of the set of epigenetic target regions is at least 10 times larger than the size of the set of sequence variable target regions.

[0044] Embodiment 34 is the method of any one of embodiments 6 to 33, wherein the footprint of the set of sequence variable target regions is at least 25 kB or 50 kB.

[0045] Embodiment 35 is the method of any one of the preceding embodiments, wherein partitioning the sample into a plurality of sub-samples comprises partitioning based on methylation levels.

[0046] Embodiment 36 is the method of embodiment 35, wherein the distributing step comprises contacting the collected cfDNA with a methyl-binding reagent immobilized on a solid support.

[0047] Embodiment 37 is a method according to any one of the preceding embodiments, comprising differentially tagging the first sub-sample and the second sub-sample.

[0048] Embodiment 38 is the method of embodiment 37, wherein the first sub-sample and the second sub-sample are differentially tagged before subjecting the first sub-sample to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first sub-sample.

[0049] Embodiment 39 is the method of embodiment 37 or 38, wherein the first sub-sample and the second sub-sample are pooled after subjecting the first sub-sample to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first sub-sample.

[0050] Embodiment 40 is the method according to any one of embodiments 37 to 39, wherein the first sub-sample and the second sub-sample are sequenced in the same sequencing cell.

[0051] Embodiment 41 is the method of any one of the preceding embodiments, wherein the plurality of sub-samples includes a third sub-sample comprising DNA with a higher proportion of nucleotide modifications (e.g., cytosine modifications) than the second sub-sample but a lower proportion than the first sub-sample.

[0052] Embodiment 42 is the method of embodiment 41, further comprising differentially tagging the third sub-sample so that it is distinguishable from the first sub-sample and the second sub-sample.

[0053] Embodiment 43 is the method of embodiment 42, wherein after subjecting the first sub-sample to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first sub-sample, the first, second, and third sub-samples are combined, and optionally the first, second, and third sub-samples are sequenced in the same sequencing cell.

[0054] Embodiment 44 is the method of any one of the preceding embodiments, wherein the procedure by which the first sub-sample is subjected alters the base-pairing specificity of the first nucleobase without substantially altering the base-pairing specificity of the second nucleobase.

[0055] Embodiment 45 is the method of any one of the preceding embodiments, wherein the first nucleobase is a modified or unmodified cytosine and the second nucleobase is a modified or unmodified cytosine.

[0056] Embodiment 46 is the method of any one of the preceding embodiments, wherein the first nucleobase comprises an unmodified cytosine (C).

[0057] Embodiment 47 is the method of any one of the preceding embodiments, wherein the second nucleobase comprises 5-methylcytosine (mC).

[0058] Embodiment 48 is the method according to any one of the preceding embodiments, wherein the procedure to which the first sub-sample is subjected comprises bisulfite conversion.

[0059] Embodiment 49 is the method of any one of embodiments 1 to 46, wherein the first nucleobase comprises mC.

[0060] Embodiment 50 is the method of any one of the preceding embodiments, wherein the second nucleobase comprises 5-hydroxymethylcytosine (hmC).

[0061] Embodiment 51 is the method according to embodiment 50, wherein the procedure to which the first sub-sample is subjected comprises protection of 5hmC.

[0062] Embodiment 52 is the method of embodiment 50, wherein the procedure to which the first sub-sample is subjected comprises Tet-assisted bisulfite conversion.

[0063] Embodiment 53 is the method of embodiment 50, wherein the procedure to which the first sub-sample is subjected comprises a Tet-assisted conversion with a substituted borane reducing agent, optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.

[0064] Embodiment 54 is the method of embodiment 53, wherein the substituted borane reducing agent is 2-picoline borane or borane pyridine.

[0065] Embodiment 55 is the method of any one of embodiments 49-51 or 53-54, wherein the second nucleobase comprises C.

[0066] Embodiment 56 is the method of any one of embodiments 49-51 or 55, wherein the procedure to which the first subsample is subjected comprises protection of hmC, followed by Tet-assisted conversion with a substituted borane reducing agent, optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.

[0067] Embodiment 57 is the method of embodiment 56, wherein the substituted borane reducing agent is 2-picoline borane or borane pyridine.

[0068] Embodiment 58 is the method of any one of embodiments 46, 47, 49-51, or 55, wherein the procedure to which the first sub-sample is subjected comprises protection of hmC, followed by deamination of mC and / or C.

[0069] Embodiment 59 is the method of embodiment 58, wherein deamination of mC and / or C comprises treatment with an AID / APOBEC family DNA deaminase enzyme.

[0070] Embodiment 60 is the method of any one of embodiments 51 or 55-59, wherein the protection of hmC comprises glycosylation of hmC.

[0071] Embodiment 61 is the method of any one of embodiments 1 to 45, 47, 49, or 55, wherein the procedure to which the first sub-sample is subjected comprises a chemically assisted conversion with a substituted borane reducing agent, optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.

[0072] Embodiment 62 is the method of embodiment 61, wherein the substituted borane reducing agent is 2-picoline borane or borane pyridine.

[0073] Embodiment 63 is the method of any one of embodiments 1-45, 47, 49, 55, or 61-62, wherein the first nucleobase comprises hmC.

[0074] Embodiment 64 is the method according to any one of embodiments 1 to 44, wherein the procedure in which the first sub-sample is provided comprises a step of separating DNA that naturally contains the first nucleobase from DNA that does not naturally contain the first nucleobase.

[0075] Embodiment 65 is the method of embodiment 64, wherein the first nucleobase is hmC.

[0076] Embodiment 66 is the method of embodiment 64 or 65, wherein separating DNA that naturally contains the first nucleobase from DNA that does not naturally contain the first nucleobase comprises labeling the first nucleobase.

[0077] Embodiment 67 is the method of embodiment 66, wherein the labeling step comprises biotinylating.

[0078] Embodiment 68 is the method of embodiment 66 or 67, wherein the labeling step comprises glycosylating.

[0079] Embodiment 69 is the method of embodiment 68, wherein the glucosylating step attaches a glucosyl-azide moiety.

[0080] Embodiment 70 is the method of embodiment 66 or 68, wherein the labeling step comprises glucosylating before biotinylating, and subsequently attaching a biotin moiety to the glucosyl.

[0081] Embodiment 71 is the method of embodiment 70, wherein the step of attaching the biotin moiety to the glucosyl comprises Huisgen cycloaddition chemistry.

[0082] Embodiment 72 is the method of any one of embodiments 54 to 61, wherein the step of separating DNA that naturally contains the first nucleobase from DNA that does not naturally contain the first nucleobase comprises binding the DNA that naturally contains the first nucleobase to a capture agent.

[0083] Embodiment 73 is the method of embodiment 72, wherein the capture agent comprises a biotin-binding agent, optionally wherein the biotin-binding agent comprises avidin or streptavidin.

[0084] Embodiment 74 is the method of any one of embodiments 64 to 73, comprising differentially tagging each of the DNA that naturally includes the first nucleobase, the DNA that does not naturally include the first nucleobase, and the DNA of the second sub-sample.

[0085] Embodiment 75 is the method of embodiment 74, comprising differentially tagging and then pooling the DNA that originally comprises the first nucleobase, the DNA that does not originally comprise the first nucleobase, and the DNA of the second sub-sample, and optionally sequencing the DNA that originally comprises the first nucleobase, the DNA that does not originally comprise the first nucleobase, and the DNA of the second sub-sample in the same sequencing cell.

[0086] Embodiment 76 is the method of any one of embodiments 1 to 44, wherein the first nucleobase is a modified or unmodified adenine and the second nucleobase is a modified or unmodified adenine.

[0087] Embodiment 77 is the method of any one of embodiments 1 to 44, wherein the first nucleobase is modified or unmodified guanine and the second nucleobase is modified or unmodified guanine.

[0088] Embodiment 78 is the method of any one of embodiments 1 to 44, wherein the first nucleobase is modified or unmodified thymine and the second nucleobase is modified or unmodified thymine.

[0089] Embodiment 79 is the method of any one of the preceding embodiments, wherein the second subpopulation is not subjected to a procedure that affects the first nucleobase differently than the second nucleobase.

[0090] Embodiment 80 is a combination comprising first and second populations of captured DNA, wherein the first population comprises or is derived from DNA having a higher proportion of nucleotide modifications (e.g., cytosine modifications) than the second population, wherein a first nucleobase type originally present in the DNA before the base-pairing specificity is altered is a modified or unmodified nucleobase, and the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, wherein the first nucleobase type originally present in the DNA before the base-pairing specificity is altered and the second nucleobase have the same base-pairing specificity, and wherein the second population does not comprise the first nucleobase type originally present in the DNA with the altered base-pairing specificity.

[0091] Embodiment 81 is the combination of embodiment 80, wherein the first population comprises sequence tags selected from a first set of one or more sequence tags, and the second population comprises sequence tags selected from a second set of one or more sequence tags, the second set of sequence tags being different from the first set of sequence tags.

[0092] Embodiment 82 is the combination of embodiment 81, wherein the sequence tag comprises a barcode.

[0093] Embodiment 83 is a combination according to any one of embodiments 80 to 82, wherein the cytosine modification is a methylation.

[0094] Embodiment 84 is a combination according to any one of embodiments 80 to 83, wherein the first nucleobase is a modified or unmodified cytosine and the second nucleobase is a modified or unmodified cytosine.

[0095] Embodiment 85 is a combination according to any one of embodiments 80 to 84, wherein the first nucleobase comprises an unmodified cytosine (C).

[0096] Embodiment 86 is a combination according to any one of embodiments 80 to 85, wherein the second nucleobase comprises one or both of 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC).

[0097] Embodiment 87 is a combination according to any one of embodiments 80 to 86, wherein the first population has been subjected to bisulfite conversion.

[0098] Embodiment 88 is a combination according to any one of embodiments 80 to 86, wherein the first nucleobase comprises mC.

[0099] Embodiment 89 is a combination according to any one of embodiments 80 to 88, wherein the second nucleobase comprises hmC.

[0100] Embodiment 90 is a combination according to any one of embodiments 80 to 89, wherein the first population comprises protected hmC.

[0101] Embodiment 91 is a combination according to embodiment 84 or 90, wherein the first population has been subjected to Tet-assisted bisulfite conversion.

[0102] Embodiment 92 is the combination of embodiment 84 or 90, wherein the first population has been subjected to a Tet-assisted conversion with a substituted borane reducing agent, optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.

[0103] Embodiment 93 is the combination of embodiment 90, wherein the first population is subjected to protection of hmC followed by Tet-assisted conversion with a substituted borane reducing agent, which is optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.

[0104] Embodiment 94 is a combination of any one of embodiments 80-82, 86, 88-90, or 92-93, wherein the second nucleobase comprises C.

[0105] Embodiment 95 is a combination according to embodiment 90, wherein the first population has been subjected to protection of hmC and subsequent deamination of mC and / or C.

[0106] Embodiment 96 is a combination according to any one of embodiments 90 to 95, wherein the protected hmC comprises glycosylated hmC.

[0107] Embodiment 97 is a combination according to any one of embodiments 80 to 83, wherein the first nucleobase comprises hmC.

[0108] Embodiment 98 is a combination of any one of embodiments 80 to 83, or 97, wherein the second nucleobase comprises mC.

[0109] Embodiment 99 is a combination according to any one of embodiments 80 to 83 or 97 to 98, wherein the second nucleobase comprises C.

[0110] Embodiment 100 is the combination of any one of embodiments 80-83 or 97-99, wherein the first population has been subjected to a chemical-assisted conversion with a substituted borane reducing agent, optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.

[0111] Embodiment 101 is a combination of any one of embodiments 80 to 83, wherein the first nucleobase is a modified or unmodified adenine and the second nucleobase is a modified or unmodified adenine.

[0112] Embodiment 102 is a combination according to any one of embodiments 80 to 83, wherein the first nucleobase is a modified or unmodified guanine and the second nucleobase is a modified or unmodified guanine.

[0113] Embodiment 103 is a combination according to any one of embodiments 80 to 83, wherein the first nucleobase is a modified or unmodified thymine and the second nucleobase is a modified or unmodified thymine.

[0114] Embodiment 104 is a combination comprising a first and a second population of captured DNA, the first population comprises or is derived from DNA having a higher proportion of nucleotide modifications (e.g., cytosine modifications) than the second population; the first population comprises first and second subpopulations; the first subpopulation comprising a higher proportion of the first nucleobase than the second subpopulation; A combination in which the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base-pairing specificity; when the first nucleobase is modified or unmodified thymine, the second nucleobase is modified or unmodified thymine, and the second population does not include the first nucleobase.

[0115] Embodiment 105 is a combination according to embodiment 104, wherein the first nucleobase is a modified or unmodified cytosine and the second nucleobase is a modified or unmodified cytosine.

[0116] Embodiment 106 is the combination of embodiment 105, wherein the first nucleobase is a protected modified cytosine.

[0117] Embodiment 107 is a combination according to embodiment 105 or 106, wherein the first nucleobase is a derivative of hmC.

[0118] Embodiment 108 is the combination of embodiment 107, wherein the first nucleobase is glycosylated hmC.

[0119] Embodiment 109 is a combination according to embodiment 107 or embodiment 108, wherein the first nucleobase is biotinylated hmC.

[0120] Embodiment 110 is a combination according to any one of embodiments 106 to 109, wherein the first nucleobase is the product of Huisgen cycloaddition to β-6-azido-glucosyl-5-hydroxymethylcytosine comprising an affinity label.

[0121] Embodiment 111 is the combination of embodiment 104, wherein the first nucleobase is a modified or unmodified adenine and the second nucleobase is a modified or unmodified adenine.

[0122] Embodiment 112 is the combination of embodiment 104, wherein the first nucleobase is a modified or unmodified guanine and the second nucleobase is a modified or unmodified guanine.

[0123] Embodiment 113 is the combination of embodiment 104, wherein the first nucleobase is a modified or unmodified thymine and the second nucleobase is a modified or unmodified thymine.

[0124] Embodiment 114 is a combination according to any one of embodiments 104 to 113, wherein the first subpopulation comprises a first sequence tag, the second subpopulation comprises a second sequence tag different from the first sequence tag, and the second population comprises a third sequence tag different from the first and second sequence tags, and optionally the first, second, and / or third tag is a barcode.

[0125] Embodiment 115 is a combination according to any one of embodiments 80 to 114, wherein the captured DNA comprises cfDNA.

[0126] Embodiment 116 is a combination according to any one of embodiments 80 to 115, wherein the captured DNA comprises a sequence variable target region and an epigenetic target region, the concentration of the sequence variable target region is greater than the concentration of the epigenetic target region, and the concentrations are normalized with respect to the footprint size of the sequence variable target region and the epigenetic target region.

[0127] Embodiment 117 is a combination according to embodiment 116, wherein the concentration of the sequence variable target region is at least two-fold higher than the concentration of the epigenetic target region.

[0128] Embodiment 118 is a combination according to embodiment 116, wherein the concentration of the sequence variable target region is at least four or five times higher than the concentration of the epigenetic target region.

[0129] Embodiment 119 is a combination according to any one of embodiments 116 to 118, wherein the concentrations are mass / volume concentrations normalized with respect to the footprint size of the target area.

[0130] Embodiment 120 is a combination of any one of embodiments 116 to 119, wherein the epigenetic target region comprises one, two, three, or four of: a hypermethylated variable target region; a hypomethylated variable target region; a transcription start site region; and a CTCF binding region; and optionally, the epigenetic target region further comprises a methylation control target region.

[0131] Embodiment 121 is a combination according to any one of embodiments 80 to 120, produced according to a method according to any one of embodiments 1 to 79.

[0132] Embodiment 122 is a communication interface that receives, via a communication network, a plurality of sequence reads generated by the nucleic acid sequencer from sequencing the DNA in the first sub-sample and the DNA in the second sub-sample according to the method of any one of embodiments 1 to 79; and When executed by at least one electronic processor, (i) receiving, via a communication network, sequence reads generated by a nucleic acid sequencer; and (ii) mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads. a controller including or having access to a computer-readable medium including non-transitory computer-executable instructions for performing a method including The system includes:

[0133] Embodiment 123 is a communication interface that receives, via a communication network, a plurality of sequence reads generated by a nucleic acid sequencer from sequencing the combination of the first and second populations of captured DNA according to any one of embodiments 80 to 121; and When executed by at least one electronic processor, (i) receiving, via a communication network, sequence reads generated by a nucleic acid sequencer; and (ii) mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads. a controller including or having access to a computer-readable medium including non-transitory computer-executable instructions for performing a method including The system includes:

[0134] Embodiment 124 is a method executed by at least one electronic processor, comprising: (iii) processing the mapped sequence reads corresponding to the set of sequence variable target regions and the set of epigenetic target regions to determine the likelihood that the subject has cancer. 124. The system of embodiment 122 or 123, further comprising:

[0135] Embodiment 125 is the method of any one of embodiments 1 to 79, further comprising determining the likelihood that the subject has cancer.

[0136] Embodiment 126 is the method according to the preceding embodiment, wherein the sequencing generates a plurality of sequence 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 corresponding to the set of sequence variable target regions and the mapped sequence reads corresponding to the set of epigenetic target regions to determine the likelihood that the subject has cancer.

[0137] Embodiment 127 is the method of any one of embodiments 1 to 79, wherein the test subject has already been diagnosed with cancer and has undergone one or more prior cancer treatments, and optionally, the cfDNA is obtained at one or more preselected time points after the one or more prior cancer treatments.

[0138] Embodiment 128 is a method according to the preceding embodiment, further comprising sequencing the captured set of cfDNA molecules, thereby producing a set of sequence information.

[0139] Embodiment 129 is the method according to the previous embodiment, wherein the captured DNA molecules of the set of sequence-variable target regions are sequenced to a higher sequencing depth than the captured DNA sequences of the set of epigenetic target regions.

[0140] Embodiment 130 is the method of embodiment 128 or 129, further comprising detecting the presence or absence of DNA originating from or derived from tumor cells at a preselected time point using the sequence information set.

[0141] Embodiment 131 is the method according to the preceding embodiments, further comprising determining a cancer recurrence score indicative of the presence or absence of DNA originating from or derived from the tumor cells of the test subject.

[0142] Embodiment 132 is the method according to the preceding embodiments, further comprising determining the cancer recurrence status based on the cancer recurrence score, wherein if the cancer recurrence score is determined to be at or above a predetermined threshold, the cancer recurrence status of the test subject is determined to be at risk of cancer recurrence, or if the cancer recurrence score is below the predetermined threshold, the cancer recurrence status of the test subject is determined to be at low risk of cancer recurrence.

[0143] Embodiment 133 is the method of embodiment 131 or 132, further comprising the step of comparing the cancer recurrence score of the test subject with a predetermined cancer recurrence threshold, wherein if the cancer recurrence score is above the cancer recurrence threshold, the test subject is classified as a candidate for subsequent cancer treatment, or if the cancer recurrence score is below the cancer recurrence threshold, the test subject is classified as not a candidate for subsequent cancer treatment.

[0144] Embodiment 134 is the method of any one of embodiments 131 to 133, wherein the test subject is classified as being at risk for cancer recurrence and a candidate for subsequent cancer treatment.

[0145] Embodiment 135 is the method of any one of embodiments 131, 133, or 134, wherein the subsequent cancer treatment comprises administration of a chemotherapy or therapeutic composition.

[0146] Embodiment 136 is the method of any one of embodiments 132 to 135, wherein the DNA originating or derived from the tumor cell is cell-free DNA.

[0147] Embodiment 137 is the method of any one of embodiments 132 to 135, wherein the DNA originating from or derived from the tumor cell is obtained from a tissue sample.

[0148] Embodiment 138 is a method according to any one of claims 129 to 137, further comprising determining the disease-free survival (DFS) period of the test subject based on the cancer recurrence score.

[0149] Embodiment 139 is the method of embodiment 138, wherein the DFS period is 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years.

[0150] Embodiment 140 is the method of any one of embodiments 128 to 139, wherein the sequence information set comprises a sequence variable target region sequence, and determining the cancer recurrence score comprises determining at least a first subscore indicative of the amount of SNV, insertion / deletion, CNV, and / or fusion present in the sequence variable target region sequence.

[0151] Embodiment 141 is the method of embodiment 140, wherein the number of mutations in the sequence variable target regions 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, optionally wherein the number of mutations is selected from 1, 2, or 3.

[0152] Embodiment 142 is a method described in any one of embodiments 128 to 141, wherein the sequence information set includes an epigenetic target region sequence, and the step of determining the cancer recurrence score includes a step of determining a second subscore indicating the amount of abnormal sequence reads in the epigenetic target region sequence.

[0153] Embodiment 143 is the method of embodiment 142, wherein the aberrant sequence reads comprise reads indicative of methylation of a hypermethylated variable target sequence and / or reads indicative of aberrant fragmentation in a fragmented variable target region.

[0154] Embodiment 144 is the method of embodiment 143, wherein a proportion of reads corresponding to the set of hypermethylated variable target regions and / or fragmented variable target regions exhibiting hypermethylation in the set of hypermethylated variable target regions and / or aberrant fragmentation in the set of fragmented variable target regions greater than or equal to a value in the range of 0.001% to 10% is sufficient to classify the second subscore as positive for cancer recurrence.

[0155] Embodiment 145 is the method of embodiment 144, wherein the range is 0.001% to 1% or 0.005% to 1%.

[0156] Embodiment 146 is the method of embodiment 144, wherein the range is 0.01% to 5% or 0.01% to 2%.

[0157] Embodiment 147 is the method of embodiment 144, wherein the range is 0.01% to 1%.

[0158] Embodiment 148 is a method described in any one of embodiments 128 to 147, further comprising a step of determining the proportion of tumor DNA from the proportion of reads in the sequence information set that exhibit one or more characteristics indicative of originating from tumor cells.

[0159] Embodiment 149 is the method of embodiment 148, wherein the one or more features indicative of origin from a tumor cell include one or more of alterations in sequence variable target regions, hypermethylation of hypermethylated variable target regions, and aberrant fragmentation of fragmented variable target regions.

[0160] Embodiment 150 further comprises determining a cancer recurrence score based at least in part on the percentage of tumor DNA, -11 ~1 or 10 -10 150. The method of embodiment 148 or 149, wherein a proportion of tumor DNA greater than or equal to a predetermined value in the range of ∼1 is sufficient to classify the Cancer Recurrence Score as positive for cancer recurrence.

[0161] Embodiment 151 is 10 -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 -1 151. The method of embodiment 150, wherein a proportion of tumor DNA greater than or equal to a predetermined value in the range of is sufficient to classify the Cancer Recurrence Score as positive for cancer recurrence.

[0162] In embodiment 152, the default value is 10 -8 ~10 -6 or in the range of 10 -7 152. The method of embodiment 150 or 151, wherein

[0163] Embodiment 153 is a method described in any one of embodiments 149 to 152, wherein the proportion of tumor DNA is determined to be greater than or equal to a predetermined value if the cumulative probability that the proportion of tumor DNA is greater than or equal to a predetermined value is at least 0.5, 0.75, 0.9, 0.95, 0.98, 0.99, 0.995, or 0.999.

[0164] Embodiment 154 is the method of embodiment 153, in which the cumulative probability is at least 0.95.

[0165] Embodiment 155 is the method described in embodiment 153, in which the cumulative probability is in the range of 0.98 to 0.995, or is 0.99.

[0166] Embodiment 156 is the method of any one of embodiments 128 to 155, wherein the sequence information set comprises a sequence variable target region sequence and an epigenetic target region sequence, and determining the cancer recurrence score comprises determining a first subscore indicative of the amount of SNVs, insertions / deletions, CNVs, and / or fusions present in the sequence variable target region sequence and a second subscore indicative of the amount of aberrant sequence reads in the epigenetic target region sequence, and combining the first and second subscores to provide the cancer recurrence score.

[0167] Embodiment 157 is the method of embodiment 156, wherein combining the first and second subscores comprises applying a threshold 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 aberrant (e.g., tumor) reads in the epigenetic target region), or comprises training a machine learning classifier to determine the status based on multiple positive and negative training samples.

[0168] Embodiment 158 ​​is the method of embodiment 157, wherein a combined score value ranging from −4 to 2 or −3 to 1 is sufficient to classify the Cancer Recurrence Score as positive for cancer recurrence.

