Tissue-informed detection / monitoring of biologically linked plasma biomarkers

Abstract

The methods disclosed herein are related to tissue-informed MRD / longitudinal monitoring assays, tracking biologically-linked biomarkers in blood.

Description

Attorney Ref. No. GH0271WOTISSUE-INFORMED DETECTION / MONITORING OF BIOLOGICALLY LINKED PLASMA BIOMARKERS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application No.63 / 755,973, filed February 7, 2025, which is incorporated by reference herein in its entirety for all purposes.FIELD OF THE INVENTION

[0002] This disclosure relates to methods of monitoring a subject for minimal residual disease (MRD) by tracking biologically-linked biomarkers in blood.BACKGROUND AND SUMMARY

[0003] Cancer is responsible for millions of deaths per year worldwide. Early cancer detection may result in improved outcomes because early-stage cancer tends to be more susceptible to treatment.

[0004] Improperly controlled cell growth is a hallmark of cancer. Cancer is usually caused by the accumulation of mutations within an individual's normal cells, at least some resulting in improperly regulated cell division. Such mutations commonly include single nucleotide variations (SNVs), gene fusions, insertions and deletions (indels), transversions, translocations, copy number variations (CNVs) and inversions. Cancers may also exhibit an accumulation of epigenetic changes, including modification of cytosine (e.g., 5-methylcytosine, 5-hydroxymethylcytosine, and other more oxidized forms) and association of DNA with chromatin proteins and transcription factors. Thus, cancer can be indicated by sequence and non-sequence modifications, such as methylation. Examples of methylation changes in cancer include local gains of DNA methylation, e.g., in the CpG islands at the transcription start sites of genes involved in normal growth control, DNA repair, cell cycle regulation, and / or cell differentiation. Hypermethylation can be associated with an aberrant loss of transcriptional capacity of involved genes and occurs at least as frequently as point mutations and deletions as a cause of altered gene expression. Tumor associated somatic biomarkers can also include messenger RNA (mRNA), microRNA (miRNA), non-coding RNA, protein, and protein fragments. Furthermore, without wishing to be bound by any particular theory,Attorney Ref. No. GH0271WOcells in or around a cancer or neoplasm may shed more DNA than cells of the same tissue type in a healthy subject. The DNA from such cells may differ epigenetically from shed DNA in a healthy subject. As such, the distribution of epigenetically modified (e.g., methylated) DNA in certain DNA samples, such as cell-free DNA(cfDNA), may change upon carcinogenesis.

[0005] Biopsies represent a traditional approach for detecting or diagnosing cancer in which cells or tissue are extracted from a possible cancer site and analyzed for relevant phenotypic and / or genotypic features. Biopsies have the drawback of being invasive. Cancer detection based on analysis of body fluids (“liquid biopsies”), such as blood, is an intriguing alternative based on the observation that DNA from cancer cells is released into body fluids. A liquid biopsy is noninvasive (sometimes requiring only a blood draw). However, it has been challenging to develop accurate and sensitive methods for analyzing liquid biopsy material in part because the amount of nucleic acids released into body fluids is low and variable, as is recovery of nucleic acids from such fluids in analyzable form. Further, the contribution of DNA from cells in or around a cancer or neoplasm to a sample may be relatively small relative to the contribution from other cells, and the DNA contributed from other cells may be uninformative as to cancer status. Isolating and processing cell-free DNA useful for further analysis in liquid biopsy procedures can be a useful part of these methods.

[0006] Current methods of cancer diagnostic assays of cell-free nucleic acids (e.g., cell-free DNA or cell-free RNA) may focus on the detection of tumor-related somatic variants, including single nucleotide variants (SNVs), copy number variations (CNVs), fusions, and indels (i.e., insertions or deletions), which are all mainstream targets for liquid biopsy. However, detection of somatic variants can be costly and / or labor-intensive given the low abundance of such variants in many samples and the need to distinguish them from germline variants. While detection can be facilitated by identifying and enriching genomic regions known to contain somatic variants in tumor DNA, validation of probes used for enrichment can still consume significant resources and effort.

[0007] Accordingly, there is a continued need for improved methods and compositions for detecting somatic variants in DNA, including cell-free DNA, e.g., in liquid biopsies.

[0008] Personalized, tissue-informed biomarkers have shown promise for improving diagnostic performance in cfDNA-based minimal residual disease (MRD) assays. A fundamental limitation in achievable sensitivity is that detection is limited by tumor shedding rate (cell apoptosis), and one tumor cell presents only one tumor-derived DNA molecule (per biomarker region).Attorney Ref. No. GH0271WO

[0009] Relative to cfDNA, other types of circulating (in blood) cancer biomarkers may have amplified signal and not dependent on tumor shedding rate (i.e. circulating proteins, exosomes). These non-DNA markers are noisy and thus are predicted to benefit from a 'tumor-informed' method, but none exist This is likely because directly measuring the circulating biomarkers in tissue (especially FFPE) is either very challenging (e.g. circulating protein) or non-existent (e g. FFPE exosomal surface proteins).

[0010] The methods disclosed herein are related to tissue-informed MRD / longitudinal monitoring assays, tracking biologically-linked biomarkers in blood. These biologically-linked be geneproducts (e.g. LINE1 ORlFp protein) associated with tissue biomarker (e.g. LINE1 DNA hypomethylation) or altered phenotypes (e.g. methylation patterns) associated with tissue detected biomarker. The biologically-linked biomarkers can be combined with any other type of biomarker in standard MRD tests. Assay could be 'tissue-enhanced' by using a fixed panel for both tissue and cfDNA samples or 'tissue-informed' by using custom panel for the cfDNA sample that is specific to the subject.

[0011] Provided herein is a method for monitoring a subject for minimal residual disease, comprising: a) obtaining a first sample from a subject; wherein the first sample comprises a tumor tissue sample; b) profiling nucleic acids obtained from the first sample to identify biologically-linked plasma biomarkers; c) identifying one or more biologically-linked plasma biomarkers specific to the subject; d) determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject; and e) classifying the subject to be positive or negative for minimal residual disease based on the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in the second sample. In some embodiments, the methods provided herein further comprise obtaining a third sample from the subject, wherein the third sample is a matched normal tissue sample and / or a buffy coat sample. In some embodiments, the third sample is a buffy coat sample. In some embodiments, the methods provided herein further comprise profiling nucleic acids obtained from the third sample. In some embodiments, the methods provided herein further comprise