[0169] Embodiment 159 is a method according to any one of embodiments 127 to 158, wherein the one or more preselected time points are selected from the group consisting of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, and 5 years after administration of the one or more previous cancer treatments.

[0170] Embodiment 160 is the method of any one of embodiments 127 to 159, wherein the cancer is colorectal cancer.

[0171] Embodiment 161 is the method of any one of embodiments 127 to 160, wherein the one or more previous cancer treatments include surgery.

[0172] Embodiment 162 is the method of any one of embodiments 127 to 161, wherein the one or more previous cancer treatments include administration of a therapeutic composition.

[0173] Embodiment 163 is the method of any one of embodiments 127 to 162, wherein the one or more previous cancer treatments include chemotherapy.

[0174] Embodiment 164 is a combination according to any one of embodiments 80 to 120, wherein the altered base specificity is produced by chemical conversion.

[0175] Embodiment 165 is the combination of embodiment 164, wherein the chemical transformation is selected from the group consisting of (i) bisulfite transformation, (ii) Tet-assisted bisulfite transformation, (iii) Tet-assisted transformation with a substituted borane reducing agent, and (iv) protection of hmC followed by Tet-assisted transformation with a substituted borane reducing agent. I. Brief description of the drawings [Brief explanation of the drawings]

[0176] [Figure 1-1] 1 illustrates an exemplary workflow according to certain embodiments of the present disclosure, starting with a blood sample, in which cfDNA is isolated from the blood sample, the cfDNA is partitioned into hypo-, moderately, and hypermethylated sub-samples using methyl-binding domain proteins (MBDs), each sub-sample is subjected to molecular barcoding to distinguishably tag DNA from the hypo-, moderately, and hypermethylated sub-samples, the hypermethylated sub-sample is treated with bisulfite (BS) to convert C to U; the samples are pooled (in any suitable order), captured, amplified, and sequenced. Methods generally similar to this exemplary method, including bisulfite conversion of the first sub-sample, which is the hypermethylated sub-sample, indicate, through conversion or its absence, which cytosine positions were unmodified cytosines relative to mC or hmC. [Figure 1-2] 1 illustrates an exemplary workflow according to certain embodiments of the present disclosure, starting with a blood sample, in which cfDNA is isolated from the blood sample, the cfDNA is partitioned into hypo-, moderately, and hypermethylated sub-samples using methyl-binding domain proteins (MBDs), each sub-sample is subjected to molecular barcoding to distinguishably tag DNA from the hypo-, moderately, and hypermethylated sub-samples, the hypermethylated sub-sample is treated with bisulfite (BS) to convert C to U; the samples are pooled (in any suitable order), captured, amplified, and sequenced. Methods generally similar to this exemplary method, including bisulfite conversion of the first sub-sample, which is the hypermethylated sub-sample, indicate, through conversion or its absence, which cytosine positions were unmodified cytosines relative to mC or hmC.

[0177] [Figure 2-1]Figure 2 illustrates an exemplary workflow according to certain embodiments of the present disclosure, starting with a blood sample, in which cfDNA is isolated from the blood sample, the cfDNA is partitioned into hypo-, moderately, and hypermethylated sub-samples using methyl-binding domain proteins (MBDs), each sub-sample is subjected to molecular barcoding to distinguishably tag DNA from the hypo-, moderately, and hypermethylated sub-samples, the hypermethylated sub-sample is treated with β-glucosyltransferase (protecting hmC via glycosylation) and APOBEC3A (A3A) (deaminating C and mC so that they sequence as U / T); the samples are pooled (in any suitable order), captured, amplified, and sequenced. A generally similar method to this exemplary method, involving protection of hmC and deamination of mC in the first sub-sample, the hypermethylated sub-sample, indicates, through conversion or absence thereof, which cytosine positions were unmodified cytosine or mC relative to hmC. [Figure 2-2] Figure 2 illustrates an exemplary workflow according to certain embodiments of the present disclosure, starting with a blood sample, in which cfDNA is isolated from the blood sample, the cfDNA is partitioned into hypo-, moderately, and hypermethylated sub-samples using methyl-binding domain proteins (MBDs), each sub-sample is subjected to molecular barcoding to distinguishably tag DNA from the hypo-, moderately, and hypermethylated sub-samples, the hypermethylated sub-sample is treated with β-glucosyltransferase (protecting hmC via glycosylation) and APOBEC3A (A3A) (deaminating C and mC so that they sequence as U / T); the samples are pooled (in any suitable order), captured, amplified, and sequenced. A generally similar method to this exemplary method, involving protection of hmC and deamination of mC in the first sub-sample, the hypermethylated sub-sample, indicates, through conversion or absence thereof, which cytosine positions were unmodified cytosine or mC relative to hmC.

[0178] [Figure 3-1]Figure 3 illustrates an exemplary workflow according to certain embodiments of the present disclosure, starting with a blood sample, in which cfDNA is isolated from the blood sample; the cfDNA is partitioned into hypo-, moderately-, and hypermethylated subsamples using a methyl-binding domain protein (MBD); the hypermethylated subsample is further separated using an hmC-sealing method involving treatment with βGT and biotinylation to separate hmC-containing DNA from other DNA; each subsample is subjected to molecular barcoding to distinguishably tag DNA from the hypomethylated, moderately-, hypermethylated, and hypermethylated + hmC subsamples; the samples are pooled (in any suitable order), captured, amplified, and sequenced. A generally similar method to this exemplary method, involving separation of hmC-containing DNA from other DNA in the hypermethylated subsample, indicates which reads of the hypermethylated DNA contained hmC. [Figure 3-2] Figure 3 illustrates an exemplary workflow according to certain embodiments of the present disclosure, starting with a blood sample, in which cfDNA is isolated from the blood sample; the cfDNA is partitioned into hypo-, moderately-, and hypermethylated subsamples using a methyl-binding domain protein (MBD); the hypermethylated subsample is further separated using an hmC-sealing method involving treatment with βGT and biotinylation to separate hmC-containing DNA from other DNA; each subsample is subjected to molecular barcoding to distinguishably tag DNA from the hypomethylated, moderately-, hypermethylated, and hypermethylated + hmC subsamples; the samples are pooled (in any suitable order), captured, amplified, and sequenced. A generally similar method to this exemplary method, involving separation of hmC-containing DNA from other DNA in the hypermethylated subsample, indicates which reads of the hypermethylated DNA contained hmC.

[0179] [Figure 4] FIG. 4 is a schematic diagram of an example system suitable for use with some embodiments of the present disclosure.

[0180] [Figure 5]Figure 5 shows CpG methylation quantification results obtained as described in Example 2 for three samples from subjects with early stage colorectal cancer ("Early CRC") and three healthy subjects ("Normals"). For the Early CRC plot, MAF indicates the proportion of mutant alleles. DETAILED DESCRIPTION OF THE INVENTION

[0181] II. 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 they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the appended claims.

[0182] Before describing the teachings of the present invention in detail, it is understood that the disclosure is not limited to specific compositions or process steps, as these may vary. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, a reference to "a nucleic acid" includes a plurality of nucleic acids, a reference to "a cell" includes a plurality of cells, etc.

[0183] Numerical ranges are inclusive of the numbers defining the range. Measured and measurable values ​​are understood to be approximations, taking into account significant digits and errors associated with measurement. Similarly, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It is understood that both the foregoing general and detailed descriptions are exemplary and explanatory only and do not limit the present teachings.

[0184] Unless specifically noted in the specification above, embodiments herein that recite various components as "comprising" are also contemplated as "consisting of" or "consisting essentially of" the recited components, and embodiments herein that recite various components as "consisting essentially of" are also contemplated as "comprising" or "consisting essentially of" the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0185] The section headings used herein are for organizational purposes only and are not to be construed as limiting the disclosed subject matter in any way. In the event that any document or other material incorporated by reference conflicts with the express contents of this specification, including definitions, the present specification will control.

[0186] A.Definition "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 originally resides 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 in the organism, and can be obtained by obtaining a sample of the fluid without the need to perform an in vitro cell lysis step.

[0187] As used herein, a modification or other feature is "present in a higher proportion" in a first subsample or population of nucleic acids than in a second subsample or population if the proportion of nucleotides having the modification or other feature is higher in the first subsample or population than in the second subsample. For example, if one in ten nucleotides in the first subsample are mC and one in twelve nucleotides in the second subsample are mC, then the first subsample contains a higher proportion of 5-methylated cytosine modifications than the second subsample.

[0188] As used herein, "without substantially changing the base-pairing specificity" of a given nucleobase means that the majority of molecules that comprise the nucleobase that can be sequenced do not have the base-pairing specificity of the second nucleobase changed compared to its base-pairing specificity when present in the original isolated sample.In some embodiments, 75%, 90%, 95% or 99% of molecules that comprise the nucleobase that can be sequenced do not have the base-pairing specificity of the second nucleobase changed compared to its base-pairing specificity when present in the original isolated sample.

[0189] As used herein, "base-pairing specificity" refers to the standard DNA base (A, C, G, or T) with which a given base pairs most preferentially. Thus, for example, unmodified cytosine and 5-methylcytosine have the same base-pairing specificity (i.e., specificity for G), but uracil and cytosine have different base-pairing specificities, since uracil has base-pairing specificity for A, whereas cytosine has base-pairing specificity for G. The ability of uracil to form a wobble base pair with G is irrelevant, since uracil nevertheless pairs most preferentially with A among the four standard DNA bases.

[0190] As used herein, a "combination" including multiple members refers to either a single composition or a set of adjacent compositions that include the members, for example, in separate containers or compartments within a larger container, such as a multi-well plate, tube rack, refrigerator, freezer, incubator, water bath, ice bucket, machine, or other storage configuration.

[0191] The "capture yield" of a collection of probes for a given target set refers to the amount of nucleic acid corresponding to the target set that the collection of probes captures under typical conditions (e.g., the amount or absolute amount relative to another target set). Exemplary typical capture conditions are incubating sample nucleic acid and probes at 65°C for 10-18 hours in a small reaction volume (approximately 20 μL) containing a stringent hybridization buffer. Capture yields can be expressed in absolute terms, or, in the case of a collection of multiple probes, in relative terms. When comparing capture yields for a set of multiple target regions, they are normalized with respect to the footprint size (e.g., on a per kilobase basis) of the target region set. Thus, for example, if the footprint sizes of the first and second target regions are 50 kb and 500 kb, respectively (using a normalization factor of 0.1), and the mass / volume concentration of the captured DNA corresponding to the first set of target regions is greater than 0.1 times the mass / volume concentration of the captured DNA corresponding to the second set of target regions, the DNA corresponding to the first set of target regions will be captured with a higher yield than the 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 / volume concentration that is 0.2 times the mass / volume concentration of the captured DNA corresponding to the second set of target regions, the DNA corresponding to the first set of target regions will be captured with a capture yield that is 2 times higher than the DNA corresponding to the second set of target regions.

[0192] "Capturing" one or more target nucleic acids refers to preferentially isolating or separating one or more target nucleic acids from non-target nucleic acids.

[0193] A "captured set" of nucleic acids refers to nucleic acids that have undergone capture.

[0194] A "target region set" or "set of target regions" refers to multiple genomic loci targeted for capture and / or targeted by a set of probes (e.g., through sequence complementarity).

[0195] "Corresponding to a set of target regions" means that a nucleic acid, e.g., cfDNA, originates from a locus in the set of target regions or specifically binds to one or more probes for the set of target regions.

[0196] "Specifically bind" in the context of a probe or other oligonucleotide and a target sequence means that, under appropriate hybridization conditions, the oligonucleotide or probe hybridizes to its target sequence or a replica thereof to form a stable probe:target hybrid such that the formation of stable probe:non-target hybrids is minimized. Thus, the probe hybridizes to the target sequence or a replica thereof to a sufficiently greater extent than non-target sequences to allow 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 determined by using routine testing methods (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, incorporated herein by reference). ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), see §§ 1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57, especially §§ 9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57.

[0197] A "sequence variable target region set" refers to a set of target regions that may exhibit sequence changes such as nucleotide substitutions (i.e., single base mutations), insertions, deletions, or gene fusions or rearrangements in neoplastic cells (e.g., tumor and cancer cells).

[0198] " Epigenetic target region set " refers to a set of target regions that can show sequence-independent alterations in neoplastic cells (e.g., tumor cells and cancer cells), or can show sequence-independent changes in cfDNA from cancer subjects compared with cfDNA from healthy subjects.Examples of sequence-independent changes include, but are not limited to, methylation changes (increase or decrease), nucleosome distribution, CTCF binding, transcription start site, and regulatory protein binding region.For the purpose of the present invention, the loci that are prone to neoplastic, tumor, or cancer-related local amplification and / or gene fusion can also be included in the epigenetic target region set, because the detection of copy number changes by sequencing or fusion sequences that map to more than one locus in the reference genome tends to be more similar to the detection of exemplary epigenetic changes discussed above than the detection of nucleotide substitutions, insertions, or deletions, in that, for example, local amplification and / or gene fusion can be detected at a relatively low sequencing depth because its detection does not depend on the accuracy of base calls at one or a few individual positions. In some embodiments, the set of epigenetic target regions includes one or more genomic regions in which the epigenetic state (e.g., methylation state) of cfDNA molecules in these regions is unchanged in cancer, but whose presence / amount in the blood indicates increased aberrant representation of cfDNA into the circulation from a particular tissue (e.g., the origin of the cancer).

[0199] The nucleic acid is "produced by the tumor" or, if it originates from a tumor cell, is ctDNA or circulating tumor DNA. A tumor cell is a neoplastic cell that originates from a tumor, whether it remains within the tumor or becomes separated from the tumor (e.g., in the case of metastatic cancer cells and circulating tumor cells).

[0200] The term "methylation" or "DNA methylation" refers to the addition of a methyl group to a nucleotide base in a nucleic acid molecule. In some embodiments, methylation refers to the addition of a methyl group to a cytosine at a CpG site (i.e., a cytosine followed by a guanine in the 5' to 3' direction of a nucleic acid sequence). In some embodiments, DNA methylation refers to the addition of a methyl group to an adenine, e.g., N 6 5-methylation refers to the addition of a methyl group to 5-methyladenine. In some embodiments, DNA methylation is 5-methylation (modification of the 5 carbon of cytosine). In some embodiments, 5-methylation refers to the addition of a methyl group to the 5C position of cytosine to produce 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 The modification is 3C methylation (modification of the 3 carbon of cytosine). In some embodiments, 3C methylation involves adding a methyl group to the 3C position of cytosine to generate 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, if DNA in a promoter region is methylated, gene transcription can be suppressed. DNA methylation is crucial for normal development, and abnormal methylation can disrupt epigenetic regulation. Disruption of epigenetic regulation, for example, suppression, can cause diseases such as cancer. Promoter methylation in DNA can indicate cancer.

[0201] The term "hypermethylated" refers to an increased level or degree of methylation of a nucleic acid molecule compared to other nucleic acid molecules within a population (e.g., a sample) of nucleic acid molecules. In some embodiments, hypermethylated DNA can 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.

[0202] The term "hypomethylated" refers to a decrease in the level or degree of methylation of a nucleic acid molecule compared to other nucleic acid molecules within a population (e.g., a sample) of nucleic acid molecules. In some embodiments, hypomethylated DNA includes unmethylated DNA molecules. In some embodiments, hypomethylated DNA can include DNA molecules that contain 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.

[0203] The terms "or combinations thereof" and "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 also includes BA, CA, CB, ACB, CBA, BCA, BAC, or CAB if order is important in the particular context. Continuing with this example, combinations containing repeats of one or more items or terms are expressly included, e.g., BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those of skill in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise apparent from the context.

[0204] "Or" is used in the inclusive sense, ie, equivalent to "and / or" unless the context requires otherwise.

[0205] B. Exemplary Methods 1. Partitioning of the sample into multiple subsamples; sample characteristics; analysis of epigenetic signatures In certain embodiments described herein, different types of nucleic acid populations (e.g., hypermethylated and hypomethylated DNA in a sample, e.g., a captured cfDNA set described herein) can be physically partitioned based on one or more nucleic acid characteristics prior to further analysis, e.g., differential modification or isolation of nucleic acid bases, tagging, and / or sequencing. This approach can be used to determine, for example, whether a particular sequence is hypermethylated or hypomethylated. In some embodiments, hypermethylated variable epigenetic target regions can be analyzed to determine whether they are indicative of a hypermethylated characteristic of tumor cells, and / or hypomethylated variable epigenetic target regions can be analyzed to determine whether they are indicative of a hypomethylated characteristic of tumor cells. In addition, partitioning heterogeneous nucleic acid populations can increase rare signals, e.g., by enriching for rare nucleic acid molecules that are more abundant in one fraction (or fractions) of the population. For example, genetic variations 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 fractions of a sample, multidimensional analysis of a single locus or nucleic acid species of the genome can be performed, thus achieving greater sensitivity.

[0206] In some examples, a heterogeneous nucleic acid sample is divided into two or more fractions (e.g., at least 3, 4, 5, 6, or 7 fractions). In some embodiments, each fraction is differentially tagged. The tagged fractions can then be pooled together for collective sample preparation and / or sequencing. The dividing-tagging-pooling step can occur more than once, with each round of dividing occurring based on different characteristics (examples provided herein) and tagged using a differential tag that distinguishes it from other fractions and dividing means.

[0207] Examples of characteristics that can be used for partitioning include sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and / or proteins binding to DNA. The resulting fractions can contain one or more of the following nucleic acid types: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), short DNA fragments, and long DNA fragments. In some embodiments, partitioning based on cytosine modification (e.g., cytosine methylation) or methylation is generally performed, optionally combined with at least one additional partitioning step that can be based on any of the aforementioned DNA characteristics or types. In some embodiments, a heterogeneous nucleic acid population is partitioned into nucleic acids with one or more epigenetic modifications and nucleic acids without 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 vs. 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 additionally, heterogeneous nucleic acid populations can be divided into nucleosome-associated nucleic acid molecules and nucleosome-free nucleic acid molecules.Alternatively or additionally, heterogeneous nucleic acid populations can be divided into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).Alternatively or additionally, heterogeneous nucleic acid populations can be divided based on nucleic acid length (for example, molecules up to 160 bp and molecules with a length greater than 160 bp).

[0208] In some instances, each fraction (representing a different nucleic acid type) is differentially labeled and the fractions are pooled together prior to sequencing. In other instances, the different types are sequenced separately.

[0209] In some embodiments, different nucleic acid populations are divided into two or more different fractions. Each fraction represents a different nucleic acid type, and the first fraction (also called a sub-sample) contains a higher proportion of DNA with cytosine modifications than the second sub-sample. Each fraction is separately tagged. The first sub-sample is subjected to a procedure that affects a first nucleobase in the DNA so that it is different from a second nucleobase in the DNA of the first sub-sample, where the first nucleobase is a modified or unmodified nucleobase, and the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity. The tagged nucleic acids are pooled together before sequencing. Sequence reads are obtained and analyzed in silico, including distinguishing the first nucleobase from the second nucleobase in the DNA of the first sub-sample. The tags are used to sort the reads from different fractions. Analysis for detecting genetic variants can be performed at the level of each fraction and at the level of the entire nucleic acid population.For example, analysis can include in silico analysis to determine genetic variants, such as CNVs, SNVs, indels, and fusions in the nucleic acids of each fraction.In some examples, in silico analysis can include determining chromatin structure.For example, the coverage of sequence reads can be used to determine nucleosome positions in chromatin.High coverage can be correlated with high nucleosome occupancy in genomic regions, while low coverage can be correlated with low nucleosome occupancy or nucleosome-depleted regions (NDRs).

[0210] The sample may contain nucleic acids that differ in modifications that include post-replication modifications to nucleotides and binding, usually non-covalent binding, to one or more proteins.

[0211] In one embodiment, the nucleic acid population is obtained from serum, plasma, or blood samples from subjects suspected of having neoplasia, tumor, or cancer, or from subjects already diagnosed with neoplasia, tumor, or cancer. The nucleic acid population comprises nucleic acids with various methylation levels. Methylation can occur from any one or more post-replication or post-transcriptional modifications. Post-replication modifications include modifications of nucleotide cytosine, particularly modifications at the 5th position of the nucleic acid base, such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine.

[0212] Affinity agents can be antibodies with the desired specificity, their natural binding partners or variants (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68-72 (2011)), or antibodies that bind to a given target, for example, by phage display. The peptide may be an artificial peptide selected to have specificity for a target.

[0213] Examples of capture moieties contemplated herein include the methyl-binding domains (MBDs) and methyl-binding proteins (MBPs) described herein, including proteins such as MeCP2 and antibodies that preferentially bind to 5-methylcytosine.

[0214] Similarly, partitioning of different types of nucleic acids can be performed using 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.

[0215] For some affinity agents and modifications, binding to the agent may occur essentially in an all-or-none manner depending on whether the nucleic acid has the modification, but the separation may be of a degree. In such cases, nucleic acids that are over-represented in the modification will bind to the agent to a greater extent than nucleic acids that are under-represented in the modification. Alternatively, nucleic acids with modifications may bind in an all-or-none manner. However, modifications of various levels may be sequentially eluted from the binding agent.

[0216] For example, in some embodiments, the partitioning can be binary or based on the degree / level of modification. For example, all methylated fragments can be partitioned from unmethylated fragments using a methyl-binding domain protein (e.g., MethylMiner Methylated DNA Enrichment Kit (ThermoFisher Scientific)). Further partitioning can then involve eluting fragments with different levels of methylation by adjusting the salt concentration in a solution containing the methyl-binding domain and bound fragments. As the salt concentration increases, fragments with greater methylation levels are eluted.

[0217] In some cases, the final fractions are representative of nucleic acids with different degrees of modification (over- or under-representation of the modification). Over- and under-representation can be defined by the number of modifications a nucleic acid has compared to the median number of modifications per strand in the population. For example, if the median number of 5-methylcytosine residues in nucleic acids in a sample is 2, nucleic acids containing more than two 5-methylcytosine residues will be over-represented in this modification, while nucleic acids with one or zero 5-methylcytosine residues will be under-represented. The effect of affinity separation is to enrich nucleic acids that are over-represented in the modification in the binding phase and under-represented in the modification in the non-binding phase (i.e., in solution). The nucleic acids in the binding phase can be eluted prior to further processing.

[0218] When using the MethylMiner Methylated DNA Enrichment Kit (ThermoFisher Scientific), various levels of methylation can be separated using sequential elution. For example, a low-methylated fraction (e.g., no methylation) can be separated from a methylated fraction by contacting a nucleic acid population with MBD from the kit bound to magnetic beads. The beads are used to separate methylated nucleic acids from unmethylated nucleic acids. One or more sequential elution steps are then performed to elute nucleic acids with different levels of methylation. For example, a first set of methylated nucleic acids can be eluted at a salt concentration of 160 mM or higher, such as at least 150 mM, at least 200 mM, at least 300 mM, at least 400 mM, at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM, at least 1000 mM, or at least 2000 mM. After eluting such methylated nucleic acids, magnetic separation is again used to separate highly methylated nucleic acids from nucleic acids with low levels of methylation. The elution and magnetic separation steps can be repeated to generate various fractions, such as a hypomethylated fraction (representative of no methylation), a methylated fraction (representative of low levels of methylation), and a hypermethylated fraction (representative of high levels of methylation).

[0219] In some methods, nucleic acids bound to the agent used for affinity separation are subjected to a washing step. The washing step washes away nucleic acids that are weakly bound to the affinity agent. Such nucleic acids can be enriched for nucleic acids with a degree of modification closer to the mean or median (i.e., intermediate between nucleic acids that remain bound to the solid phase and nucleic acids that do not bind to the solid phase when the agent is first contacted with the sample).

[0220] Affinity separation results in at least two, sometimes three or more fractions of nucleic acids with different degrees of modification.Although the fractions are still separated, the nucleic acids of at least one fraction, usually two or three (or more) fractions, are usually linked to nucleic acid tags provided as components of adapters, and the nucleic acids in different fractions are tagged with different tags that distinguish the members of one fraction from the members of another fraction.The tags linked to the nucleic acid molecules of the same fraction can be the same or different from each other.However, when different from each other, the tags can have part of their code in common, so that the molecules to which they are linked are identified as molecules of a specific fraction.

[0221] For further details regarding partitioning nucleic acid samples based on characteristics such as methylation, see WO2018 / 119452, which is incorporated herein by reference.

[0222] In some embodiments, the nucleic acid molecules can be fractionated into different fractions based on nucleic acid molecules that are bound to a particular protein or fragment thereof and nucleic acid molecules that are not bound to a particular protein or fragment thereof.

[0223] Nucleic acid molecules can be fractionated based on DNA-protein binding. Protein-DNA complexes can be fractionated based on specific protein properties. Examples of such properties include various epitopes, modifications (e.g., histone methylation or acetylation), or enzymatic activity. Examples of proteins that bind to DNA and can serve as the basis for fractionation include, but are not limited to, protein A and protein G. Any suitable method can be used to fractionate nucleic acid molecules based on protein-bound regions. Examples of methods used to fractionate nucleic acid molecules based on protein-bound regions include, but are not limited to, SDS-PAGE, chromatin-immunoprecipitation (ChIP), heparin chromatography, and asymmetric field-flow fractionation (AF4).

[0224] In some embodiments, nucleic acid partitioning is performed by contacting the nucleic acid with the methylation binding domain ("MBD") of methylation binding protein ("MBP"). The MBD binds to 5-methylcytosine (5mC). The MBD is linked 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.

[0225] Examples of MBPs contemplated herein include, but are not limited to: (a) MeCP2 is a protein that preferentially binds 5-methyl-cytosine compared to unmodified cytosine. (b) RPL26, PRP8, and the DNA mismatch repair protein MHS6 preferentially bind 5-hydroxymethyl-cytosine compared to unmodified cytosine. (c) FOXK1, FOXK2, FOXP1, FOXP4, and FOXI3 preferentially bind 5-formyl-cytosine compared to unmodified cytosine (Iurlaro et al., Genome Biol. 14: R119 (2013)). (d) an antibody specific for one or more methylated nucleotide bases; Examples include:

[0226] Generally, elution is a function of the number of methylation sites per molecule, with molecules with more methylation eluting at increasing salt concentrations. A series of elution buffers with increasing NaCl concentrations can be used to elute DNA into distinct populations based on the degree of methylation. Salt concentrations can range from about 100 nM to about 2500 mM NaCl. In one embodiment, the process results in three fractions. Molecules are contacted with a solution at a first salt concentration containing molecules containing a methyl-binding domain, allowing the molecules to bind to a capture moiety such as streptavidin. At the first salt concentration, some population of molecules bind to the MBD, while others remain unbound. The unbound population can be separated as a "hypomethylated" population. For example, the first fraction, representing hypomethylated DNA, is the fraction that remains unbound at a low salt concentration, e.g., 100 mM or 160 mM. The second fraction, representing intermediate methylated DNA, is eluted using an intermediate salt concentration, e.g., 100 mM to 2000 mM. This is also separated from the sample. A third fraction, representing hypermethylated DNA, is eluted using a high salt concentration, for example at least about 2000 mM.

[0227] a. Fraction tagging In some embodiments, two or more fractions, e.g., each fraction, are differentially tagged. The tag or index can be a molecule, such as a nucleic acid, that contains information that indicates the characteristics of the molecule with which the tag is associated. For example, a molecule can have a sample tag or sample index (that distinguishes molecules in one sample from molecules in a different sample), a fraction tag (that distinguishes molecules in one fraction from molecules in a different fraction), and / or a molecular tag / molecular barcode / barcode (that distinguishes different molecules from each other (in both unique and non-unique tagging scenarios)). In certain embodiments, a tag can include one or a combination of barcodes. As used herein, the term "barcode" refers to a nucleic acid molecule having a specific nucleotide sequence or the nucleotide sequence itself, depending on the context. A barcode can have, for example, between 10 and 100 nucleotides. A collection of barcodes can have a degenerate sequence or can have sequences with a certain Hamming distance if desired for a particular purpose. Thus, for example, a molecular barcode can be composed of one barcode or a combination of two barcodes, each attached to a different end of the molecule. Additionally or alternatively, different sets of molecular barcodes, molecular tags, or molecular indexes can be used for different fractions and / or samples, such that the barcodes serve as molecular tags through their individual sequences and serve to identify the fractions and / or samples to which they correspond based on the sets of which they are members.

[0228] Tags can be used to label fractions of individual polynucleotide populations in order to correlate the tag (or tags) with a particular fraction. Alternatively, tags can be used in embodiments of the invention that do not use a partitioning step. In some embodiments, a single tag can be used to label a particular fraction. In some embodiments, multiple different tags can be used to label a particular fraction. In embodiments that use multiple different tags to label a particular fraction, the set of tags used to label one fraction can be easily distinguished from the set of tags used to label other fractions. In some embodiments, tags may have additional functions, for example, they can be used to index the source of a sample, or as unique molecular identifiers (which can be used to improve the quality of sequencing data by distinguishing sequencing errors from mutations, e.g., as described in Kinde et al., Proc Nat'l Acad Sci USA 108: 9530-9535 (2011), Kou et al., PLoS ONE,11: e0146638 (2016)), or as non-unique molecular identifiers, e.g., as described in U.S. Pat. No. 9,598,731. Similarly, in some embodiments, tags may have additional functions, for example, they can be used to index the source of a sample, or as non-unique molecular identifiers (which can be used to improve the quality of sequencing data by distinguishing sequencing errors from mutations).

[0229] In one embodiment, tagging the fractions includes tagging the molecules in each fraction with a fraction tag. After the fractions are recombined (e.g., to reduce the number of required sequencing runs and avoid unnecessary costs) and the molecules are sequenced, the fraction tag identifies the source fraction. In another embodiment, different fractions are tagged with different molecular tag sets, including, for example, barcode pairs. In this way, each molecular barcode is useful for indicating the source fraction and distinguishing molecules within the fraction. For example, a first set of 35 barcodes can be used to tag molecules in a first fraction, and a second set of 35 barcodes can be used to tag molecules in a second fraction.

[0230] In some embodiments, after being divided and tagged by fraction tag, molecules can be pooled for sequencing in one run.In some embodiments, sample tag is added to molecule, for example, in the step after adding fraction tag and after pooling.Sample tag can facilitate the pooling of the material generated from multiple samples for sequencing in one run.

[0231] Alternatively, in some embodiments, fraction tags can be associated with samples and fractions.As a simple example, a first tag can indicate the first fraction of a first sample, a second tag can indicate the second fraction of a first sample, a third tag can indicate the first fraction of a second sample, and a fourth tag can indicate the second fraction of a second sample.

[0232] Tags may be attached to molecules that have already been sorted 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 sorted and tagged, but the final tagged molecules in the library will likely be double-stranded.Similarly, DNA may be sorted based on different methylation levels, but the tagged molecules derived from these molecules in the final library will likely be unmethylated.Therefore, the tags attached to molecules in the library typically represent the characteristics of the "parent molecule" from which the final tagged molecules are derived, and are not necessarily the characteristics of the tagged molecules themselves.

[0233] For example, use barcode 1, 2, 3, 4 etc. to tag and label the molecules in the first fraction; use barcode A, B, C, D etc. to tag and label the molecules in the second fraction; and use barcode a, b, c, d etc. to tag and label the molecules in the third fraction.Differentially tagged fractions can be pooled before sequencing.Differentially tagged fractions can be sequenced separately, or can be sequenced together simultaneously, for example, in the same flow cell of Illumina sequencer.

[0234] After sequencing, analysis of reads to detect genetic variants can be performed at the level of each fraction and at the level of the entire nucleic acid population. Tags are used to sort reads from different fractions. Analysis can include in silico analysis to determine genetic and epigenetic variations (one or more of methylation, chromatin structure, etc.) using sequence information, genomic coordinate length, coverage, and / or copy number. In some embodiments, high coverage can be correlated with high nucleosome occupancy in genomic regions, while low coverage can be correlated with low nucleosome occupancy or nucleosome-depleted regions (NDRs).

[0235] b. Alternative methods for analysis of modified nucleic acids In some embodiments, adapters may be added to nucleic acids after partitioning, while in other embodiments, adapters may be added to nucleic acids before partitioning. In some such methods, a population of nucleic acids with different degrees of modification (e.g., 0, 1, 2, 3, 4, 5, or more methyl groups per nucleic acid molecule) is contacted with adapters before fractionating the population according to the degree of modification. The adapters are attached to either one or both ends of the nucleic acid molecules in the population. Preferably, the adapters contain a sufficient number of different tags such that the number of tag combinations results in a low probability, e.g., 95, 99, or 99.9%, that two nucleic acids with the same start and stop points will receive the same combination of tags. Although adapters may contain the same or different primer binding sites, regardless of whether they have the same or different tags, preferably, the adapters contain the same primer binding sites. After adapter binding, the nucleic acids are contacted with an agent (e.g., an agent previously described) that preferentially binds to nucleic acids with modifications. The nucleic acids are partitioned into at least two subsamples that differ in the degree to which the nucleic acids have modifications due to binding to the agent. For example, if the agent has affinity for nucleic acids with a modification, nucleic acids with an overrepresented modification (compared to the median occurrence in the population) will preferentially bind to the agent, while nucleic acids with an underrepresented modification will not bind to the agent or will be more easily eluted from the agent. After partitioning, the first subsample is subjected to a procedure that affects a first nucleobase in the DNA so that it differs from a second nucleobase in the DNA of the first subsample, where the first nucleobase is a modified or unmodified nucleobase, and the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and the first and second nucleobases have the same base pairing specificity. The nucleic acid is then amplified from a primer that binds to the primer binding site in the adapter. After amplification, different fractions can be subjected to further processing steps, which typically include further amplification (e.g., clonal amplification) and sequence analysis in parallel but separately. The sequence data from different fractions can then be compared.

[0236] In another embodiment, the partitioning scheme can be implemented using the following exemplary procedure: A nucleic acid is ligated to both ends of a Y-shaped adapter containing a primer binding site and a tag. The molecule is amplified. The amplified molecule is then fractionated by contacting with an antibody that preferentially binds to 5-methylcytosine to produce two fractions. One fraction contains the original molecule lacking methylation and the amplified copy that has lost methylation. The other fraction contains the original DNA molecule with methylation. The fraction containing the original DNA molecule with methylation is subjected to a procedure that affects a first nucleobase in the DNA so that it differs from a second nucleobase in the DNA of a first subsample, where the first nucleobase is a modified or unmodified nucleobase, and the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and the first and second nucleobases have the same base-pairing specificity. The two fractions are then processed and sequenced separately, with further amplification of the methylated fraction. The sequence data of the two fractions can then be compared. In this example, the tags are not used to distinguish between methylated and unmethylated DNA, but rather to distinguish between different molecules within those fractions, so that it can be determined whether reads with the same start and stop points are based on the same or different molecules.

[0237] The present disclosure provides further methods for analyzing a nucleic acid population, at least some of whose nucleic acids contain one or more modified cytosine residues, such as 5-methylcytosine and any of the other modifications already described. In these methods, after partitioning, a subsample of nucleic acids is contacted with an adapter containing one or more cytosine residues modified at the 5C position, such as 5-methylcytosine. Preferably, all cytosine residues in such adapters are also modified, or all such cytosines in the primer binding region of the adapter are modified. The adapters are attached to both ends of the nucleic acid molecules in the population. Preferably, the adapters contain a sufficient number of different tags such that the number of tag combinations results in a low probability, e.g., 95, 99, or 99.9%, that two nucleic acids with the same start and stop points will receive the same combination of tags. The primer binding sites in such adapters can be the same or different, but are preferably the same. After adapter binding, the nucleic acids are amplified from primers that bind to the primer binding sites of the adapters. The amplified nucleic acids are divided into first and second aliquots. The first aliquot is assayed for sequence data, with or without further processing. The sequence data of the molecules in the first aliquot is thus determined regardless of the initial methylation state of the nucleic acid molecule. The nucleic acid molecules in the second aliquot are subjected to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA, where the first nucleobase contains a modified cytosine at position 5 and the second nucleobase contains an unmodified cytosine. This procedure can be bisulfite treatment or another procedure that converts the unmodified cytosine to uracil. The nucleic acids that have been subjected to the procedure are then amplified with a primer directed to the original primer binding site of the adapter ligated to the nucleic acid. These nucleic acids retain a cytosine at the primer binding site of the adapter, but the amplification products have been converted to uracil in the bisulfite treatment and have lost the methylation of these cytosine residues, so only the nucleic acid molecules originally ligated to the adapter (different from the amplification products) can be amplified. In this way, only the original molecules in the population, at least some of which are methylated, are amplified. After amplification, these nucleic acids are subjected to sequence analysis.Comparison of the sequences determined from the first and second aliquots can indicate, among other things, which cytosines in the nucleic acid population have been subjected to methylation.

[0238] Such analysis can be performed using the following exemplary procedure: After partitioning, the methylated DNA is ligated to Y-shaped adapters at both ends, each containing a primer binding site and a tag. The cytosine in the adapter is modified at position 5 (e.g., 5-methylated). The adapter modification serves to protect the primer binding site in a subsequent conversion step (e.g., bisulfite treatment, TAP conversion, or any other conversion that does not affect modified cytosines but affects unmodified cytosines). After adapter ligation, the DNA molecule is amplified. The amplification product is divided into two aliquots for sequencing with and without conversion. The aliquot that is not subjected to conversion can be subjected to sequence analysis with or without further processing. The other aliquot is subjected to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA, such that the first nucleobase contains a cytosine modified at position 5 and the second nucleobase contains an unmodified cytosine. This procedure can be bisulfite treatment or another procedure that converts unmodified cytosines to uracil. Only primer binding sites protected by cytosine modifications can support amplification when contacted with a primer specific for the original primer binding site. Thus, only the original molecules, not copies of the first amplification, are subjected to further amplification. The amplified molecules are then subjected to sequence analysis. The sequences from the two aliquots can then be compared. As with the separation scheme described above, the nucleic acid tags in the adapters are not used to distinguish between methylated and unmethylated DNA, but rather to distinguish between nucleic acid molecules within the same fraction.

[0239] 2. Subjecting the first sub-sample to a procedure that affects a first nucleobase in the DNA differently than a second nucleobase in the DNA of the first sub-sample. The methods disclosed herein include subjecting a first sub-sample to a procedure that affects a first nucleobase in DNA differently from a second nucleobase in the DNA of the first sub-sample, wherein the first nucleobase is a modified or unmodified nucleobase and the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and wherein the first nucleobase and the second nucleobase have the same base-pairing specificity. In some embodiments, when the first nucleobase is modified or unmodified adenine, the second nucleobase is modified or unmodified adenine; when the first nucleobase is modified or unmodified cytosine, the second nucleobase is modified or unmodified cytosine; when the first nucleobase is modified or unmodified guanine, the second nucleobase is modified or unmodified guanine; when the first nucleobase is modified or unmodified thymine, the second nucleobase is modified or unmodified thymine (modified and unmodified uracil are encompassed within modified thymine for the purposes of this step).

[0240] In some embodiments, when the first nucleobase is modified or unmodified cytosine, the second nucleobase is modified or unmodified cytosine.For example, the first nucleobase can comprise unmodified cytosine (C), and the second nucleobase can comprise one or more of 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC).Alternatively, the second nucleobase can comprise C, and the first nucleobase can comprise one or more of mC and hmC.For example, as shown in the summary above and the following description, other combinations are also possible, such as when one of the first and second nucleobases comprises mC, and the other comprises hmC.

[0241] In some embodiments, the procedure that affects the first nucleic acid base in DNA differently from the second nucleic acid base in the DNA of the first sub-sample comprises bisulfite conversion.Bisulfite treatment converts unmodified cytosine and certain modified cytosine nucleotides (for example, 5-formylcytosine (fC) or 5-carboxylcytosine (caC)) to uracil, while other modified cytosines (for example, 5-methylcytosine, 5-hydroxymethylcytosine) are not converted.Thus, when using bisulfite conversion, the first nucleic acid base comprises one or more of unmodified cytosine, 5-formylcytosine, 5-carboxylcytosine, or other cytosine types that are affected by bisulfite, and the second nucleic acid base can comprise one or more of mC and hmC, for example, mC and optionally hmC.Sequencing of bisulfite-treated DNA identifies the position that is read as cytosine as mC or hmC position. On the other hand, positions that are read as T are identified as T or bisulfite-sensitive forms of C, such as unmodified cytosine, 5-formylcytosine, or 5-carboxylcytosine. Thus, performing bisulfite conversion on a first subsample as described herein facilitates identifying positions containing mC or hmC using sequence reads obtained from the first subsample. For an exemplary description of bisulfite conversion, see, e.g., Moss et al., Nat Commun. 2018; 9: 5068. Hypermethylation An exemplary workflow for performing bisulfite conversion on a first sub-sample having:

[0242] In some embodiments, the procedure that affects the first nucleobase in DNA differently from the second nucleobase in the DNA of the first subsample includes oxidative bisulfite (Ox-BS) conversion. This procedure first converts hmC to fC, which is bisulfite-sensitive, and then performs bisulfite conversion. Thus, when using oxidative bisulfite conversion, the first nucleobase includes one or more of unmodified cytosine, fC, caC, hmC, or other cytosine types that are affected by bisulfite, and the second nucleobase includes mC. Sequencing the Ox-BS-converted DNA identifies positions that are read as cytosine as mC positions. Meanwhile, positions that are read as T are identified as T, hmC, or bisulfite-sensitive C, such as unmodified cytosine, fC, or hmC. Thus, performing Ox-BS conversion on a first sub-sample as described herein facilitates identifying mC-containing positions using sequence reads obtained from the first sub-sample. For an exemplary description of oxidative bisulfite conversion, see, e.g., Booth et al., Science 2012; 336: 934-937.

[0243] In some embodiments, the procedure for affecting a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first sub-sample comprises Tet-assisted bisulfite (TAB) conversion. In TAB conversion, hmC is protected from conversion and mC is oxidized prior to bisulfite treatment, thereby converting the position originally occupied by mC to U and leaving the position originally occupied by hmC as a protected form of cytosine. See, e.g., Yu et al., As described in Cell 2012; 149: 1368-80, after using β-glucosyltransferase to protect hmC (forming 5-glucosylhydroxymethylcytosine (ghmC)), a TET protein such as mTet1 can be used to convert mC to caC, and then bisulfite treatment can be used to convert C and caC to U, while leaving ghmC unaffected. Thus, when using TAB conversion, the first nucleobase contains one or more of unmodified cytosine, fC, caC, mC, or other types of cytosine affected by bisulfite, and the second nucleobase contains hmC. Sequencing of TAB-converted DNA identifies positions that read as cytosine as hmC positions, while positions that read as T are identified as T, mC, or bisulfite-sensitive types of C, such as unmodified cytosine, fC, or caC. Thus, performing TAB conversion on a first sub-sample as described herein facilitates identifying positions containing hmC using sequence reads obtained from the first sub-sample.

[0244] In some embodiments, the step of affecting a first nucleobase in DNA differently from a second nucleobase in the DNA of the first sub-sample comprises Tet-assisted conversion with a substituted borane reducing agent, optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. In the Tet-assisted pic-borane conversion with a substituted borane reducing agent conversion, a TET protein is used to convert mC and hmC to caC without affecting unmodified C, and then caC and, if present, fC are converted to dihydrouracil (DHU) by treatment with 2-picoline borane (pic-borane) or another substituted borane reducing agent, such as borane pyridine, tert-butylamine borane, or ammonia borane, also without affecting unmodified C. For example, Liu See Liu et al., Nature Biotechnology 2019; 37:424-429 (e.g., Supplementary Figure 1 and Supplementary Note 7). DHU is read as T in sequencing. Thus, when using this type of conversion, the first nucleobase includes one or more of mC, fC, caC, or hmC, and the second nucleobase includes an unmodified cytosine. Sequencing of the converted DNA identifies positions that read as cytosine as unmodified C positions. Meanwhile, positions that read as T are identified as T, mC, fC, caC, or hmC. Thus, performing TAP conversion on a first subsample as described herein facilitates identifying positions containing unmodified C using sequence reads obtained from the first subsample. This procedure encompasses Tet-assisted pyridine borane sequencing (TAPS), as described in further detail in Liu et al. 2019, supra.

[0245] Alternatively, protection of hmC (e.g., using βGT) can be combined with Tet-assisted conversion using a substituted borane reducing agent. hmC can be protected as described above through glucosylation using βGT to form ghmC. Treatment with a TET protein, such as mTet1, then converts mC to caC, but not C or ghmC. caC is then converted to DHU by treatment with pic-borane or another substituted borane reducing agent, such as borane pyridine, tert-butylamine borane, or ammonia borane, without affecting unmodified C or ghmC. Thus, when using Tet-assisted conversion using a substituted borane reducing agent, the first nucleobase comprises mC, and the second nucleobase comprises unmodified cytosine or hmC, e.g., unmodified cytosine, and optionally one or more of hmC, fC, and / or caC. Sequencing of the converted DNA identifies positions that read as cytosine as hmC or unmodified C positions. On the other hand, positions read as T are identified as T, fC, caC, or mC. Thus, performing TAPSβ conversion on a first sub-sample as described herein facilitates distinguishing positions containing either unmodified C or hmC from positions containing mC using sequence reads from the first sub-sample. For an exemplary description of this type of conversion, see, e.g., Liu et al., Nature Biotechnology 2019; 37:424-429.

[0246] In some embodiments, the procedure for affecting a first nucleobase in DNA differently from a second nucleobase in the DNA of the first sub-sample comprises chemical-assisted conversion with a substituted borane reducing agent, optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. In the chemical-assisted conversion with a substituted borane reducing agent, an oxidizing agent, such as potassium perruthenate (KRuO4) (also suitable for use in the ox-BS conversion), is used to specifically oxidize hmC to fC. Treatment with pic-borane or another substituted borane reducing agent, such as borane pyridine, tert-butylamine borane, or ammonia borane, converts fC and caC to DHU, but does not affect mC or unmodified C. Thus, when using this type of conversion, the first nucleobase comprises one or more of hmC, fC, and caC, and the second nucleobase comprises one or more of unmodified cytosine or mC, for example, unmodified cytosine and, optionally, mC. Sequencing the converted DNA identifies positions that read as cytosine as mC or unmodified C positions, while positions that read as T are identified as T, fC, caC, or hmC. Thus, performing this type of conversion on a first sub-sample as described herein facilitates distinguishing positions containing unmodified C or mC from positions containing hmC using sequence reads from the first sub-sample. For an exemplary description of this type of conversion, see, for example, Liu et al., Nature Biotechnology 2019; 37:424-429.

[0247] In some embodiments, the procedure for affecting a first nucleobase in DNA differently from a second nucleobase in the DNA of a first subsample comprises APOBEC-linked epigenetic (ACE) conversion. ACE conversion uses an AID / APOBEC family DNA deaminase enzyme, such as APOBEC3A (A3A), to deaminate unmodified cytosine and mC without deaminating hmC, fC, or caC. Thus, when using ACE conversion, the first nucleobase comprises unmodified C and / or mC (e.g., unmodified C and optionally mC), and the second nucleobase comprises hmC. Sequencing of the ACE-converted DNA identifies positions that read as cytosine as hmC, fC, or caC positions. Meanwhile, positions that read as T are identified as T, unmodified C, or mC. Thus, performing ACE conversion on a first sub-sample as described herein facilitates using sequence reads from the first sub-sample to distinguish positions containing hmC from positions containing mC or unmodified C. For an exemplary description of ACE conversion, see, e.g., Schutsky et al., Nature Biotechnology 2018; 36: 1083-1090.

[0248] In some embodiments, the procedure affecting a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first sub-sample comprises enzymatic conversion of the first nucleobase, e.g., enzymatic conversion in 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 / 2019.12.20.884692, available at www.biorxiv.org / content / 10.1101 / 2019.12.20.884692v1. For example, TET2 and T4-βGT can be used to convert 5mC and 5hmC into substrates that cannot be deaminated by a deaminase (e.g., APOBEC3A), which can then be used to deaminate unmodified cytosines, converting them to uracil.

[0249] In some embodiments, the procedure for affecting the first nucleobase in DNA so that it differs from the second nucleobase in the DNA of the first subsample comprises separating DNA that originally contains the first nucleobase from DNA that does not originally contain the first nucleobase. In some such embodiments, the first nucleobase is hmC. DNA that originally contains the first nucleobase can be separated from other DNA using a labeling procedure that includes a biotinylation site that originally contains the first nucleobase. In some embodiments, the first nucleobase is first derivatized with an azide-containing moiety, for example, a glucosyl-azide-containing moiety. The azide-containing moiety can then serve as a reagent for binding biotin, for example, through Huisgen cycloaddition chemistry. The DNA that originally contained the now biotinylated first nucleobase can then be separated from DNA that did not originally contain the first nucleobase using a biotin-binding agent, such as avidin, neutravidin (a deglycosylated avidin with an isoelectric point of about 6.3), or streptavidin. An example of a procedure for separating DNA that originally contained the first nucleobase from DNA that did not originally contain the first nucleobase is hmC-seal, which involves labeling hmC to form β-6-azido-glucosyl-5-hydroxymethylcytosine, then attaching a biotin moiety via Huisgen cycloaddition, and then separating the biotinylated DNA from other DNA using a biotin-binding agent. For an exemplary description of hmC-seal, see, e.g., Han et al. See, e.g., J. et al., Mol. Cell 2016; 63: 711-719. This approach It is useful for identifying fragments containing one or more hmC nucleobases.

[0250] In some embodiments, after such separation, method further comprises differentially tagging the DNA that originally comprises the first nucleobase, the DNA that does not originally comprise the first nucleobase, and the DNA of second sub-sample.Method can further comprise pooling the DNA that originally comprises the first nucleobase, the DNA that does not originally comprise the first nucleobase, and the DNA of second sub-sample after differentially tagging.Then, the DNA that originally comprises the first nucleobase, the DNA that does not originally comprise the first nucleobase, and the DNA of second sub-sample can be sequenced in the same sequencing cell using differential tags, while retaining the ability to determine whether a given read originates from the molecule of DNA that originally comprises the first nucleobase, originates from the DNA that does not originally comprise the first nucleobase, or originates from the DNA of second sub-sample.

[0251] In some embodiments, the first nucleobase is a modified or unmodified adenine and the second nucleobase is a modified or unmodified adenine. In some embodiments, the modified adenine is N 6 In some embodiments, the modified adenine is N-methyladenine (mA). 6 -Methyladenine (mA), N 6 -hydroxymethyladenine (hmA), or N 6 -formyl adenine (fA).

[0252] Techniques including methylated DNA immunoprecipitation (MeDIP) can be used to separate DNA containing modified bases, such as mA, from other DNA. See, e.g., Kumar et al., Frontiers Genet. 2018; 9: 640; Greer et al., Cell 2015; 161: 868-878. Antibodies specific for mA are described in Sun et al., Bioessays 2015; 37: 1155-62. Various modified nucleobases, such as 5-bromouracil, can be isolated. Antibodies against thymine / uracil forms, including oxygenated forms, are commercially available. Various modified bases can also be detected based on changes in their base pairing specificity. For example, hypoxanthine is a modified form of adenine that can result from deamination and is read as G in sequencing. See, e.g., U.S. Patent No. 8,486,630; Brown, Genomes, 2 nd Ed., John Wiley & Sons, Inc., New York, NY, 2002, chapter 14, "Mutation, Repair, and Recombination."

[0253] 3. Enrichment / Capture Step; Amplification; Adapters; Barcoding In some embodiments, the method disclosed herein comprises capturing one or more target regions of DNA, for example, cfDNA.Capturing can be carried out by any suitable method known in the art.

[0254] In some embodiments, the capturing step comprises contacting the DNA to be captured with a target-specific probe set. The target-specific probe set may have any of the features described herein for target-specific probe sets, including, but not limited to, the embodiments above and the probe-related section below. The capturing step may be performed on one or more sub-samples prepared during the methods disclosed herein. In some embodiments, DNA is captured from at least a first sub-sample or a second sub-sample, for example, at least a first sub-sample and a second sub-sample. If the first sub-sample undergoes a separation step (e.g., a step of separating DNA originally containing a first nucleobase (e.g., hmC) from DNA not originally containing the first nucleobase, e.g., hmC-seal), the capturing step may be performed on any, any two, or all of the DNA originally containing the first nucleobase (e.g., hmC), the DNA not originally containing the first nucleobase, and the DNA of the second sub-sample. In some embodiments, the sub-samples are differentially tagged (eg, as described herein) and then pooled before undergoing capture.

[0255] The capturing step can be carried out using conditions suitable for specific nucleic acid hybridization, which generally depend in part on probe characteristics such as length, base composition, etc. Those skilled in the art will be familiar with appropriate conditions given their general knowledge in the art of nucleic acid hybridization. In some embodiments, a complex between the target-specific probe and DNA is formed.

[0256] In some embodiments, the methods described herein include capturing cfDNA obtained from a test subject for a set of multiple target regions. The target regions include epigenetic target regions, which may exhibit differences in methylation levels and / or fragmentation patterns depending on whether they originate from tumors or healthy cells. The target regions may also include sequence-variable target regions, which may exhibit sequence differences depending on whether they originate from tumors or healthy cells. The capturing step produces a set of captured cfDNA molecules, and cfDNA molecules corresponding to the set of sequence-variable target regions are captured with a higher capture yield in the set of captured cfDNA molecules than cfDNA molecules corresponding to the set of epigenetic target regions. For further discussion of the capturing step, capture yield, and related aspects, see WO2020 / 160414, which is incorporated herein by reference for all purposes.

[0257] In some embodiments, the methods described herein include contacting cfDNA obtained from a test subject with a target-specific probe set, wherein the target-specific probe set is configured to capture cfDNA corresponding to a set of sequence-variable target regions with a higher capture yield than cfDNA corresponding to a set of epigenetic target regions.

[0258] Because analyzing sequence variable target regions with sufficient reliability or accuracy may require a higher sequencing depth than that required for analyzing epigenetic target regions, it may be beneficial to capture the cfDNA corresponding to the sequence variable target region set with a higher capture yield than the cfDNA corresponding to the epigenetic target region set.The amount of data required to determine fragmentation patterns (for example, to test for perturbations in transcription start sites or CTCF binding sites) or fragment abundance (for example, in hypermethylated and hypomethylated fractions) is generally less than the amount of data required to determine the presence or absence of sequence mutations related to cancer.Capturing target region sets with different yields can facilitate sequencing target regions to different sequencing depths in the same sequencing run (for example, using pooled mixtures and / or in the same sequencing cell).

[0259] In various embodiments, the method further comprises sequencing the captured cfDNA to different degrees of sequencing depth, e.g., with respect to the set of epigenetic and sequence-variable target regions, consistent with the discussion herein.

[0260] In some embodiments, the complex between the target-specific probe and DNA is separated from the DNA that is not bound to the target-specific probe.For example, if the target-specific probe is covalently or non-covalently bound to a solid support, washing or suction steps can be used to separate the unbound material.Alternatively, if the complex has different chromatographic properties from the unbound material (for example, if the probe contains a ligand that binds to a chromatography resin), chromatography can be used.

[0261] As discussed in detail elsewhere herein, a target-specific probe set may include multiple sets, such as probes for a set of sequence-variable target regions and probes for a set of epigenetic target regions. In some such embodiments, the capturing step is performed simultaneously in the same container for the probes for the set of sequence-variable target regions and the probes for the set of epigenetic target regions, e.g., the probes for the set of sequence-variable target regions and the probes for the set of epigenetic target regions are in the same composition. This approach provides a relatively streamlined workflow. In some embodiments, the concentration of the probes for the set of sequence-variable target regions is higher than the concentration of the probes for the set of epigenetic target regions.

[0262] Alternatively, the capturing step is carried out using a sequence variable target region probe set in a first container and an epigenetic target region probe set in a second container, or the contacting step is carried out using a sequence variable target region probe set at a first time and in the first container and an epigenetic target region probe set at a second time before or after the first time.This approach allows the first and second compositions to be prepared separately, each containing the captured DNA corresponding to the sequence variable target region set and the captured DNA corresponding to the epigenetic target region set.If desired, the compositions can be processed separately (for example, to fractionate based on methylation as described elsewhere herein), and recombined at the appropriate ratio to provide material for further processing and analysis, such as sequencing.

[0263] In some embodiments, the DNA is amplified. In some embodiments, the amplification is performed before the capturing step. In some embodiments, the amplification is performed after the capturing step.

[0264] In some embodiments, the adapters are included in the DNA. This can be done simultaneously with the amplification procedure, for example, by providing the adapters at the 5' portion of the primers as described above. Alternatively, the adapters can be added by other approaches, such as ligation.

[0265] In some embodiments, the DNA contains a tag that may be or include a barcode. The tag can facilitate identification of the origin of the nucleic acid. For example, the barcode can be used to identify the source (e.g., subject) from which the DNA originates 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 tag / barcode are provided by the same primer or primer set. For example, the barcode can be located 3' of the adapter and 5' of the target-hybridizing portion of the primer. Alternatively, the barcode can be added by other approaches, such as ligation, optionally with the adapter in the same ligation substrate.

[0266] Additional details regarding amplification, tags, and barcodes are discussed below in the "General Features of the Method" section, which can be combined to the extent practicable with any of the preceding embodiments and those described in the Introduction and Summary sections.

[0267] 4. Captured Set In some embodiments, a set of captured DNA (for example, cfDNA) is provided.For example, in the method of the present disclosure, a set of captured DNA can be provided by carrying out a capturing step after the distributing step described herein.The captured set can comprise the DNA corresponding to a set of sequence variable target regions, a set of epigenetic target regions, or a combination thereof.In some embodiments, when normalized for the difference in the size (footprint size) of target regions, the amount of captured sequence variable target region DNA is greater than the amount of captured epigenetic target region DNA.

[0268] Alternatively, first and second captured sets may be provided, each containing DNA corresponding to the set of sequence variable target regions and DNA corresponding to the set of epigenetic target regions, and the first and second captured sets may be combined to provide a combined captured set.

[0269] In some embodiments, where the captured set includes DNA corresponding to the set of sequence variable target regions and the set of epigenetic target regions, including combinations of the captured sets discussed above, the DNA corresponding to the set of sequence variable target regions is present at a higher concentration than the DNA corresponding to the set of epigenetic target regions, e.g., 1.1 to 1.2 times higher, 1.2 to 1.4 times higher, 1.4 to 1.6 times higher, 1.6 to 1.8 times higher, 1.8 to 2.0 times higher, 2.0 to 2.2 times higher, 2.2 to 2.4 times higher, 2.4 to 2.6 times higher, 2.6 to 2.8 times higher, 2.8 to 3.0 times higher, 3.0 to 3.5 times higher, 3.5 to 4.0 times higher, 4.0 to 4.5 times higher, 4.5 to 5.0 times higher, 5.0 to 5.5 times higher, 5.5 to 6.0 times higher, or 6.0 to 6.5 times higher. Degree, 6.5-7.0 times higher, 7.0-7.5 times higher concentration, 7.5-8.0 times higher concentration, 8.0-8.5 times higher concentration, 8.5-9.0 times higher concentration, 9.0-9.5 times higher concentration, 9.5-10.0 times higher concentration, 10-11 times higher concentration, 11-12 times higher concentration, 12-13 times higher concentration, 13-14 times higher concentration, 14-15 times higher concentration, 15-16 times higher concentration, 16-17 times higher concentration, 17-18 times higher concentration The concentration difference can be 18-19 times higher, 19-20 times higher, 20-30 times higher, 30-40 times higher, 40-50 times higher, 50-60 times higher, 60-70 times higher, 70-80 times higher, 80-90 times higher, 90-100 times higher, 10-20 times higher, 10-40 times higher, 10-50 times higher, 10-70 times higher, or 10-100 times higher. The degree of concentration difference accounts for normalization with respect to the footprint size of the target region, as discussed in the definition section.

[0270] a. Epigenetic target region set Epigenetic target region set can comprise one or more types of target region that may distinguish the DNA from neoplastic (for example, tumor or cancer) cells from the DNA from healthy cells, for example, non-neoplastic circulating cells.The exemplary types of such regions are discussed in detail herein.Epigenetic target region set can also comprise one or more control regions, for example, as described herein.

[0271] In some embodiments, the set of epigenetic target regions has a footprint of at least 100 kb, e.g., at least 200 kb, at least 300 kb, or at least 400 kb. In some embodiments, the set of epigenetic target regions has a footprint in the range of 100 to 1,000 kb, e.g., 100 to 200 kb, 200 to 300 kb, 300 to 400 kb, 400 to 500 kb, 500 to 600 kb, 600 to 700 kb, 700 to 800 kb, 800 to 900 kb, and 900 to 1,000 kb.

[0272] i. Hypermethylated variable target regions In some embodiments, the set of epigenetic target regions includes one or more hypermethylated variable target regions. Generally, a hypermethylated variable target region refers to a region, e.g., in a cfDNA sample, where an increased observed methylation level indicates an increased likelihood that the sample (e.g., a cfDNA sample) contains DNA produced by neoplastic cells, e.g., tumor or cancer cells. For example, hypermethylation of promoters of tumor suppressor genes has been repeatedly observed. See, e.g., Kang et al., Genome Biol. 18:53 (2017) and references cited therein. In one example, a hypermethylated variable target region does not necessarily differ in methylation in cancerous tissue compared to DNA from healthy tissue of the same type, but may include a region that differs in methylation (e.g., has more methylation) compared to cfDNA typical of healthy subjects. For example, if the presence of cancer results in increased cell death, such as apoptosis, of cells of the tissue type corresponding to the cancer, such cancer can be detected, at least in part, using such hypermethylated variable target regions. In some embodiments, the hypermethylated variable target region comprises one or more genomic regions in which cfDNA molecules in those regions do not differ in methylation status in cancer subjects compared to cfDNA from healthy subjects, but the presence / increased amount of hypermethylated cfDNA in those regions is indicative of a particular tissue type (e.g., cancer of origin) and presents as cfDNA with increased apoptosis (e.g., tumor shedding) entering circulation.

[0273] A comprehensive review of methylation variable target regions in colorectal cancer is provided in Lam et al., Biochim Biophys Acta. 1866:106-20 (2016). These are available in VIM, SE These include PT9, ITGA4, OSM4, GATA4, and NDRG4. An exemplary set of hypermethylated variable target regions based on colorectal cancer (CRC) studies is provided in Table 1. Many of these genes likely have relevance to cancers other than colorectal cancer; for example, TP53 is a critical tumor suppressor, and it is widely recognized that hypermethylation-based inactivation of this gene may be a common mechanism of tumorigenesis. [Table 1-1] [Table 1-2]

[0274] In some embodiments, the hypermethylated variable target region includes multiple 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. For example, for each locus included as a target region, there may be one or more probes having hybridization sites that bind between the transcription start site and the stop codon of the gene (or the final stop codon in the case of an alternatively spliced ​​gene) or in the promoter region of the gene. In some embodiments, the one or more probes bind within 300 bp, e.g., within 200 or 100 bp, of the transcription start site of a gene in Table 1.

[0275] Methylation-variable target regions in various types of lung cancer are described, for example, in Ooki et al., Clin. Cancer Res. 23:7141-52 (2017); Belinksy, Annu. Rev. Physiol. 77:453-74 (2015); Hulbert et al., Clin. Cancer Res. 23:1998-2005 (2017); Shi et al., BMC Genomics 18:901 (2017); Schneider et al., BMC Cancer. 11:102 (2011); Lissa et al., Transl Lung Cancer Res 5(5):492-504 (2016); Skvortsova et al., Br. J. Cancer. 94(10):1492-1495 (2006); Kim et al., Cancer Res. 61:3419-3424 (2001); Furonaka et al., Pathology International 55:303-309 (2005); Gomes et al., Rev. Port. Pneumol. 20:20-30 (2014); Kim et al., Oncogene. 20:1765-70 (2001); Hopkins-Donaldson et al., Cell Death Differ. 10:356-64 (2003); Kikuchi et al., Clin. Cancer Res. 11:2954-61 (2005); Heller et al., Oncogene 25:959-968 (2006); Licchesi et al., Carcinogenesis. 29:895-904 (2008); Guo et al., Clin. Cancer Res. 10:7917-24 (2004); Palmisano et al., Cancer Res. 63:4620-4625 (2003); and Toyooka et al., Cancer Res. 61:4556-4560, (2001) have been discussed in detail. They have been discussed in detail.

[0276] An exemplary set of hypermethylated variable target regions based on lung cancer studies is provided in Table 2. Many of these genes may also have relevance to cancers other than lung cancer; for example, Casp8 (caspase 8) is a key enzyme in programmed cell death, and hypermethylation-based inactivation of this gene may be a common tumorigenesis mechanism not limited to lung cancer. In addition, several genes appear in both Tables 1 and 2, indicating generality. [Table 2]

[0277] Any of the foregoing embodiments relating to target regions identified in Table 2 may be combined with any of the above embodiments relating to target regions identified in Table 1. In some embodiments, the hypermethylated variable target regions include 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.

[0278] Additional hypermethylated target regions may be obtained, for example, from the Cancer Genome Atlas. Kang et al., Genome Biology 18:53 (2017) describe the construction of a probabilistic method called CancerLocator using hypermethylated target regions from breast, colon, kidney, liver, and lung. In some embodiments, the hypermethylated target regions may be specific to one or more types of cancer. Thus, in some embodiments, the hypermethylated target regions include one, two, three, four, or five subsets of hypermethylated target regions that collectively exhibit hypermethylation in one, two, three, four, or five of breast cancer, colon cancer, kidney cancer, liver cancer, and lung cancer.

[0279] ii. Hypomethylated variable target regions Global hypomethylation is a common phenomenon observed in various cancers. For example, Hon et al., Genome Res. 22:246-258 (2012) (breast cancer); Ehrlich, Epigenomics 1:239-259 (2009) (colon cancer, ovarian cancer, prostate cancer, leukemia, hepatocellular carcinoma, and See the review article describing findings regarding hypomethylation in cervical cancer and cervical cancer. For example, regions such as repetitive elements, e.g., LINE1 elements, Alu elements, centromeric tandem repeats, paracentromeric tandem repeats, and satellite DNA, as well as intergenic regions that are normally methylated in healthy cells, may exhibit reduced methylation in tumor cells. Thus, in some embodiments, the set of epigenetic target regions includes hypomethylated variable target regions, and a decrease in the observed methylation level indicates an increased likelihood that the sample (e.g., a cfDNA sample) contains DNA produced by neoplastic cells, e.g., tumor cells or cancer cells. In one example, hypomethylated variable target regions do not necessarily have a different methylation status in cancerous tissue compared to DNA from healthy tissue of the same type, but may include regions that are differentially methylated (e.g., hypomethylated) compared to cfDNA typical of healthy subjects. For example, if the presence of cancer results in increased cell death, such as apoptosis, of cells of the tissue type corresponding to the cancer, such cancer can be detected, at least in part, using such hypomethylated variable target regions. In some embodiments, the hypomethylated variable target region comprises one or more genomic regions in which cfDNA molecules in those regions do not differ in methylation status in cancer subjects compared to cfDNA from healthy subjects, but the presence / increased amount of hypomethylated cfDNA in those regions is indicative of a particular tissue type (e.g., cancer of origin) and presents as cfDNA with increased apoptosis (e.g., tumor shedding) entering circulation.

[0280] In some embodiments, the hypomethylated variable target region comprises a repetitive element and / or an intergenic region, hi some embodiments, the repetitive element comprises one, two, three, four, or five of a LINE1 element, an Alu element, a centromeric tandem repeat, a paracentromeric tandem repeat, and / or satellite DNA.

[0281] Exemplary specific genomic regions that exhibit cancer-associated hypomethylation include nucleotides 8403565-8953708 and 151104701-151106035 of human chromosome 1. In some embodiments, the hypomethylated variable target region overlaps with or includes one or both of these regions.

[0282] iii.CTCF binding region CTCF is a DNA-binding protein that contributes to chromatin organization and often colocalizes with cohesin. Perturbations of CTCF binding sites have been reported in a variety of different cancers. See, for example, Katainen et al., Nature Genetics, doi:10.1038 / ng.3335; Guo et al., Nat. Commun. 9:1520 (2018), published online June 8, 2015. CTCF binding can be monitored by sequencing, e.g., through fragment length analysis. Further details regarding sequencing-based fragment length analysis can be found in Snyder et al., Cell 164:57-68 (2016); WO2018 / 009723; and US Pat. No. 6,233,999, each of which is incorporated herein by reference. Provided in issue 20170211143A1.

[0283] Thus, perturbation of CTCF binding leads to variations in the fragmentation pattern of cfDNA, and therefore, CTCF binding sites represent one type of fragmentation variable target region.

[0284] There are many known CTCF binding sites, see, for example, CTCFBSDB (CTCF Binding Site Database), available online at insulatordb.uthsc.edu / ; Cuddapah et al., Genome Res. 19:24-32 (2009); Martin See, e.g., Rhee et al., Nat. Struct. Mol. Biol. 18:708-14 (2011); Rhee et al., Cell. 147:1408-19 (2011), each of which is incorporated herein by reference. Exemplary CTCF binding sites are nucleotides 56014955-56016161 on chromosome 8 and nucleotides 95359169-95360473 on chromosome 13.

[0285] Thus, in some embodiments, the set of epigenetic target regions comprises CTCF binding regions, hi some embodiments, the CTCF binding regions comprise 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 listed above or in the CTCFBSDB or one or more of the above-cited articles by Cuddapah et al., Martin et al., or Rhee et al.

[0286] In some embodiments, at least a portion of the CTCF sites can be methylated or unmethylated, and the methylation status correlates with whether the cell is a cancer cell. In some embodiments, the set of epigenetic target regions includes regions 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 upstream and downstream of the CTCF binding site.

[0287] iv. Transcription start site Transcription start site can also show perturbation in neoplastic cells.For example, the nucleosome organization at various transcription start sites in healthy cells of hematopoietic lineage contributes substantially to cfDNA in healthy individuals, but can be different from the nucleosome organization at those transcription start sites in neoplastic cells.This leads to different cfDNA patterns, which are generally described in, for example, Snyder et al., Cell 164:57-68 (2016); WO2018 / 009 As discussed in US20170211143A1 and US20170211143A1, transcription start site can be detected by sequencing.In another example, the transcription start site is not necessarily epigenetically different in cancerous tissue compared with the DNA from the same type of healthy tissue, but it is epigenetically different (for example, in terms of nucleosome structure) compared with the cfDNA that is typical in healthy subjects.For example, if the existence of cancer leads to increased cell death, such as apoptosis, of the cells of the tissue type corresponding to cancer, this cancer can be at least partially detected using this transcription start site.

[0288] Thus, perturbations in transcription start sites also result in variations in cfDNA fragmentation patterns, and therefore represent a type of variable fragmentation target region.

[0289] Human transcription start sites are available from DBTSS (DataBase of Human Transcription Start Sites), available on the internet at dbtss.hgc.jp, and are described in Yamashita et al., Nucleic Acids Res. 34(Database issue): D86-D89 (2006), which is incorporated herein by reference.

[0290] Thus, in some embodiments, the set of epigenetic target regions includes a transcription start site. In some embodiments, the transcription start sites include 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, e.g., transcription start sites described in the DBTSS. In some embodiments, at least a portion of the transcription start sites can be methylated or unmethylated, and the methylation status correlates with whether the cell is a cancer cell. In some embodiments, the set of epigenetic target regions includes regions 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 upstream and downstream of the transcription start site.

[0291] v. Local amplification Although local amplification is somatic mutation, it can be detected by sequencing based on read frequency in a similar manner to the method of detecting certain epigenetic changes, such as methylation changes.Therefore, the region that may show local amplification in cancer can be included in epigenetic target region set, and they can include one or more 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, epigenetic target region set comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 of the above-mentioned targets.

[0292] vi. Methylation control region It may be useful to include a control region to facilitate data validation. In some embodiments, the set of epigenetic target regions includes a control region that is expected to be methylated or unmethylated in essentially all samples, regardless of whether the DNA is derived from cancer cells or normal cells. In some embodiments, the set of epigenetic target regions includes a control hypomethylated region that is expected to be hypomethylated in essentially all samples. In some embodiments, the set of epigenetic target regions includes a control hypermethylated region that is expected to be hypermethylated in essentially all samples.

[0293] b. Set of sequence-variable target regions In some embodiments, the set of sequence variable target regions comprises multiple regions known to undergo somatic mutation in cancer.

[0294] In some embodiments, the sequence variable target region set targets a plurality of different genes or genomic regions ("panel") that are selected so that a predetermined proportion 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 limit the sequencing region to a fixed number of base pairs. The panel can 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 can also be selected to achieve a desired depth of sequence reads. The panel can be selected to achieve a desired depth or coverage of sequence reads in terms of the amount of sequenced base pairs. The panel can be selected to achieve a theoretical sensitivity, specificity, and / or accuracy for detecting one or more genetic variants in a sample.

[0295] The probes for detecting the panel of regions can include probes for detecting genomic regions of interest (hotspot regions) and nucleosome recognition probes (e.g., KRAS codons 12 and 13), and can be designed to optimize capture based on the analysis of cfDNA coverage and fragment size variation, which are affected by nucleosome binding patterns and GC sequence composition.Regions as used herein can also include non-hotspot regions that are optimized based on nucleosome position and GC model.

[0296] Examples of lists of genomic locations of interest can be found in Tables 3 and 4. In some embodiments, the set of sequence variable target regions used in the methods of the disclosure includes 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 in Table 3. In some embodiments, the set of sequence variable target regions used in the methods of the disclosure includes 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 in Table 3. In some embodiments, the set of sequence variable target regions used in the methods of the disclosure includes at least 1, at least 2, at least 3, at least 4, at least 5, or 6 of the fusions in Table 3. In some embodiments, the set of sequence variable target regions used in the methods of the disclosure includes at least a portion of at least 1, at least 2, or 3 of the indels in Table 3. In some embodiments, the set of sequence variable target regions used in the methods of the disclosure includes 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 in Table 4. In some embodiments, the set of sequence variable target regions used in the methods of the disclosure includes 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 in Table 4.In some embodiments, the set of sequence variable target regions used in the methods of the disclosure includes 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 set of sequence variable target regions used in the methods of the disclosure includes at least a portion of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or 18 of the indels in Table 4. Each of these genomic locations of interest can be identified as a scaffold region or a hotspot region for a given panel. An example list of hotspot genomic locations of interest can be found in Table 5. In some embodiments, the set of sequence variable target regions used in the methods of the present disclosure includes 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 in Table 5. Each hotspot genomic region is described along with several characteristics, including the associated gene, the chromosome on which it resides, the start and stop positions in the genome representing the locus, the base pair length of the locus, the exons covered by the gene, and important features (e.g., types of mutations) that a given genomic region of interest may seek to capture. [Table 3] [Table 4] [Table 5-1] [Table 5-2]

[0297] Additionally or alternatively, suitable target region set can be obtained from literature.For example, Gale et al., PLoS One 13: e0194630 (2018), which is incorporated herein by reference, describes a panel of 35 cancer-related gene targets that can be used as part or all of 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.

[0298] In some embodiments, the set of sequence variable target regions includes target regions from at least 10, 20, 30, or 35 genes associated with cancer, such as those genes associated with cancers listed above.

[0299] 5. Target In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject having cancer. In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject suspected of having cancer. In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject having a tumor. In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject suspected of having a tumor. In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject having a neoplasm. In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject suspected of having a neoplasm. In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject in remission from a tumor, cancer, or neoplasm (e.g., after chemotherapy, surgical resection, radiation, or a combination thereof). In any of the foregoing embodiments, the cancer, tumor, or neoplasm, or suspected cancer, tumor, or neoplasm, is a cancer, tumor, or neoplasm of the lung, colon, rectum, kidney, breast, prostate, or liver. In some embodiments, the cancer, tumor, or neoplasm, or suspected cancer, tumor, or neoplasm, is a lung cancer, tumor, or neoplasm. In some embodiments, the cancer, tumor, or neoplasm, or suspected cancer, tumor, or neoplasm, is a colon or rectal cancer, tumor, or neoplasm. In some embodiments, the cancer, tumor, or neoplasm, or suspected cancer, tumor, or neoplasm, is a breast cancer, tumor, or neoplasm. In some embodiments, the cancer, tumor, or neoplasm, or suspected cancer, tumor, or neoplasm, is a prostate cancer, tumor, or neoplasm. In any of the foregoing embodiments, the subject may be a human subject.

[0300] 6. Sequencing The adaptor-flanked sample nucleic acid is generally subjected to sequencing, with or without prior amplification. 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 sequencing, and the like. These include Gene Expression (Helicos), next-generation sequencing (NGS), single molecule sequencing by synthesis (SMSS) (Helicos), massively parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Ion Torrent, Oxford Nanopore, Roche Genia, Maxam-Gilbert sequencing, primer walking, and sequencing using PacBio, SOLiD, Ion Torrent, or Nanopore platforms. Sequencing reactions can be performed in various sample processing units, which can include multiple lanes, multiple channels, multiple wells, or other means for processing multiple sets of samples substantially simultaneously. Sample processing units can also include multiple sample chambers that can process multiple runs simultaneously.

[0301] Sequencing reactions can be performed on one or more types of nucleic acids, at least one of which is known to contain a marker for cancer or other diseases.Sequencing reactions can also be performed on any nucleic acid fragments present in a 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, sequencing reactions can provide at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% sequence coverage of the genome. Sequence coverage can be performed for at least 5, 10, 20, 70, 100, 200, or 500 different genes, or for at most 5000, 2500, 1000, 500, or 100 different genes.

[0302] Simultaneous sequencing reaction can be carried out using multiplex sequencing.In some examples, cell-free nucleic acid can be sequenced by at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions.In other examples, cell-free nucleic acid can be sequenced by less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions.Sequencing reaction can be carried out sequentially or simultaneously.Subsequent data analysis can be carried out for all or part of sequencing reaction. In some examples, data analysis can be performed for at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions. In other examples, data analysis can be performed for less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions. An exemplary read depth is about 1000 to about 50,000 reads per locus (base).

[0303] a. Differential depth of sequencing In some embodiments, nucleic acids corresponding to the set of sequence variant target regions are sequenced to a higher sequencing depth than nucleic acids corresponding to the set of epigenetic target regions. For example, the sequencing depth for nucleic acids corresponding to the set of sequence variant 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 1 less than the sequencing depth for nucleic acids corresponding to the set of epigenetic target regions. 5-fold higher, or 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, 14-15, or 15-100 times higher. In some embodiments, the sequencing depth is at least 2-fold higher. In some embodiments, the sequencing depth is at least 5-fold higher. In some embodiments, the sequencing depth is at least 10-fold higher. In some embodiments, the sequencing depth is 4-10-fold higher. In some embodiments, the sequencing depth is 4-100-fold higher. Each of these embodiments refers to the extent to which nucleic acids corresponding to the set of sequence variable target regions are sequenced to a greater sequencing depth than nucleic acids corresponding to the set of epigenetic target regions.

[0304] In some embodiments, the captured cfDNA corresponding to the set of sequence variable target regions and the captured cfDNA corresponding to the set of epigenetic target regions are sequenced simultaneously, e.g., in the same sequencing cell (e.g., the flow cell of an Illumina sequencer) and / or in the same composition, which may be a pooled composition obtained by recombining separately captured sets, or a composition obtained by capturing cfDNA corresponding to the set of sequence variable target regions and the captured cfDNA corresponding to the set of epigenetic target regions in the same container.

[0305] 7.Analysis In some embodiments, the methods described herein include identifying the presence or absence of DNA produced by a tumor (or neoplastic or cancer cell).

[0306] The present method can be used to diagnose the presence or absence of a condition, particularly cancer, in a subject, characterize the condition (e.g., stage the cancer or determine the heterogeneity of the cancer), monitor the response to treatment of the condition, and provide a prognostic risk for the development 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 treatment is successful, as more cancer cells can die and shed DNA, a successful treatment option may increase the amount of copy number variations or rare mutations detected in the subject's blood. In other examples, this may not occur. In another example, perhaps a particular treatment option can be correlated with the genetic profile of the cancer over time. This correlation can be useful for selecting a therapy.

[0307] Additionally, if the cancer is observed to be in remission after treatment, the method can be used to monitor for residual disease or recurrence of the disease.

[0308] The types and number of cancers that can be detected can include blood cancer, brain cancer, lung cancer, skin cancer, nasal cancer, pharyngeal cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, intestinal cancer, rectal cancer, thyroid cancer, bladder cancer, kidney cancer, oral cancer, stomach cancer, solid tumors, heterogeneous tumors, homogeneous tumors, etc. The type and / or stage of cancer can be detected by genetic variations including mutations, rare mutations, indels, copy number variations, transversions, translocations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, changes in chromosomal structure, gene fusions, chromosomal fusions, gene truncations, gene amplification, gene duplications, chromosomal damage, DNA damage, abnormal changes in chemical modifications of nucleic acids, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5-methylcytosine.

[0309] Genetic data can also be used to characterize specific forms of cancer. Cancers are often heterogeneous in both their composition and stage classification. Genetic profile data can enable characterization of specific subtypes of cancer, which can be important in diagnosing or treating that specific subtype. This information provides subjects or clinicians with clues regarding the prognosis of specific types of cancer and allows them to adapt treatment options as the disease progresses. Some cancers may progress to become more aggressive and genetically unstable. Other cancers may remain benign, inactive, or dormant. The systems and methods of the present disclosure can be useful in determining disease progression.

[0310] Furthermore, the methods of the present disclosure can be used to characterize the heterogeneity of an abnormal condition in a subject. Such a method can include, for example, generating a genetic profile of extracellular polynucleotides derived from the subject, the genetic profile including multiple data resulting from analysis of copy number variations and rare mutations. In some embodiments, the abnormal condition is cancer. In some embodiments, the abnormal condition can be a condition that results 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 foci of disease. Again, in the example of cancer, there are multiple tumor foci, and perhaps one or more foci are the result of metastasis that has spread from the primary site.

[0311] The method can be used to generate or profile a fingerprint or dataset that is the sum of genetic information derived from different cells in a heterogeneous disease, which dataset may include analysis of copy number variations, epigenetic variations, and mutations, either alone or in combination.

[0312] This method can be used to diagnose, prognose, monitor or observe cancer or other diseases.In some embodiments, the method herein does not include the diagnosis, prognosis or monitoring of fetus, and therefore is not intended for non-invasive prenatal testing.In other embodiments, these methodologies can be used in pregnant subjects to diagnose, prognose, monitor or observe cancer or other diseases in unborn subjects, whose DNA and other polynucleotides may circulate with maternal molecules.

[0313] An exemplary method for molecular tag identification of an MBD-bead partitioned library through NGS, comprising subjecting a first sub-sample to a procedure that affects a first nucleobase in the DNA differently than a second nucleobase in the DNA of the first sub-sample, is as follows: 1. Physical partitioning of extracted DNA samples (e.g., plasma DNA extracted from human samples that have been subjected to target capture as described herein, if desired) using a methyl-binding domain protein-bead purification kit, preserving all eluate from the process for downstream processing. 2. Parallel application of differential molecular tags and NGS-enabling adapter sequences to each fraction. For example, hypermethylated, residually methylated ("wash"), and hypomethylated fractions are ligated with molecular tags to NGS-adapters. 3. Subjecting the hypermethylated fraction to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA, such as any of the procedures described herein. 4. All molecularly tagged fractions are recombined and then amplified using adaptor-specific DNA primer sequences. 5. Capture / hybridization of the recombined amplified total library targeting genomic regions of interest (e.g., cancer-specific gene variants and differentially methylated regions). 6. Re-amplification of the captured DNA library to add sample tags. Different samples are pooled and assayed multiplexed on an NGS instrument. 7. Bioinformatics analysis of NGS data, where molecular tags are used to identify unique molecules and deconvolve samples into differentially distributed molecules. This analysis can yield information about relative 5-methylcytosine content across genomic regions in parallel with standard gene sequencing / variant detection.

[0314] In some embodiments of the methods described herein, including but not limited to those set forth above, a molecular tag consists of nucleotides that are not altered by a procedure that affects a first nucleobase in DNA differently from a second nucleobase in the DNA, such as any of the procedures described herein (e.g., mC with A, T, and G if the procedure is a bisulfite conversion or any other conversion that does not affect mC; hmC with A, T, and G if the procedure is a conversion that does not affect hmC, etc.). In some embodiments described herein, including but not limited to those set forth above, a molecular tag does not include nucleotides that are not altered by a procedure that affects a first nucleobase in DNA differently from a second nucleobase in the DNA, such as any of the procedures described herein (e.g., if the procedure is a bisulfite conversion or any other conversion that affects C, the tag does not include an unmodified C; if the procedure is a conversion that affects mC, the tag does not include mC; if the procedure is a conversion that affects hmC, the tag does not include hmC, etc.).

[0315] Generally, the procedure that affects the first nucleic acid base in DNA differently from the second nucleic acid base in DNA can be carried out before the step of applying the adapter sequence that allows for differential molecular tagging and NGS to each fraction in parallel instead.For example, this can be done when the procedure that affects the first nucleic acid base in DNA differently from the second nucleic acid base in DNA is separation, such as hmC-seal, and in such cases, the separated populations can be differentially tagged compared to each other.Such an exemplary method is as follows: 1. Physical partitioning of extracted DNA samples (e.g., plasma DNA extracted from human samples that have been subjected to target capture as described herein, if desired) using a methyl-binding domain protein-bead purification kit, preserving all eluate from the process for downstream processing. 2. Subjecting the hypermethylated fraction to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA, such as any of the procedures described herein. 3. Parallel application of differential molecular tags and NGS-enabling adapter sequences to each fraction: For example, the highly methylated fraction (or, if applicable, two or more subfractions of the highly methylated fraction), the residually methylated ("wash") fraction, and the hypomethylated fraction are ligated to NGS-adapters along with molecular tags. 4. All molecularly tagged fractions are recombined and then amplified using adaptor-specific DNA primer sequences. 5. Capture / hybridization of the recombined amplified total library targeting genomic regions of interest (e.g., cancer-specific gene variants and differentially methylated regions). 6. Re-amplification of the captured DNA library to add sample tags. Different samples are pooled and assayed multiplexed on an NGS instrument. 7. Bioinformatics analysis of NGS data, where molecular tags are used to identify unique molecules and deconvolve samples into differentially distributed molecules. This analysis can yield information about relative 5-methylcytosine content across genomic regions in parallel with standard gene sequencing / variant detection.

[0316] 8. Example Workflow Exemplary workflows for distribution and library preparation are provided herein. In some embodiments, some or all features of the distribution and library preparation workflows may be used in combination.

[0317] a. Distribution In some embodiments, sample DNA (e.g., between 5 ng and 200 ng) is mixed with a methyl-binding domain (MBD) buffer and magnetic beads conjugated to MBD protein and incubated overnight. Methylated DNA (hypermethylated DNA) binds to the MBD protein on the magnetic beads during this incubation. Unmethylated (hypomethylated DNA) or less methylated DNA (intermediate methylation) is washed off the beads with buffers containing increasing salt concentrations. For example, one, two, or more fractions containing unmethylated, hypomethylated, and / or intermediate methylated DNA can be obtained by such washing. Finally, a high salt buffer is used to elute highly methylated DNA (hypermethylated DNA) from the MBD protein. In some embodiments, these washes result in three fractions of DNA with increasing methylation levels: a hypomethylated fraction, an intermediate methylated fraction, and a hypermethylated fraction.

[0318] In some embodiments, three fractions of DNA are desalted and concentrated in preparation for the enzymatic steps for library preparation.

[0319] b. Library preparation In some embodiments (e.g., after enriching DNA in a fraction), the divided DNA is made ligatable, for example, by extending the overhangs at the ends of the DNA molecules, adding adenosine residues to the 3' ends of the fragments, and phosphorylating the 5' ends of each DNA fragment. DNA ligase and adapters are added to ligate adapters to each end of each divided DNA molecule. These adapters contain fraction tags (e.g., non-random, non-unique barcodes) that are distinguishable from the fraction tags in the adapters used in other fractions. Before or after making the divided DNA ligatable and performing ligation, the hypermethylated fraction is subjected to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA, such as any of the procedures described herein. If a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA further divides the hypermethylated fraction, adapter ligation must be performed after the procedure so that subfractions of the hypermethylated fraction can be differentially tagged. The three (or more) fractions are then pooled together and amplified (eg, by PCR using primers specific for the adapters).

[0320] After PCR, the amplified DNA may be purified and concentrated before enrichment. The amplified DNA is contacted with a collection of probes (e.g., biotinylated RNA probes) described herein that target specific regions of interest. The mixture is incubated, for example, overnight, in a salt buffer. The probes are captured (e.g., using streptavidin magnetic beads) and separated from uncaptured amplified DNA, for example, by a series of salt washes, thereby enriching the sample. After enrichment, the enriched sample is amplified by PCR. In some embodiments, the PCR primers contain sample tags, thereby incorporating the sample tags into DNA molecules. In some embodiments, DNA from different samples is pooled together and then multiplexed sequenced, for example, using an Illumina NovaSeq sequencer.

[0321] C. Additional Features of Certain Disclosed Methods 1. Sample The sample may be any biological sample isolated from a subject. The sample may be a bodily sample. The sample may include bodily tissues such as known or suspected solid tumors, whole blood, platelets, serum, plasma, feces, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies, cerebrospinal fluid, synovial fluid, lymphatic fluid, ascites, interstitial or extracellular fluids, interstitial space fluids such as endodontic crevicular fluid, bone marrow, pleural effusion, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, and urine. The sample is preferably a bodily fluid, particularly blood and its fractions, and urine. The sample may be in the original form isolated from the subject, or may have undergone further processing to remove or add components such as cells or to enrich one component for another. Thus, preferred bodily fluids for analysis are plasma or serum, which contain cell-free nucleic acids. The sample can be isolated or obtained from the subject and transported to a site for sample analysis. Samples may be stored and shipped at a desired temperature, e.g., room temperature, 4°C, -20°C, and / or -80°C. Samples may be isolated or obtained from a subject at the site of sample analysis. Subjects may include humans, mammals, animals, companion animals, service animals, etc. The subject may be a human, animal, or pet. The subject may have cancer. The subject may not have cancer or detectable symptoms of cancer. The subject may be being treated with one or more cancer therapies, such as any one or more of chemotherapy, antibodies, vaccines, or biologics. The subject may be in remission. The subject may or may not have been diagnosed with cancer or a predisposition to any cancer-associated genetic mutation / disorder. In some embodiments, the sample is a polynucleotide sample obtained from a tumor tissue biopsy.

[0322] The volume of plasma can depend on the desired read depth of the region being sequenced. Exemplary volumes are 0.4-40 ml, 5-20 ml, and 10-20 ml. For example, the volume can be 0.5 ml, 1 ml, 5 ml, 10 ml, 20 ml, 30 ml, or 40 ml. The volume of sampled plasma can be 5-20 ml.

[0323] A sample can contain various amounts of nucleic acid, including genome equivalents. For example, a sample of about 30 ng of DNA contains about 10,000 (10 4 ) haploid human genome equivalents, and in the case of cfDNA, approximately 200 billion (2 × 10 11 ) individual polynucleotide molecules. Similarly, a sample of about 100 ng of DNA may contain about 30,000 haploid human genome equivalents, or about 600 billion individual molecules in the case of cfDNA.

[0324] A sample may contain nucleic acids from different sources, e.g., cells and acellular samples from the same subject, or cells and acellular samples from different subjects. A sample may contain nucleic acids having mutations. For example, a sample may contain DNA having germline mutations and / or somatic mutations. A germline mutation refers to a mutation present in a subject's germline DNA. A somatic mutation refers to a mutation originating in a subject's somatic cells, e.g., cancer cells. A sample may contain DNA having a cancer-associated mutation (e.g., a cancer-associated somatic mutation). A sample may contain 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, a sample that does not contain a genetic variant contains an epigenetic variant associated with the presence of the genetic variant.

[0325] 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, or 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 from 1 femtogram (fg) to 200 ng of cell-free nucleic acid molecules from a sample.

[0326] Cell-free nucleic acids are nucleic acids that are not contained within or otherwise associated with cells, or in other words, nucleic acids that remain in a sample after the removal of intact cells. 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-interacting 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 body fluids through 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 body 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, cell-free nucleic acids are produced from a mixture of tumor cells and non-tumor cells.

[0327] The cell-free nucleic acids have an exemplary size distribution of about 100-500 nucleotides, with molecules of 110 to about 230 nucleotides representing about 90% of the molecules, a mode at about 168 nucleotides, and a second minor peak in the range of 240-440 nucleotides.

[0328] Cell-free nucleic acids can be isolated from bodily fluids through fractionation or partitioning steps, in which cell-free nucleic acids found in solution are separated from intact cells or other insoluble components of the bodily fluid. Partitioning can include techniques such as centrifugation or filtration. Alternatively, cells in the bodily fluid can be lysed, and both cell-free and cellular nucleic acids can be processed. Generally, after the addition of buffer and washing steps, nucleic acids can be precipitated with alcohol. Additional cleanup steps, such as silica-based columns to remove contaminants or salts, can also be used. To optimize certain aspects of the procedure, such as yield, nonspecific bulk carrier nucleic acids, such as C1 DNA, DNA, or proteins for bisulfite sequencing, hybridization, and / or ligation, can be added to the overall reaction.

[0329] After such processing, the sample may contain various forms of nucleic acids, including double-stranded DNA, single-stranded DNA, and single-stranded RNA. In some embodiments, single-stranded DNA and RNA are converted to double-stranded forms, which can be included in subsequent processing and analysis steps.

[0330] Double-stranded DNA molecules in a sample and single-stranded nucleic acid molecules converted to double-stranded DNA molecules can be ligated to adapters at one or both ends. Typically, double-stranded molecules are blunt-ended by treatment with a polymerase containing a 5'-3' polymerase and a 3'-5' exonuclease (or proofreading function) in the presence of all four standard nucleotides. Klenow large fragment and T4 polymerase are examples of suitable polymerases. The blunt-ended DNA molecules can be ligated to at least partially double-stranded adapters (e.g., Y-shaped or bell-shaped adapters). Alternatively, complementary nucleotides can be added to the blunt ends of the sample nucleic acid and adapter to facilitate ligation. Both blunt-end and sticky-end ligation are contemplated herein. In blunt-end ligation, both the nucleic acid molecule and the adapter tag have blunt ends. In sticky-end ligation, the nucleic acid molecule typically has an "A" overhang and the adapter has a "T" overhang.

[0331] 2. Amplification Adapter-flanked sample nucleic acids can be amplified by PCR and other amplification methods. Amplification is typically initiated by primers that bind to primer-binding sites in the adapters adjacent to the DNA molecule to be amplified. Amplification methods may involve cycles of denaturation, annealing, and extension due to thermal cycling, or may be isothermal, as in transcription-mediated amplification. Other amplification methods include ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and sequence-based self-sustained replication.

[0332] In some embodiments, the method performs dsDNA ligation using T-tailed and C-tailed adapters, which results in at least 50, 60, 70, or 80% amplification of double-stranded nucleic acids. Preferably, the method increases the amount or number of amplified molecules by at least 10, 15, or 20% compared to a control method performed using only T-tailed adapters.

[0333] 3. Tags Tags containing barcodes can be incorporated into or otherwise attached to the adapters. Tags can be incorporated by ligation, overlap extension PCR, among other methods.

[0334] a. Molecular tagging strategies In some embodiments, nucleic acid molecules (derived from a sample of polynucleotides) may be tagged with a sample index and / or a molecular barcode (commonly referred to as a "tag"). The tag may be incorporated into or otherwise linked to an adapter by chemical synthesis, ligation (e.g., blunt-end ligation or sticky-end ligation), or overlap-extension polymerase chain reaction (PCR), among other methods. Such an adapter may ultimately be ligated to a target nucleic acid molecule. In other embodiments, one or more rounds of amplification cycles (e.g., PCR amplification) are applied to introduce a sample index into the nucleic acid molecule, typically using conventional nucleic acid amplification methods. Amplification may be performed in one or more reaction mixtures (e.g., multiple microwells in an array). The molecular barcode and / or sample index may be introduced simultaneously or in any sequential order. In some embodiments, the molecular barcode and / or sample index are introduced before and / or after performing a sequence capture step. In some embodiments, only the molecular barcode is introduced before the probe capture step, and the sample index is introduced after performing the sequence capture step. In some embodiments, both the molecular barcode and the sample index are introduced before performing a probe-based capture step. In some embodiments, the sample index is introduced after the sequence capture step is performed. In some embodiments, the molecular barcode is incorporated into the nucleic acid molecule (e.g., cfDNA) in the sample through an adapter via ligation (e.g., blunt-end ligation or cohesive-end ligation). In some embodiments, the sample index is incorporated into the nucleic acid molecule (e.g., cfDNA) in the sample through overlap-extension polymerase chain reaction (PCR). Typically, the sequence capture protocol involves introducing a single-stranded nucleic acid molecule complementary to a targeted nucleic acid sequence (e.g., a coding sequence of a genomic region), where mutations in such a region are associated with a type of cancer.

[0335] In some embodiments, the tag can be located at one end or both ends of the sample nucleic acid molecule.In some embodiments, the tag is a predetermined, or random or semi-random sequence oligonucleotide.In some embodiments, the tag can be less than about 500, 200, 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide in length.The tag can be randomly or non-randomly linked to the sample nucleic acid.

[0336] In some embodiments, each sample is uniquely tagged with a sample index or a combination of sample indexes. In some embodiments, each nucleic acid molecule of a sample or sub-sample is uniquely tagged with a molecular barcode or a combination of molecular barcodes. In other embodiments, multiple molecular barcodes may be used, such that the molecular barcodes are not necessarily unique to each other among the multiple molecular barcodes (e.g., non-unique molecular barcodes). In these embodiments, molecular barcodes are typically attached (e.g., by ligation) to individual molecules, and the combination of the molecular barcode and the sequence to which it can be attached generates a unique sequence that can be individually tracked. Detection of the non-unique molecular barcode in combination with endogenous sequence information (e.g., the start (start) and / or end (stop) genomic location / location corresponding to the sequence of the original nucleic acid molecule in the sample, the start and stop genomic locations corresponding to the sequence of the original nucleic acid molecule in the sample, the start (start) and / or end (stop) genomic location / location of the sequence read mapped to the reference sequence, the start and stop genomic locations of the sequence read mapped to the reference sequence, subsequences of the sequence read at one or both ends, the length of the sequence read, and / or the length of the original nucleic acid molecule in the sample) typically allows assignment of a unique identity to a particular molecule. In some embodiments, the start region comprises the first 1 base position, the first 2 base positions, the first 5 base positions, the first 10 base positions, the first 15 base positions, the first 20 base positions, the first 25 base positions, the first 30 base positions, or at least the first 30 base positions at the 5' end of the sequencing read that aligns with the reference sequence. In some embodiments, the termination region comprises the last 1 base position, the last 2 base positions, the last 5 base positions, the last 10 base positions, the last 15 base positions, the last 20 base positions, the last 25 base positions, the last 30 base positions, or at least the last 30 base positions at the 3' end of the sequencing read that aligns with the reference sequence. The length of an individual sequence read, or its number of base pairs, is also used to assign a unique identity to a given molecule, as needed.As described herein, fragments from a single strand of nucleic acid that have been assigned a unique identity may thereby allow subsequent identification of the fragment from the parental and / or complementary strand.

[0337] In certain embodiments, the number of different tags used to uniquely identify a number z of molecules in a class is 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., lower bound) and 100,000 * z, 10,000 * z, 1000 * z or 100 *z (e.g., upper limit). In some embodiments, molecular barcodes are introduced in an expected ratio to molecules of the identifier set (e.g., a combination of unique or non-unique molecular barcodes) in the sample. An example format uses about 2 to about 1,000,000 different molecular barcode sequences, or about 5 to about 150 different molecular barcode sequences, or about 20 to about 50 different molecular barcode sequences ligated to both ends of the target molecule. Alternatively, about 25 to about 1,000,000 different molecular barcode sequences may be used. For example, 20-50 x 20-50 molecular barcode sequences (i.e., one of 20-50 different molecular barcode sequences can be attached to each end of the target molecule) can be used. Such a number of identifiers is typically sufficient so that different molecules with the same start and stop points have a high probability (e.g., at least 94%, 99.5%, 99.99%, or 99.999%) of receiving different combinations of identifiers. In some embodiments, about 80%, about 90%, about 95%, or about 99% of the molecules have the same combination of molecular barcodes.

[0338] In some embodiments, the assignment of unique or non-unique molecular barcodes in the reactions is performed using, for example, the methods and systems described in U.S. Patent Applications Nos. 20010053519, 20030152490, and 20110160078, and U.S. Patent Nos. 6,582,908, 7,537,898, 9,598,731, and 9,902,992, each of which is incorporated by reference in its entirety. Alternatively, in some embodiments, only intrinsic sequence information (e.g., start and / or stop positions, subsequences at one or both ends of the sequence, and / or length) may be used to identify different nucleic acid molecules in a sample.

[0339] 4.Bait set; capture part As discussed above, nucleic acids in a sample can be subjected to a capture step, in which molecules having target sequences are captured for subsequent analysis. Target capture can include the use of a bait set containing oligonucleotide baits labeled with a capture moiety, such as biotin or other examples described below. The probes can have sequences, e.g., genes, selected to span a panel of regions. In some embodiments, the bait set can have a higher or lower capture yield for a set of target regions, such as the capture yield for a set of sequence-variable target regions and a set of epigenetic target regions, as discussed elsewhere herein. Such a bait set is combined with the sample under conditions that allow hybridization of the target molecules with the baits. The captured molecules are then isolated using a capture moiety, e.g., a bead-based streptavidin capture moiety, using a biotin capture moiety. Such methods are further described, for example, in U.S. Patent 9,850,523, issued December 26, 2017, which is incorporated herein by reference.

[0340] Capture moieties include, without limitation, biotin, avidin, streptavidin, nucleic acids containing specific nucleotide sequences, haptens recognized by antibodies, and magnetically attractable particles. Extraction moieties can be members of a binding pair, such as biotin / streptavidin or hapten / antibody. In some embodiments, the capture moiety bound to the analyte is captured by a binding pair bound to an isolable moiety, such as a magnetically attractable particle or a larger particle that can be sedimented by centrifugation. The capture moiety can be any type of molecule that allows affinity separation of nucleic acids bearing the capture moiety from nucleic acids lacking the capture moiety. Exemplary capture moieties are biotin, which allows affinity separation by binding to streptavidin bound or ligatable to a solid phase, or oligonucleotides, which allow affinity separation by binding to complementary oligonucleotides bound or ligatable to a solid phase.

[0341] D. Collection of target-specific probes 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 for a set of sequence-variable target regions and target-binding probes specific for a set of epigenetic target regions. In some embodiments, the capture yield of the target-binding probes specific for the set of sequence-variable target regions is higher (e.g., at least two-fold higher) than the capture yield of the target-binding probes specific for the set of epigenetic target regions. In some embodiments, the collection of target-specific probes is configured to have a capture yield specific for the set of sequence-variable target regions that is higher (e.g., at least two-fold higher) than its capture yield specific for the set of epigenetic target regions.

[0342] In some embodiments, the capture yield of target binding probes specific for the set of sequence variable target regions is at least 1.25x, 1.5x, 1.75x, 2x, 2.25x, 2.5x, 2.75x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, or 15x 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 fold, 1.5-1.75 fold, 1.75-2 fold, 2-2.25 fold, 2.25-2.5 fold, 2.5-2.75 fold, 2.75-3 fold, 3-3.5 fold, 3.5-4 fold, 4-4.5 fold, 4.5-5 fold, 5-5.5 fold, 5.5-6 fold, 6-7 fold, 7-8 fold, 8-9 fold, 9-10 fold, 10-11 fold, 11-12 fold, 13-14 fold, or 14-15 fold higher than the capture yield of target binding probes specific for the set of epigenetic target regions.

[0343] 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 1.25x, 1.5x, 1.75x, 2x, 2.25x, 2.5x, 2.75x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, or 15x higher than the capture yield of the 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 times, 1.5-1.75 times, 1.75-2 times, 2-2.25 times, 2.25-2.5 times, 2.5-2.75 times, 2.75-3 times, 3-3.5 times, 3.5-4 times, 4-4.5 times, 4.5-5 times, 5-5.5 times, 5.5-6 times, 6-7 times, 7-8 times, 8-9 times, 9-10 times, 10-11 times, 11-12 times, 13-14 times, or 14-15 times higher than its capture yield specific for the set of epigenetic target regions.

[0344] A collection of probes can be configured to provide higher capture yields for a set of sequence-variable target regions in a variety of ways, including enrichment, varying length and / or chemistry (e.g., to affect affinity), and combinations thereof. Affinity can be modulated by adjusting the length of the probe and / or by including nucleotide modifications as discussed below.

[0345] In some embodiments, the target-specific probes specific for the set of sequence variable target regions are present at a higher concentration than the target-specific probes specific for the set of epigenetic target regions, hi some embodiments, the concentration of target-binding probes specific for the set of sequence variable target regions is at least 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold higher than the concentration of target-binding probes specific for the set of epigenetic target regions. In some embodiments, the concentration of target-binding probes specific for the set of sequence-variable target regions is 1.25-1.5x, 1.5-1.75x, 1.75-2x, 2-2.25x, 2.25-2.5x, 2.5-2.75x, 2.75-3x, 3-3.5x, 3.5-4x, 4-4.5x, 4.5-5x, 5-5.5x, 5.5-6x, 6-7x, 7-8x, 8-9x, 9-10x, 10-11x, 11-12x, 13-14x, or 14-15x higher than the concentration of target-binding probes specific for the set of epigenetic target regions. In such embodiments, concentration may refer to the average mass / volume concentration of the individual probes in each set.

[0346] In some embodiments, target-specific probes specific to a set of sequence-variable target regions have higher affinity for the target than target-specific probes specific to a set of epigenetic target regions. Affinity can be modulated by any method known to those skilled in the art, including using different probe chemistries. For example, certain nucleotide modifications, such as cytosine 5-methylation (in the context of a particular sequence), modifications that introduce heteroatoms at the 2' sugar position, and LNA nucleotides, can increase the stability of double-stranded nucleic acids, and oligonucleotides with such modifications have been shown to have relatively high affinity for 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. Additionally, longer sequence lengths generally provide increased affinity. Other nucleotide modifications, such as substitution of guanine with the nucleobase hypoxanthine, reduce affinity by reducing the amount of hydrogen bonding between the oligonucleotide and its complementary sequence. In some embodiments, target-specific probes specific for a set of sequence-variable target regions have modifications that increase affinity for their targets. In some embodiments, instead or in addition, target-specific probes specific for a set of epigenetic target regions have modifications that decrease affinity for their targets. In some embodiments, target-specific probes specific for a set of sequence-variable target regions have a longer average length and / or a higher average melting temperature than target-specific probes specific for a set of epigenetic target regions. These embodiments can be combined with each other and / or with concentration differences as discussed above to achieve a desired fold difference in capture yield, for example, any of the fold differences or ranges described above.

[0347] In some embodiments, the target-specific probe comprises a capture moiety. The capture moiety can be any of the capture moieties described herein, for example, biotin. In some embodiments, the target-specific probe is linked to a solid support, for example, covalently or non-covalently, such as by interaction of the capture moiety's binding pair. In some embodiments, the solid support is a bead, for example, a magnetic bead.

[0348] In some embodiments, the target-specific probes specific for the set of sequence-variable target regions and / or the target-specific probes specific for the set of epigenetic target regions are probes that comprise capture moieties and sequences selected to span a panel of regions, such as the bait set discussed above, e.g., genes.

[0349] In some embodiments, the target-specific probes are provided in a single composition. The single composition may be in solution (liquid or frozen). Alternatively, the composition may be lyophilized.

[0350] Alternatively, target-specific probes can be provided as multiple compositions, including, for example, a first composition containing probes specific to a set of epigenetic target regions and a second composition containing probes specific to a set of sequence-variable target regions. These probes can be mixed in appropriate ratios to provide a combined probe composition having any of the above-mentioned fold differences in concentration and / or capture yield. Alternatively, these probes can be used in separate capture procedures (e.g., on aliquots of a sample or sequentially on the same sample) to provide first and second compositions containing captured epigenetic and sequence-variable target regions, respectively.

[0351] 1. Probes specific to epigenetic target regions The probes for the set of epigenetic target regions can include probes specific for one or more types of target regions that may distinguish DNA from 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 section above regarding captured sets. The probes for the set of epigenetic target regions can also include probes for one or more control regions, e.g., as described herein.

[0352] In some embodiments, the probes of the epigenetic target region probe set have a footprint of at least 100 kb, e.g., at least 200 kb, at least 300 kb, or at least 400 kb. In some embodiments, the probes of the epigenetic target region set have a footprint in the range of 100 to 1,000 kb, e.g., 100 to 200 kb, 200 to 300 kb, 300 to 400 kb, 400 to 500 kb, 500 to 600 kb, 600 to 700 kb, 700 to 800 kb, 800 to 900 kb, and 900 to 1,000 kb. In some embodiments, the probes of the epigenetic target region probe set have a footprint of less than 5 kb, at least 5 kb, e.g., at least 10, 20, or 50 kb.

[0353] a. Hypermethylated variable target region 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 can be any of the target regions described 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, probes specific for hypermethylated variable target regions include probes specific for multiple loci listed in Table 1 or Table 2, e.g., 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 having hybridization sites that bind between the transcription start site of the gene and the stop codon (or the final stop codon for alternatively spliced ​​genes). In some embodiments, the one or more probes bind within 300 bp, e.g., within 200 or 100 bp, of the listed position. In some embodiments, the probes have hybridization sites that overlap with the above-listed positions. In some embodiments, the probes specific for hypermethylated variable target regions comprise probes specific for a subset of one, two, three, four, or five hypermethylated target regions that collectively exhibit hypermethylation in one, two, three, four, or five of breast cancer, colon cancer, renal cancer, liver cancer, and lung cancer.

[0354] b. Hypomethylated variable target region In some embodiments, the probes for the set of epigenetic target regions include probes specific for one or more hypomethylated variable target regions. The hypomethylated variable target regions may be any of the target regions described above. For example, the probes specific for one or more hypomethylated variable target regions may include probes for repetitive elements, such as LINE1 elements, Alu elements, centromeric tandem repeats, paracentromeric tandem repeats, and satellite DNA, as well as intergenic regions that are normally methylated in healthy cells but show reduced methylation in tumor cells.

[0355] In some embodiments, the probes specific for the hypomethylated variable target region comprise probes specific for repetitive elements and / or intergenic regions, hi some embodiments, the probes specific for repetitive elements comprise probes specific for one, two, three, four, or five of the following: a LINE1 element, an Alu element, a centromeric tandem repeat, a paracentromeric tandem repeat, and / or satellite DNA.

[0356] 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.

[0357] c.CTCF binding region 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 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 listed above or in the CTCFBSDB or one or more of the above-cited articles by Cuddapah et al., Martin et al., or Rhee et al. 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 upstream and downstream of the CTCF binding site.

[0358] d. transcription start site In some embodiments, the probes for the set of epigenetic target regions include probes specific for transcription start sites. In some embodiments, the probes specific for transcription start sites include probes specific for 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 sequences 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 upstream and downstream of the transcription start site.

[0359] e. Local amplification As mentioned above, local amplification is a somatic mutation, but it can be detected by sequencing based on read frequency in a similar manner to the approach for detecting certain epigenetic changes, such as methylation changes.Therefore, as discussed above, the region that may show local amplification in cancer can be included in the epigenetic target region set.In some embodiments, the probe specific to the epigenetic target region set comprises the probe specific to local amplification.In some embodiments, the probe specific to local amplification comprises the probe specific to one or more 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, 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.

[0360] f. Control area It may be useful to include control regions to facilitate data validation. In some embodiments, the probes specific for the set of epigenetic target regions include probes specific for control methylated regions that are expected to be methylated in essentially all samples. In some embodiments, the probes specific for the set of epigenetic target regions include probes specific for control hypomethylated regions that are expected to be hypomethylated in essentially all samples.

[0361] 2. Probes specific to sequence-variable target regions The probes for the set of sequence variable target regions may include probes specific for multiple regions known to undergo somatic mutation in cancer. The probes may be specific for any of the 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 captured sets.

[0362] In some embodiments, sequence-variable target region probe sets have 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, epigenetic target region probe sets have a footprint in the range of 0.5 to 100 kb, e.g., 0.5 to 2 kb, 2 to 10 kb, 10 to 20 kb, 20 to 30 kb, 30 to 40 kb, 40 to 50 kb, 50 to 60 kb, 60 to 70 kb, 70 to 80 kb, 80 to 90 kb, and 90 to 100 kb.

[0363] 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 in 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 in 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 in 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 1, at least 2, or 3 of the indels in 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 in 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 in Table 4. 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 in Table 4.In some embodiments, the probes specific for the set of sequence variable target regions comprise 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 comprise 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.

[0364] 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.

[0365] E. Compositions Containing Captured DNA Provided herein is a combination comprising a first and a second group of captured DNA.The first group can comprise or be derived from DNA with a higher proportion of cytosine modification than the second group.The first group can comprise a first type of nucleobase originally present in DNA with altered base pairing specificity and a second nucleobase that does not have altered base pairing specificity, wherein the first type of nucleobase originally present in DNA before altered base pairing specificity is a modified nucleobase or an unmodified nucleobase, and the second nucleobase is a modified nucleobase or an unmodified nucleobase different from the first nucleobase, and the first type of nucleobase originally present in DNA before altered base pairing specificity and the second nucleobase have the same base pairing specificity.The second group does not comprise the first type of nucleobase originally present in DNA with altered base pairing specificity.In some embodiments, the cytosine modification is cytosine methylation. In some embodiments, the first nucleobase is a modified or unmodified cytosine, and the second nucleobase is a modified or unmodified cytosine.The first and second nucleobases are any of the nucleobases discussed in the summary of this specification, or the nucleobases discussed in connection with subjecting a first sub-sample to a procedure that affects the first nucleobase in DNA differently from the second nucleobase in the DNA of the first sub-sample.

[0366] In some embodiments, the first population comprises sequence tags selected from a first set of one or more sequence tags, and the second population comprises sequence tags selected from a second set of one or more sequence tags, the second set of sequence tags being different from the first set of sequence tags. The sequence tags may comprise barcodes.

[0367] In some embodiments, the first population comprises protected hmC, eg, glycosylated hmC.

[0368] In some embodiments, the first population has been subjected to any of the conversion procedures discussed herein, e.g., bisulfite conversion, Ox-BS conversion, TAB conversion, ACE conversion, TAP conversion, TAPSβ conversion, or CAP conversion. In some embodiments, the first population has been subjected to protection of hmC followed by deamination of mC and / or C.

[0369] In some embodiments of the combination, the first population comprises or is derived from DNA with a higher proportion of cytosine modifications than the second population, the first population comprising a first and a second subpopulation, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity. In some embodiments, the second population does not comprise the first nucleobase. In some embodiments, the first nucleobase is a modified or unmodified cytosine, and the second nucleobase is a modified or unmodified cytosine, optionally the modified cytosine is mC or hmC. In some embodiments, the first nucleobase is a modified or unmodified adenine, and the second nucleobase is a modified or unmodified adenine, optionally the modified adenine is mA.

[0370] In some embodiments, the first nucleobase (e.g., modified cytosine) is biotinylated. In some embodiments, the first nucleobase (e.g., modified cytosine) is the product of Huisgen cycloaddition to β-6-azido-glucosyl-5-hydroxymethylcytosine containing an affinity label (e.g., biotin).

[0371] In any of the combinations described herein, the captured DNA can include cfDNA.

[0372] The captured DNA may have any of the characteristics described herein for the captured set, including, for example, a higher concentration of DNA corresponding to the set of sequence-variable target regions (normalized for footprint size as discussed above) than DNA corresponding to the set of epigenetic target regions. In some embodiments, the DNA of the captured set includes a sequence tag, which may be added to the DNA as described herein. Generally, the inclusion of a sequence tag results in a DNA molecule that is different from its naturally occurring, untagged form.

[0373] The combination may further include a probe set described herein or a sequencing primer, each of which may be different from a naturally occurring nucleic acid molecule. For example, a probe set described herein may include a capture moiety, and a sequencing primer may include a non-naturally occurring label.

[0374] F. Computer Systems The method of the present disclosure can be carried out by using or with the help of computer system.For example, this method can include the following steps: divide sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample comprises the DNA with cytosine modification at a higher ratio than the second sub-sample; subject the first sub-sample to a procedure that affects the first nucleobase in DNA so that it is different from the second nucleobase in the DNA of the first sub-sample, wherein the first nucleobase is modified nucleobase or unmodified nucleobase, and the second nucleobase is modified nucleobase or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity; and sequence the DNA in the first sub-sample and the DNA in the second sub-sample, so as to distinguish the first nucleobase in the DNA of the first sub-sample from the second nucleobase.

[0375] 4 illustrates a computer system 401 programmed or otherwise configured to perform the methods of the present disclosure. The computer system 401 can coordinate various aspects of sample preparation, sequencing, and / or analysis. In some examples, the computer system 401 is configured to perform sample preparation and sample analysis, including nucleic acid sequencing, for example, according to any of the methods disclosed herein.

[0376] The computer system 401 includes a central processing unit (CPU, also referred to herein as a "processor" and a "computer processor") 405, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 401 also includes memory or memory locations 410 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 415 (e.g., a hard disk), a communication interface 440 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 425, such as cache, other memory, data storage, and / or an electronic display adapter. The memory 410, the storage unit 415, the interface 420, and the peripheral devices 425 communicate with the CPU 405 through a communication network or bus (solid lines), such as a motherboard. The storage unit 415 may be a data storage unit (or data repository) for storing data. The computer system 401 can be operably coupled to a computer network 430 with the aid of the communication interface 420. The computer network 430 may be the Internet, an Internet and / or extranet, or an intranet and / or extranet in communication with the Internet. The computer network 430, in some examples, is a telecommunications and / or data network. The computer network 430 may include one or more computer servers, which may enable distributed computing, such as cloud computing. The computer network 430, in some examples, with the help of computer system 401, may implement a peer-to-peer network, which may allow devices coupled to the computer system 401 to act as clients or servers.

[0377] CPU 405 may execute sequences of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in memory locations, such as memory 410. Examples of operations performed by CPU 405 may include fetch, decode, execute, and writeback.

[0378] The storage unit 415 can store files, such as drivers, libraries, and saved programs. The storage unit 415 can store user-generated programs and recorded sessions, as well as program-related output. The storage unit 415 can store user data, such as user preferences and user programs. The computer system 401 in some examples can include one or more additional data storage units, such as those located outside the computer system 401, e.g., on a remote server that communicates with the computer system 401 through an intranet or the Internet. Data may 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).

[0379] Computer system 401 can communicate with one or more remote computer systems through network 430. For an embodiment, computer system 401 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 mobile PC), a slate or tablet PC (e.g., an Apple® iPad®, a Samsung® Galaxy Tab), a telephone, a smartphone (e.g., an Apple® iPhone®, an Android-enabled device, a Blackberry®), or a personal digital assistant. A user can access computer system 401 through network 430.

[0380] The methods described herein can be performed by machine (e.g., a computer processor) executable code stored in an electronic storage location of the computer system 401, such as memory 410 or electronic storage unit 415. The machine-executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 405. In some examples, the code is retrieved from the storage unit 415 and stored in memory 410 for ready access by the processor 405. In some situations, the electronic storage unit 415 can be omitted, and the machine-executable instructions are stored in memory 410.

[0381] 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, perform at least a portion of a method comprising: collecting cfDNA from a test subject; capturing a set of multiple target regions from the cfDNA, wherein the set of multiple target regions comprises a set of sequence variable target regions and a set of epigenetic target regions, to produce a set of captured cfDNA molecules; sequencing the captured cfDNA molecules, wherein the captured cfDNA molecules of the set of sequence variable target regions are sequenced to a higher sequencing depth than the captured cfDNA molecules of the set of epigenetic target regions; obtaining, from the sequencing of the captured cfDNA molecules, a plurality of sequence reads generated by a nucleic acid sequencer; mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads; and processing the mapped sequence reads corresponding to the set of sequence variable target regions and the set of epigenetic target regions to determine a likelihood that the subject has cancer.

[0382] The code may be precompiled 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 supplied precompiled or written in a programming language that may be selected to enable the code to be executed as it is compiled.

[0383] Aspects of the systems and methods provided herein, e.g., computer system 401, may be embodied in programming. Various aspects of the present technology can be thought of as "products" or "articles of manufacture," typically in the form of machine (or processor) executable code and / or associated data contained or embodied in a type of machine-readable medium. The machine-executable code can be stored in an electronic storage unit, e.g., memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media includes any or all of a computer's tangible memory, processor, or the like, or its associated modules, e.g., various semiconductor memories, tape drives, disk drives, and the like, which may provide non-transitory storage at any time for software programming.

[0384] All or a portion of the software may sometimes be communicated over the Internet or various other telecommunications networks. Such communication may, for example, enable loading of the software from one computer or processor to another, e.g., from a management server or host computer to an application server computer platform. Accordingly, other types of media that may bear software elements include optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical terrestrial communications networks, and over various air links. Physical elements that carry such waves, e.g., wired or wireless links, optical links, or the like, may also be considered media bearing the software. As used herein, without limitation to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that contributes to providing instructions to a processor for execution.

[0385] Thus, machine-readable media, e.g., computer-executable code, may take many forms, including, but not limited to, tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices of any computer, such as those used to execute the databases shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables, copper wire, and optical fibers, including the wires that comprise 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. Common forms of computer readable media thus include, for example, floppy disks, flexible disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punch cards, paper tape, any other physical storage media with a pattern of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, any other memory chip or cartridge, a carrier wave transporting data or instructions, a cable or link transporting 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 may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0386] The computer system 401 may include or be in communication with an electronic display that includes, for example, a user interface (UI) for providing one or more results of a sample analysis. Examples of UIs include, without limitation, graphical user interfaces (GUIs) and web-based user interfaces.

[0387] Further details regarding computer systems and networks, databases, and computer program products can be found, for example, in 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 It is also provided in Computing Architected: Solution Design Handbook, Recursive Press (2011).

[0388] G. Application 1. Cancer and other diseases The present method can be used to diagnose the presence of a condition, particularly cancer, in a subject, characterize the condition (e.g., stage the cancer or determine the heterogeneity of the cancer), monitor the response to treatment of the condition, and achieve a prognostic risk of the progression 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 option is successful, more cancer cells may die and shed DNA, so that successful treatment may increase the amount of copy number variations or rare mutations detected in the subject's blood. In other examples, this may not occur. In another example, perhaps a particular treatment option can be correlated with the genetic profile of the cancer over time. This correlation can be useful for selecting a therapy.

[0389] Additionally, if the cancer is observed to be in remission after treatment, the method can be used to monitor for residual disease or recurrence of the disease.

[0390] In some embodiments, the methods and systems disclosed herein can be used to identify customized or targeted therapies for treating a given disease or condition in a patient based on classifying nucleic acid variants as being of somatic or germline origin. Typically, the disease under consideration is a type of cancer. Non-limiting examples of such cancers include cholangiocarcinoma, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, glioma, astrocytoma, breast cancer, metaplastic carcinoma, cervical cancer, and the like. 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 lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), liver cancer, liver cancer, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, germline tumor, 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 by genetic variations including mutations, rare mutations, indels, copy number variations, transversions, translocations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, alterations in chromosomal structure, gene fusions, chromosomal fusions, gene truncations, gene amplification, gene duplications, chromosomal damage, DNA damage, abnormal changes in chemical modifications of nucleic acids, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5-methylcytosine.

[0391] Genetic data can also be used to characterize specific forms of cancer. Cancers are often heterogeneous in composition and stage classification. Genetic profile data can enable characterization of specific subtypes of cancer, which can be important in diagnosing or treating specific subtypes. This information can provide subjects or clinicians with clues regarding the prognosis of specific types of cancer and allow them to tailor treatment options as the disease progresses. Some cancers can progress to 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.

[0392] Furthermore, the methods of the present disclosure can be used to characterize the heterogeneity of an abnormal condition in a subject. Such a method can include, for example, generating a genetic profile of extracellular polynucleotides derived from the subject, the genetic profile including multiple data resulting from analysis of copy number variations and rare mutations. In some embodiments, the abnormal condition is cancer. In some embodiments, the abnormal condition can be a condition that results in a heterogeneous genomic population. In the example of cancer, it is known that some tumors contain tumor cells at different cancer stages. In other examples, the heterogeneity can include multiple disease foci. Again, in the example of cancer, there are multiple tumor foci, and perhaps one or more foci are the result of metastasis that has spread from the primary site.

[0393] The method can be used to generate or profile a fingerprint or dataset that is the sum of genetic information derived from different cells in a heterogeneous disease, which dataset may include analysis of copy number variations, epigenetic variations, and mutations, either alone or in combination.

[0394] This method can be used to diagnose, prognose, monitor or observe cancer or other diseases.In some embodiments, the method herein does not include the diagnosis, prognosis or monitoring of fetus, and therefore is not related to non-invasive prenatal testing.In other embodiments, these methodologies can be used in pregnant subjects to diagnose, prognose, monitor or observe cancer or other diseases in unborn subjects, whose DNA and other polynucleotides may circulate with maternal molecules.

[0395] Non-limiting examples of other genetic diseases, disorders, or conditions that may be evaluated, if desired, 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), cricket cricket, Crohn's disease, cystic fibrosis, Dercum's disease, Down's syndrome, Duanne's syndrome, Duchenne muscular dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, and Goniohashi disease. Examples of conditions that may be present include: rhesus disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter's syndrome, Marfan's syndrome, myotonic dystrophy, neurofibroma, Noonan's syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland variant, 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, Wilson's disease, or the like.

[0396] In some embodiments, the methods described herein include detecting the presence or absence of DNA originating from or derived from tumor cells at a preselected time point following a previous cancer treatment in a subject previously diagnosed with cancer using a set of sequence information obtained as described herein. The methods may further include determining a cancer recurrence score for the test subject that indicates the presence or absence of DNA originating from or derived from tumor cells.

[0397] When a cancer recurrence score is determined, this score can be further used to determine a cancer recurrence status. The cancer recurrence status is, for example, a risk of cancer recurrence when the cancer recurrence score is above a predetermined threshold. The cancer recurrence status is, for example, a 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 may result in a cancer recurrence status of a risk of cancer recurrence, or a low or lower risk of cancer recurrence.

[0398] 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 test subject is classified as a candidate for subsequent cancer treatment, or if the cancer recurrence score is below the cancer recurrence threshold, the test subject is classified as not being a candidate for treatment. In certain embodiments, a cancer recurrence score equal to the cancer recurrence threshold may result in classification as being a candidate for subsequent cancer treatment or not being a candidate for treatment.

[0399] The methods discussed above may further include any suitable feature(s) described elsewhere herein, including the sections relating to methods for determining the risk of cancer recurrence in a test subject and / or methods for classifying a test subject as a candidate for subsequent cancer treatment.

[0400] 2. Methods for determining the risk of cancer recurrence in a test subject and / or for classifying test subjects as candidates for subsequent cancer treatment In some embodiments, the methods provided herein are methods for determining the risk of cancer recurrence in a test subject. In some embodiments, the methods provided herein are methods for classifying a test subject as a candidate for subsequent cancer treatment.

[0401] Any of these methods may include collecting DNA (e.g., originating from or derived from tumor cells) from a test subject diagnosed with cancer at one or more preselected time points after one or more previous cancer treatments for the test subject. The subject may be any of the subjects described herein. The DNA may be cfDNA. The DNA may be obtained from a tissue sample.

[0402] Any such method may include capturing a set of multiple target regions from DNA from a subject, the set of multiple target regions including a set of sequence variable target regions and a set of epigenetic target regions, to produce a set of captured DNA molecules. The capturing step may be performed according to any of the embodiments described elsewhere herein.

[0403] In any of such methods, the prior cancer treatment may include surgery, administration of a therapeutic composition, and / or chemotherapy.

[0404] Any of these methods includes sequencing the captured DNA molecules, thereby generating a set of sequence information. The captured DNA molecules of the set of sequence-variable target regions can be sequenced to a higher sequencing depth than the captured DNA molecules of the set of epigenetic target regions.

[0405] Any such method may include using the set of sequence information to detect the presence or absence of DNA originating from or derived from the tumor cell at a preselected time point. Detecting the presence or absence of DNA originating from or derived from the tumor cell may be performed according to any of the embodiments described elsewhere herein.

[0406] The method for determining the risk of cancer recurrence in a test subject can include determining a cancer recurrence score for the test subject, which indicates the presence or absence or amount of DNA originating from or derived from tumor cells.The cancer recurrence score can be further used to determine a cancer recurrence status.The cancer recurrence status can be, for example, a risk of cancer recurrence when the cancer recurrence score is above a predetermined threshold.The cancer recurrence status can be, for example, a 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 a risk of cancer recurrence, or a low or lower risk of cancer recurrence.

[0407] A method for classifying a test subject as a candidate for subsequent cancer treatment includes comparing the test subject's cancer recurrence score with a predetermined cancer recurrence threshold, and classifying the test subject as a candidate for subsequent cancer treatment if the cancer recurrence score is above the cancer recurrence threshold, or as not a candidate for treatment 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 classification as a candidate for subsequent cancer treatment or as not a candidate for treatment. In some embodiments, the subsequent cancer treatment includes administration of chemotherapy or a therapeutic composition.

[0408] Any such method may include determining a disease-free survival (DFS) period for the test 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.

[0409] 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 the amount of SNV, insertion / deletion, CNV, and / or fusion present in the sequence variable target region sequence.

[0410] In some embodiments, the number of mutations in the sequence variable target regions 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.

[0411] In some embodiments, the set of sequence information includes epigenetic target region sequences, and determining the cancer recurrence score includes determining a second subscore indicating the amount of molecules (e.g., obtained from the epigenetic target region sequences) that exhibit an epigenetic state different from that of DNA found in a corresponding sample from a healthy subject (e.g., cfDNA found in a blood sample from the healthy subject, or DNA found in a tissue sample from the healthy subject if the tissue sample is of the same tissue type as that obtained from the test subject). These aberrant molecules (i.e., molecules with an epigenetic state different from that of DNA found in a corresponding sample from the healthy subject) may be consistent with cancer-associated epigenetic changes, such as methylation of hypermethylated variable target regions and / or perturbed fragmentation of fragmented variable target regions, where "perturbed" means different from that of DNA found in a corresponding sample from a healthy subject.

[0412] In some embodiments, a proportion of molecules corresponding to the set of hypermethylated and / or fragmented variable target regions exhibiting hypermethylation in the set of hypermethylated variable target regions and / or aberrant fragmentation in the set of fragmented variable target regions greater than or equal to a value in the range of 0.001% to 10% is sufficient to classify the second subscore as positive for cancer recurrence, which range may be 0.001% to 1%, 0.005% to 1%, 0.01% to 5%, 0.01% to 2%, or 0.01% to 1%.

[0413] In some embodiments, any of these methods may include determining the proportion of tumor DNA from the proportion of molecules in the set of sequence information that exhibit one or more features indicative of origin from tumor cells. This may be performed for molecules corresponding to some or all of the epigenetic target regions, including, for example, hypermethylated variable target regions and / or fragmented variable target regions (hypermethylation of hypermethylated variable target regions and / or aberrant fragmentation of fragmented variable target regions may be considered indicative of origin from tumor cells). This may be performed for molecules corresponding to sequence variable target regions, for example, molecules containing alterations consistent with cancer, such as SNVs, indels, CNVs, and / or fusions. The proportion of tumor DNA may be determined based on a combination of molecules corresponding to epigenetic target regions and molecules corresponding to sequence variable target regions.

[0414] The determination of the cancer recurrence score may be based at least in part on the proportion of tumor DNA, -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 in the range of at least 10 is sufficient to classify the Cancer Recurrence Score as positive for cancer recurrence. -7A tumor DNA proportion greater than this threshold is sufficient to classify the cancer recurrence score as positive for cancer recurrence.The determination that the tumor DNA proportion is greater than a threshold, for example, a threshold corresponding to any of the above embodiments, can be based on cumulative probability.For example, if the cumulative probability that the tumor proportion is greater than any of the thresholds in the above 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, for example 0.99.

[0415] In some embodiments, the set of sequence information includes a sequence variable target region sequence and an epigenetic target region sequence, 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 sequence and a second subscore indicative of the amount of aberrant molecules in the epigenetic target region sequence, and combining the first and second subscores to provide the cancer recurrence score. When combining the first and second subscores, they can be combined by applying thresholds (e.g., greater than a predetermined number of mutations in the sequence variable target region (e.g., >1) and greater than a predetermined proportion of aberrant molecules in the epigenetic target region (i.e., molecules with an epigenetic state different from that found in a corresponding sample from a healthy subject; e.g., tumor)) to each subscore independently, or by training a machine learning classifier to determine the status based on multiple positive and negative training samples.

[0416] 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.

[0417] 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.

[0418] In some embodiments, the cancer is any one of the types of cancer described elsewhere herein, for example, colorectal cancer.

[0419] 3. Treatment and Related Administration In certain embodiments, the methods disclosed herein relate to identifying and administering customized therapies to patients given the status of nucleic acid variants of somatic or germline origin. In some embodiments, essentially any cancer therapy (e.g., surgery, radiation, chemotherapy, and / or the like) can be included as part of these methods. Typically, customized therapies include at least one immunotherapy (or immunotherapeutic agent). Immunotherapy generally refers to methods that enhance immune responses against a given type of cancer. In certain embodiments, immunotherapy refers to methods that enhance T-cell responses against tumors or cancers.

[0420] In certain embodiments, the nucleic acid variant status of the sample from the subject that is the origin of somatic or germline cell lineage is compared with the database of comparator results from a reference population, and identify the customized or targeted therapy for the subject.Typically, the reference population comprises patients with the same type of cancer or disease as the test subject, and / or patients who are undergoing or have undergone the same therapy as the test subject.If the nucleic acid variant and the comparator results meet certain classification criteria (for example, are substantially or approximately the same), customized or targeted therapy (one or more therapies) can be identified.

[0421] In certain embodiments, the customized therapies described herein are typically administered parenterally (e.g., intravenously or subcutaneously). Pharmaceutical compositions containing immunotherapeutic agents are typically administered intravenously. Certain therapeutic agents are administered orally. However, customized therapies (e.g., immunotherapeutic agents, etc.) can be administered by methods such as buccal, sublingual, rectal, vaginal, intraurethral, ​​topical, intraocular, intranasal, and / or intraauricular administration, and administration can include tablets, capsules, granules, aqueous suspensions, gels, sprays, suppositories, salves, ointments, or the like.

[0422] 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. It is not intended that the present invention be limited by the specific examples provided herein. While the present invention has been described with reference to the above specification, the descriptions and explanations of the embodiments herein are not meant to be construed in a limiting sense. Numerous modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It is to be understood that various alternatives to the disclosed embodiments described herein may be employed in practicing the invention. It is therefore intended that the present disclosure should embrace any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

[0423] 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 purview of the appended claims. For example, all features, steps, elements, or other aspects of the methods, systems, computer-readable media, and / or components may be used in various combinations.

[0424] H.Kit Also provided are kits containing the compositions described herein. The kits can be useful for carrying out the methods described herein. In some embodiments, the kits include a first reagent for dividing a sample into multiple subsamples as described herein, such as any of the division reagents described elsewhere herein. In some embodiments, the kits include a second reagent for subjecting the first subsample to a procedure that affects a first nucleobase in DNA differently from a second nucleobase in the DNA of the first subsample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity (e.g., any of the reagents described elsewhere herein for converting a nucleobase, such as cytosine or methylated cytosine, to a different nucleobase). The kits can include the first and second reagents and additional elements discussed below and / or elsewhere herein.

[0425] The kit includes ALK, APC, BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN, RBI, TP53, MET, AR, ABL1, AKT1, ATM, CDH1, CSFIR, CTNNBl, ERBB4, EZH2, FGFRl, FGFR2, FGFR3, FLT3, GNA11, GNAQ, G It may further comprise a plurality of oligonucleotide probes that selectively hybridize to at least 5, 6, 7, 8, 9, 10, 20, 30, 40 or all genes selected from the group consisting of NAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, PTPN11, RET, SMAD4, SMARCB1, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAF1, BRCA1, CCND2, CDK6, NF1, TP53, ARID 1 A, BRCA2, CCNE1, ESR1, RIT1, GATA3, MAP2K1, RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA and NTRK1.The number of genes that oligonucleotide probes can selectively hybridize to can vary. For example, the number of genes can 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 can include a container comprising a plurality of oligonucleotide probes and instructions for performing any of the methods described herein.

[0426] The oligonucleotide probe can selectively hybridize to the exon region of a gene, for example, at least five genes. In some examples, the oligonucleotide probe can selectively hybridize to at least 30 exons of a gene, for example, at least five genes. In some examples, multiple probes can selectively hybridize to each of the at least 30 exons. The probe hybridizing to each exon can have a sequence that overlaps with at least one other probe. In some embodiments, the oligo probe can selectively hybridize to the non-coding region of the gene disclosed herein, for example, the intron region of the gene. The oligo probe can also selectively hybridize to the region of the gene, including both the exon and intron regions of the gene disclosed herein.

[0427] Any number of exons can be targeted by the oligonucleotide probe, for example, 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, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 400, 500, 600, 700, 800, 900, 1,000 or more exons can be targeted.

[0428] The kit may include at least 4, 5, 6, 7, or 8 different library adapters with distinct molecular barcodes and identical sample barcodes. The library adapters may not be sequencing adapters. For example, the library adapters do not include sequences that enable the formation of flow cell sequences or hairpin loops for sequencing. Different variations and combinations of molecular barcodes and sample barcodes are described throughout and are applicable to the kits. Furthermore, in some examples, the adapters are not sequencing adapters. Furthermore, the adapters provided in the kit may also 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 polynucleotide fragments. In some examples, the kit may include at least 8 different library adapters with distinct molecular barcodes and identical sample barcodes. The library adapters may not be sequencing adapters. The kit may further comprise 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 comprise a complementary double-stranded portion and a loop portion, and the double-stranded portion can be bound (e.g., ligated) to a double-stranded polynucleotide.The hairpin-shaped sequencing adaptor can be bound to both ends of a polynucleotide fragment to generate a circular molecule, which can be sequenced multiple times.Sequencing adapters may be arranged end-to-end in the following order: up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25, up to 26, up to 27, up to 28, up to 29, up to 30, up to 31, up to 32, up to 33, up to 34, up to 35, up to 36, up to 37, up to 38, up to 39, up to 40, up to 41, up to 42, up to 43, up to 44, up to 45, up to 46, up to 47, up to 48, up to 49, up to 50, up to 51, up to 52, up to 53, up to 54, up to 55, up to 56, up to 57, up to 58, up to 59, up to 60, up to 61, up to 62, up to 63, up to 64, up to 65, up to 66, up to 67, up to 68, up to 69, up to 70, up to 71, up to 72, up to 73, up to 74, up to 75, up to 76, up to 77, up to 78, up to 79, up to 80, up to 81, up to 82, up to 83, up to 84, up to 85, up to 86, up to 87, up to 88, up to 89, up to 90, up to 91, up There can be up to 5, up to 56, up to 57, up to 58, up to 59, up to 60, up to 61, up to 62, up to 63, up to 64, up to 65, up to 66, up to 67, up to 68, up to 69, up to 70, 71, up to 72, up to 73, up to 74, 75, up to 76, up to 77, up to 78, 79, up to 80, 81, up to 82, 83, up to 84, 85, up to 86, up to 87, up to 88, up to 89, up to 90, 91, up to 92, 93, up to 94, 95, up to 96, up to 97, up to 98, 99, up to 100 or more bases. The sequencing adapter can comprise 20-30, 20-40, 30-50, 30-60, 40-60, 40-70, 50-60, or 50-70 bases from end to end. In a particular example, the sequencing adapter can comprise 20-30 bases from end to end. In another example, the sequencing adapter can comprise 50-60 bases from end to end. The sequencing adapter can comprise one or more barcodes. For example, the sequencing adapter can comprise a sample barcode. The sample barcode can comprise a predetermined sequence. The sample barcode can be used to identify the source of the polynucleotide.A sample barcode can 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 nucleic acid bases (or any length described throughout) such as at least 8 bases. The barcode can be a continuous or discontinuous sequence as described above.

[0429] The library adaptors can be blunt-ended and Y-shaped and are less than or equal to 40 nucleobases in length. Other variations can be found throughout the specification and are applicable to the kits.

[0430] All patents, patent applications, websites, other publications and documents, accession numbers, and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each separate item were specifically and separately indicated to be incorporated by reference. Where different versions of a sequence are associated with accession numbers at different times, the version associated with the accession number as of the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or, if applicable, the filing date of the priority application that references the accession number. Similarly, where different versions of a publication, website, or the like were published at different times, the version published closest to the effective filing date of this application is meant, unless otherwise indicated. [Example]

[0431] III. Working Examples Example 1: Analysis of cfDNA to detect the presence / absence of tumor Patient sample sets are analyzed at Guardant Health (Redwood City, CA, USA) using a blood-based NGS assay to detect the presence or absence of cancer. cfDNA is extracted from the patient's plasma. The patient sample's cfDNA is then combined with methyl-binding domain (MBD) buffer and magnetic beads conjugated with MBD protein and incubated overnight. Methylated cfDNA (if present in the cfDNA sample) binds to the MBD protein during this incubation. Unmethylated or less methylated DNA is washed from the beads with buffers containing increasing concentrations of salt. Finally, hypermethylated DNA is washed from the MBD protein using a high-salt buffer. These washes result in three fractions of cfDNA with increasing methylation: a hypomethylated fraction, a residually methylated fraction, and a hypermethylated fraction. In this example, cfDNA molecules in the highly methylated fraction are subjected to a procedure called hmC-Seal / 5hmC-Seal (Han, D. A highly sensitive and robust method for genome-wide 5hmC profiling of rare cell populations. Mol Cell. 2016;63(4):711-719), which labels hmC residues (in cfDNA molecules in the highly methylated fraction) to form β-6-azido-glucosyl-5-hydroxymethylcytosine, then attaches a biotin moiety via Huisgen cycloaddition. A biotin-binding agent is then used to separate the biotinylated DNA from other DNA, thereby separating cfDNA molecules bearing hmC residues in the highly methylated fraction into a fourth fraction, the hmC fraction. After separation, the cfDNA molecules in the four fractions are purified to remove salts and concentrated in preparation for the enzymatic steps of library preparation.

[0432] After the cfDNA is enriched in the fractions, the overhangs at the ends of the divided cfDNA are extended, and during extension, adenosine residues are added to the 3' ends of the cfDNA fragments by polymerase. The 5' end of each fragment is phosphorylated. These modifications make the divided cfDNA ligable. DNA ligase and adapters are added to ligate adapters at each end of each divided cfDNA molecule. These adapters contain non-unique molecular barcodes, and each fraction is ligated with an adapter having a non-unique molecular barcode that is distinguishable from the barcodes in the adapters used in other fractions. After ligation, the four fractions are pooled together and amplified by PCR.

[0433] After PCR, the amplified DNA is washed and concentrated before enrichment. Once concentrated, the amplified DNA is combined with a salt buffer and biotinylated RNA probes containing probes for a set of sequence variable target regions and probes for epigenetic target regions, and the mixture is incubated overnight. The probes for the set of sequence variable target regions have a footprint of approximately 50 kb, and the probes for the epigenetic target regions have a footprint of approximately 500 kb. The probes for the set of sequence variable target regions include oligonucleotides targeting at least a subset of the genes identified in Tables 3-5, and the probes for the set of epigenetic target regions include oligonucleotides targeting selected from hypermethylated variable target regions, hypomethylated variable target regions, CTCF binding target regions, transcription start site target regions, local amplification target regions, and methylation control regions.

[0434] Biotinylated RNA probes (hybridized to DNA) are captured by streptavidin magnetic beads and separated from uncaptured 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 and deconvolute the sample into differentially distributed molecules. The method described in this example not only provides information about the overall methylation level of molecules based on that fraction (i.e., methylated cytosine residues), but can also provide higher-resolution information about the identity and / or location of the type of methylated cytosine (i.e., mC or hmC) based on further partitioning of the highly methylated fraction to yield the hmC fraction. The sequences of sequence-variable target regions are analyzed to detect genomic alterations, such as SNVs, insertions, deletions, and fusions, that can be called with sufficient confidence to distinguish true tumor variants from technical errors (e.g., PCR errors, sequencing errors). The sequences of epigenetic target regions are then independently analyzed to detect methylated cfDNA molecules in regions shown to be differentially methylated in cancer compared to normal cells. Finally, the results of both analyses are combined to generate a final tumor presence / absence call.

[0435] Example 2: Analysis of methylation at single-base resolution in cfDNA samples from healthy subjects and subjects with early-stage colorectal cancer cfDNA samples from healthy subjects and subjects with early-stage colorectal cancer were analyzed as follows. cfDNA was partitioned using MBD to provide hypermethylated, intermediate, and hypomethylated fractions. The partitioned DNA from each fraction was ligated with adapters (the adapters contain methylated cytosines to protect against EM-seq conversion and maintain compatibility with NGS library preparation) and subjected to an EM-seq conversion procedure, whereby unmodified cytosines undergo deamination but mC and hmC do not. After such deamination, the fractions were prepared for sequencing and subjected to whole-genome sequencing. While each fraction was sequenced separately, in an alternative procedure, fractions can be differentially tagged (e.g., after partitioning but before EM-seq conversion, or after partitioning and EM-seq conversion but before further preparation for sequencing), pooled, processed, and sequenced in parallel.

[0436] Sequence data from hypermethylated variable target regions were isolated bioinformatically; alternative procedures allow target regions to be enriched in vitro prior to sequencing. Per-base methylation of hypermethylated variable target regions was quantified as shown in Figure 5, which shows the number of methylated CpGs per molecule in hypermethylated variable target regions from the hypermethylated fraction. The x-axis indicates the total number of CpGs per molecule, and points along the diagonal represent molecules with per-CpG methylation. In this way, it was possible to analyze methylation at single-base resolution and quantify per-base and fractional molecule methylation in MBD-distributed material. Samples from subjects with colorectal cancer showed significantly higher overall methylation in these regions than samples from healthy subjects. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for analyzing DNA in a sample, comprising: a) partitioning the sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample contains a higher proportion of DNA with cytosine modifications than the second sub-sample; b) subjecting said first sub-sample to a procedure that affects a first nucleobase in DNA differently from a second nucleobase in the DNA of said first sub-sample, wherein said first nucleobase is a modified or unmodified nucleobase, said second nucleobase is a modified or unmodified nucleobase that is different from said first nucleobase, and said first nucleobase and and said second nucleobase have the same base-pairing specificity; and c) sequencing the DNA in said first sub-sample and the DNA in said second sub-sample so as to distinguish said first nucleobase from said second nucleobase in the DNA of said first sub-sample. A method comprising: (Item 2) 2. The method of claim 1, wherein the DNA comprises cell-free DNA (cfDNA) obtained from the test subject. (Item 3) 1. A method for analyzing DNA in a sample containing cell-free DNA (cfDNA) obtained from a test subject in the sample, comprising: a) partitioning the sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample contains a higher proportion of DNA with cytosine modifications than the second sub-sample; b) subjecting said first sub-sample to a procedure that affects a first nucleobase in DNA differently from a second nucleobase in the DNA of said first sub-sample, wherein said first nucleobase is a modified or unmodified nucleobase, said second nucleobase is a modified or unmodified nucleobase that is different from said first nucleobase, and said first nucleobase and said second nucleobase have the same base-pairing specificity; and c) sequencing the DNA in at least said first sub-sample so as to distinguish said first nucleobase from said second nucleobase in the DNA of said first sub-sample; A method comprising: (Item 4) 4. The method of claim 3, wherein step c) comprises sequencing DNA in at least the first sub-sample. (Item 5) 1. A method for analyzing a sample containing cell-free DNA (cfDNA), comprising: a) partitioning the sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample comprises cfDNA with a higher proportion of cytosine modifications than the second sub-sample; b) subjecting the first sub-sample to a procedure that affects a first nucleobase in the cfDNA differently from a second nucleobase in the cfDNA of the first sub-sample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base-pairing specificity; c) capturing at least a set of epigenetic target regions of cfDNA from said first sub-sample and said second sub-sample, thereby providing captured cfDNA; and d) sequencing the captured cfDNA to distinguish the first nucleobase from the second nucleobase in the cfDNA of the first sub-sample. (Item 6) 1. A method for analyzing a sample containing cell-free DNA (cfDNA), comprising: a) partitioning the sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample comprises cfDNA with a higher proportion of cytosine modifications than the second sub-sample; b) subjecting said first sub-sample to a procedure that affects a first nucleobase in said cfDNA differently from a second nucleobase in said cfDNA of said first sub-sample, wherein said first nucleobase is a modified or unmodified nucleobase and said second nucleobase is a modified or unmodified nucleobase that is different from said first nucleobase; wherein the one nucleobase and the second nucleobase have the same base-pairing specificity; c) capturing a set of multiple target regions of cfDNA from said first sub-sample and said second sub-sample, thereby providing captured cfDNA, the plurality of sets of target regions comprises a set of sequence variable target regions and a set of epigenetic target regions; and d) sequencing the captured cfDNA to distinguish the first nucleobase from the second nucleobase in the captured cfDNA of the first sub-sample. (Item 7) 7. The method of claim 6, wherein cfDNA molecules corresponding to the set of sequence variable target regions are captured in the sample with a higher capture yield than cfDNA molecules corresponding to the set of epigenetic target regions. (Item 8) 1. A method for isolating cell-free DNA (cfDNA) from a sample, comprising: a) partitioning the sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample comprises cfDNA with a higher proportion of cytosine modifications than the second sub-sample; b) subjecting the first sub-sample to a procedure that affects a first nucleobase in the cfDNA differently from a second nucleobase in the cfDNA of the first sub-sample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base-pairing specificity; c) contacting the cfDNA of the first sub-sample and the second sub-sample with a target-specific probe set; the target-specific probe set comprises target-binding probes specific for a sequence-variable target set and target-binding probes specific for an epigenetic target set; thereby forming a complex between the target-specific probe and the cfDNA; separating the complexes from cfDNA that did not bind to the target-specific probes, thereby providing captured cfDNA corresponding to the sequence-variable target set and cfDNA corresponding to the epigenetic target set; and d) sequencing the captured cfDNA to distinguish the first nucleobase from the second nucleobase in the cfDNA of the first sub-sample. A method comprising: (Item 9) 9. The method of claim 8, wherein the target-specific probe set is configured to capture cfDNA corresponding to the sequence-variable target set with a higher capture yield than cfDNA corresponding to the epigenetic target set. (Item 10) 1. A method for identifying the presence of DNA produced by a tumor, comprising: a) collecting a cfDNA sample from a test subject; b) partitioning the cfDNA sample into a plurality of sub-samples, including a first sub-sample and a second sub-sample, wherein the first sub-sample comprises captured cfDNA with a higher proportion of cytosine modifications than the second sub-sample; c) subjecting the first sub-sample to a procedure that affects a first nucleobase in the cfDNA differently from a second nucleobase in the cfDNA of the first sub-sample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base-pairing specificity; d) capturing a set of multiple target regions from the cfDNA of the first sub-sample and the second sub-sample, thereby providing a sample comprising captured cfDNA. There was, the plurality of sets of target regions comprises a set of sequence variable target regions and a set of epigenetic target regions; and e) sequencing the captured cfDNA in the first sub-sample and the captured cfDNA in the second sub-sample to distinguish the first nucleobase from the second nucleobase in the cfDNA of the first sub-sample. A method comprising: (Item 11) 11. The method of claim 10, wherein cfDNA molecules corresponding to the set of sequence variable target regions are captured in the sample with a higher capture yield than cfDNA molecules corresponding to the set of epigenetic target regions. (Item 12) 12. The method of any one of items 6 to 11, comprising sequencing the cfDNA molecules corresponding to the set of sequence variable target regions to a higher sequencing depth than the cfDNA molecules corresponding to the set of epigenetic target regions. (Item 13) 13. The method of claim 12, wherein the captured cfDNA molecules of the sequence-variable target set are sequenced to a sequencing depth that is at least two times higher than the captured cfDNA molecules of the epigenetic target region set. (Item 14) 13. The method of claim 12, wherein the captured cfDNA molecules of the sequence-variable target set are sequenced to a sequencing depth that is at least three times higher than the captured cfDNA molecules of the epigenetic target region set. (Item 15) 13. The method of claim 12, wherein the captured cfDNA molecules of the sequence-variable target set are sequenced to a sequencing depth that is 4 to 10 times higher than the captured cfDNA molecules of the epigenetic target region set. (Item 16) 13. The method of claim 12, wherein the captured cfDNA molecules of the sequence-variable target set are sequenced to a sequencing depth that is 4 to 100 times higher than the captured cfDNA molecules of the epigenetic target region set. (Item 17) 17. The method of any one of items 6 to 16, wherein the captured cfDNA molecules of the sequence-variable target set and the captured cfDNA molecules of the epigenetic target region set are sequenced in the same sequencing cell. (Item 18) 10. The method of any one of the preceding items, wherein the DNA is amplified prior to sequencing, or wherein the method comprises a capture step and the DNA is amplified prior to the capture step. (Item 19) 19. The method of items 5 to 18, further comprising ligating a barcode-containing adapter to the DNA prior to capture, optionally wherein the ligation step occurs before or simultaneously with amplification. (Item 20) 20. The method of any one of items 5 to 19, wherein the set of epigenetic target regions comprises a set of hypermethylated variable target regions. (Item 21) 21. The method of any one of items 5 to 20, wherein the set of epigenetic target regions comprises a set of hypomethylated variable target regions. (Item 22) 22. The method of claim 20 or 21, wherein the set of epigenetic target regions comprises a set of methylation control target regions. (Item 23) 23. The method of any one of items 5 to 22, wherein the set of epigenetic target regions comprises a set of fragmented variable target regions. (Item 24) 24. The method of claim 23, wherein the set of fragmented variable target regions comprises a transcription start site region. (Item 25) 25. The method of claim 23 or 24, wherein the set of fragmented variable target regions comprises a CTCF binding region. (Item 26) 26. The method of any one of items 5 to 25, wherein capturing the set of multiple target regions of cfDNA comprises contacting the cfDNA with target binding probes specific for the set of sequence variable target regions and target binding probes specific for the set of epigenetic target regions. (Item 27) 27. The method of claim 26, wherein target binding probes specific for the set of sequence variable target regions are present in a higher concentration than target binding probes specific for the set of epigenetic target regions. (Item 28) 27. The method of claim 26, wherein the target binding probes specific for the set of sequence variable target regions are present at a concentration at least two-fold higher than the target binding probes specific for the set of epigenetic target regions. (Item 29) 27. The method of claim 26, wherein the target binding probes specific for the set of sequence variable target regions are present at a concentration at least 4-fold or 5-fold greater than the target binding probes specific for the set of epigenetic target regions. (Item 30) 27. The method of claim 26, wherein the target binding probes specific for the set of sequence variable target regions are present at a concentration that is at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than the target binding probes specific for the set of epigenetic target regions, or the target binding probes specific for the set of sequence variable target regions are present at a concentration in the range of 2 to 3, 3 to 4, 4 to 5, 5 to 7, 7 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 times greater than the concentration of the target binding probes specific for the set of epigenetic target regions. (Item 31) 31. The method of any one of items 26 to 30, wherein target binding probes specific for the set of sequence variable target regions have higher target binding affinity than target binding probes specific for the set of epigenetic target regions. (Item 32) 33. The method of any one of items 6 to 32, wherein the footprint of the set of epigenetic target regions is at least two times larger than the size of the set of sequence variable target regions. (Item 33) 33. The method of claim 32, wherein the footprint of the set of epigenetic target regions is at least 10 times larger than the size of the set of sequence variable target regions. (Item 34) 34. The method of any one of items 6 to 33, wherein the footprint of the set of sequence variable target regions is at least 25 kb or 50 kb. (Item 35) 10. The method of any one of the preceding items, wherein (a) partitioning the sample into a plurality of sub-samples comprises partitioning based on methylation levels; and / or (b) partitioning the sample into a plurality of sub-samples comprises partitioning based on binding to proteins, optionally wherein the proteins are methylated proteins, acetylated proteins, unmethylated proteins, unacetylated proteins; and / or optionally wherein the proteins are histones. (Item 36) 36. The method of claim 35, wherein (a) the distributing step comprises contacting the collected cfDNA with a methyl-binding reagent immobilized on a solid support; and / or (b) the distributing step comprises contacting the collected cfDNA with a binding reagent specific for the protein, the binding reagent being immobilized on a solid support. (Item 37) 10. The method of any one of the preceding items, comprising differentially tagging the first sub-sample and the second sub-sample. (Item 38) 38. The method of claim 37, wherein the first sub-sample and the second sub-sample are differentially tagged before subjecting the first sub-sample to a procedure that affects the first nucleobase in DNA differently than the second nucleobase in the DNA of the first sub-sample. (Item 39) 39. The method of claim 37 or 38, wherein the first sub-sample and the second sub-sample are pooled after subjecting the first sub-sample to a procedure that affects the first nucleobase in DNA differently than the second nucleobase in the DNA of the first sub-sample. (Item 40) 40. The method of any one of items 37 to 39, wherein the first sub-sample and the second sub-sample are sequenced in the same sequencing cell. (Item 41) 2. The method of any one of the preceding items, wherein the plurality of sub-samples comprises a third sub-sample comprising a higher proportion of DNA with cytosine modifications than the second sub-sample but a lower proportion than the first sub-sample. (Item 42) 42. The method of claim 41, further comprising differentially tagging the third sub-sample such that the first sub-sample and the second sub-sample are distinguishable. (Item 43) 43. The method of claim 42, wherein after subjecting the first sub-sample to a procedure that affects a first nucleobase in DNA differently from the second nucleobase in the DNA of the first sub-sample, the first sub-sample, the second sub-sample, and the third sub-sample are combined, and optionally, the first sub-sample, the second sub-sample, and the third sub-sample are sequenced in the same sequencing cell. (Item 44) 10. The method of any one of the preceding items, wherein the procedure by which the first sub-sample is subjected alters the base-pairing specificity of the first nucleobase without substantially altering the base-pairing specificity of the second nucleobase. (Item 45) The method of any one of the preceding items, wherein the first nucleobase is a modified or unmodified cytosine and the second nucleobase is a modified or unmodified cytosine. (Item 46) The method of any one of the preceding items, wherein the first nucleobase comprises an unmodified cytosine (C). (Item 47) Any one of the preceding items, wherein the second nucleobase comprises 5-methylcytosine (mC). The method described in paragraph . (Item 48) 3. The method of any one of the preceding items, wherein the procedure to which the first sub-sample is subjected comprises bisulfite conversion. (Item 49) 47. The method of any one of items 1 to 46, wherein said first nucleobase comprises mC. (Item 50) The method of any one of the preceding items, wherein the second nucleobase comprises 5-hydroxymethylcytosine (hmC). (Item 51) 51. The method of item 50, wherein the procedure to which the first sub-sample is subjected comprises protection of 5hmC. (Item 52) 51. The method of claim 50, wherein the procedure to which the first sub-sample is subjected comprises Tet-assisted bisulfite conversion. (Item 53) 51. The method of claim 50, wherein the procedure to which the first sub-sample is subjected comprises a Tet-assisted conversion with a substituted borane reducing agent, optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. (Item 54) 54. The method of claim 53, wherein the substituted borane reducing agent is 2-picoline borane or borane pyridine. (Item 55) 55. The method of any one of items 49 to 51 or 53 to 54, wherein the second nucleobase comprises C. (Item 56) 56. The method of any one of items 49 to 51, or 55, wherein the procedure to which the first sub-sample is subjected comprises protection of hmC followed by Tet-assisted conversion, optionally with a substituted borane reducing agent, which is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. (Item 57) 57. The method of claim 56, wherein the substituted borane reducing agent is 2-picoline borane or borane pyridine. (Item 58) 56. The method of any one of items 46, 47, 49 to 51, or 55, wherein the procedure to which the first sub-sample is subjected comprises protection of hmC, followed by deamination of mC and / or C. (Item 59) 59. The method of item 58, wherein the deamination of mC and / or C comprises treatment with an AID / APOBEC family DNA deaminase enzyme. (Item 60) 60. The method of any one of items 51 or 55 to 59, wherein the protection of hmC comprises glycosylation of hmC. (Item 61) 56. The method of any one of items 1 to 45, 47, 49, or 55, wherein the procedure to which the first sub-sample is subjected comprises chemically assisted conversion with a substituted borane reducing agent, optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. (Item 62) The substituted borane reducing agent is 2-picoline borane or borane pyridine. 61. The method according to claim 61. (Item 63) 63. The method of any one of items 1 to 45, 47, 49, 55, or 61 to 62, wherein said first nucleobase comprises hmC. (Item 64) 45. The method of any one of items 1 to 44, wherein the procedure in which the first sub-sample is provided comprises a step of separating DNA that naturally contains the first nucleobase from DNA that does not naturally contain the first nucleobase. (Item 65) Item 65. The method of item 64, wherein the first nucleobase is hmC. (Item 66) 66. The method of claim 64 or 65, wherein the step of separating DNA that naturally contains the first nucleobase from DNA that does not naturally contain the first nucleobase comprises the step of labeling the first nucleobase. (Item 67) Item 67. The method of item 66, wherein the labeling step comprises biotinylation. (Item 68) 68. The method of claim 66 or 67, wherein the labeling step comprises glycosylation. (Item 69) Item 69. The method of item 68, wherein the glycosylating step attaches a glucosyl-azide moiety. (Item 70) 69. The method of claim 66 or 68, wherein the labeling step comprises a glucosylating step before biotinylating, followed by attaching a biotin moiety to the glucosyl. (Item 71) 71. The method of claim 70, wherein the step of attaching the biotin moiety to the glucosyl comprises Huisgen cycloaddition chemistry. (Item 72) 62. The method of any one of items 54 to 61, wherein separating DNA that naturally contains the first nucleobase from DNA that does not naturally contain the first nucleobase comprises binding DNA that naturally contains the first nucleobase to a capture agent. (Item 73) 73. The method of claim 72, wherein the capture agent comprises a biotin-binding agent, optionally wherein the biotin-binding agent comprises avidin or streptavidin. (Item 74) 74. The method of any one of items 64 to 73, comprising differentially tagging each of the DNA that naturally comprises the first nucleobase, the DNA that does not naturally comprise the first nucleobase, and the DNA of the second sub-sample. (Item 75) 75. The method of claim 74, comprising differentially tagging and then pooling the DNA that originally comprises the first nucleobase, the DNA that does not originally comprise the first nucleobase, and the DNA of the second sub-sample, wherein, optionally, the DNA that originally comprises the first nucleobase, the DNA that does not originally comprise the first nucleobase, and the DNA of the second sub-sample are sequenced in the same sequencing cell. (Item 76) 45. The method of any one of items 1 to 44, wherein the first nucleobase is a modified or unmodified adenine and the second nucleobase is a modified or unmodified adenine. (Item 77) 45. The method of any one of items 1 to 44, wherein said first nucleobase is modified or unmodified guanine and said second nucleobase is modified or unmodified guanine. (Item 78) 45. The method of any one of items 1 to 44, wherein said first nucleobase is modified or unmodified thymine and said second nucleobase is modified or unmodified thymine. (Item 79) The method of any one of the preceding items, wherein the second subpopulation has not been subjected to a procedure that affects said first nucleobases differently than said second nucleobases. (Item 80) 1. A combination comprising a first population and a second population of captured DNA, wherein the first population comprises or is derived from DNA having a higher proportion of cytosine modifications than the second population, the first population comprises a first nucleobase type originally present in DNA with altered base-pairing specificity and a second nucleobase that does not have the altered base-pairing specificity, the first nucleobase type originally present in DNA before the base-pairing specificity is altered or unaltered nucleobase, the second nucleobase is a modified nucleobase or unaltered nucleobase different from the first nucleobase, the first nucleobase type originally present in DNA before the base-pairing specificity is altered and the second nucleobase have the same base-pairing specificity, and the second population does not comprise the first nucleobase type originally present in DNA with the altered base-pairing specificity. (Item 81) 81. The combination of item 80, wherein the first population comprises sequence tags selected from a first set of one or more sequence tags, and the second population comprises sequence tags selected from a second set of one or more sequence tags, wherein the second set of sequence tags is different from the first set of sequence tags. (Item 82) 82. The combination of item 81, wherein the sequence tag comprises a barcode. (Item 83) 83. The combination according to any one of items 80 to 82, wherein the cytosine modification is methylation. (Item 84) 84. The combination of any one of items 80 to 83, wherein the first nucleobase is a modified or unmodified cytosine and the second nucleobase is a modified or unmodified cytosine. (Item 85) 85. The combination of any one of items 80 to 84, wherein the first nucleobase comprises an unmodified cytosine (C). (Item 86) 86. The combination of any one of items 80 to 85, wherein the second nucleobase comprises one or both of 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC). (Item 87) 87. The combination of any one of items 80 to 86, wherein the first population has been subjected to bisulfite conversion. (Item 88) 87. The combination according to any one of items 80 to 86, wherein the first nucleobase comprises mC. (Item 89) 89. The combination of any one of items 80 to 88, wherein the second nucleobase comprises hmC. (Item 90) 90. The combination of any one of items 80 to 89, wherein the first population comprises protected hmC. (Item 91) 91. The combination of item 84 or 90, wherein the first population has been subjected to Tet-assisted bisulfite conversion. (Item 92) 91. The combination of item 84 or 90, wherein the first population has been subjected to a Tet-assisted conversion with a substituted borane reducing agent that is optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. (Item 93) Item 91. The combination of item 90, wherein the first population has been subjected to protection of hmC followed by Tet-assisted conversion, optionally with a substituted borane reducing agent, which is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. (Item 94) 94. The combination of any one of items 80 to 82, 86, 88 to 90, or 92 to 93, wherein the second nucleobase comprises C. (Item 95) 91. The combination of item 90, wherein the first population has been subjected to protection of hmC and subsequent deamination of mC and / or C. (Item 96) 96. The combination of any one of items 90 to 95, wherein the protected hmC comprises glycosylated hmC. (Item 97) 84. The combination of any one of items 80 to 83, wherein the first nucleobase comprises hmC. (Item 98) 98. The combination of any one of items 80 to 83, or 97, wherein the second nucleobase comprises mC. (Item 99) 99. The combination of any one of items 80 to 83 or 97 to 98, wherein the second nucleobase comprises C. (Item 100) 99. The combination of any one of items 80 to 83 or 97 to 99, wherein the first population has been subjected to chemical-assisted conversion with a substituted borane reducing agent, optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. (Item 101) 84. The combination of any one of items 80 to 83, wherein the first nucleobase is a modified or unmodified adenine and the second nucleobase is a modified or unmodified adenine. (Item 102) 84. The combination of any one of items 80 to 83, wherein the first nucleobase is modified or unmodified guanine and the second nucleobase is modified or unmodified guanine. (Item 103) 84. The combination of any one of items 80 to 83, wherein the first nucleobase is modified or unmodified thymine and the second nucleobase is modified or unmodified thymine. (Item 104) a combination comprising a first population and a second population of captured DNA, the first population comprises or is derived from DNA with a higher proportion of cytosine modifications than the second population; the first population comprises a first subpopulation and a second subpopulation; said first subpopulation comprising a higher proportion of the first nucleobase than said second subpopulation; A combination wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase that is different from the first nucleobase, the first nucleobase and the second nucleobase have the same base-pairing specificity, and the second population does not include the first nucleobase. (Item 105) 105. The combination of item 104, wherein the first nucleobase is a modified or unmodified cytosine and the second nucleobase is a modified or unmodified cytosine. (Item 106) 106. The combination of item 105, wherein the first nucleobase is a protected modified cytosine. (Item 107) 107. The combination according to item 105 or 106, wherein the first nucleobase is a derivative of hmC. (Item 108) 108. The combination of item 107, wherein the first nucleobase is glycosylated hmC. (Item 109) 109. The combination according t...

Claims

[Claim 1] The invention described in the specification.

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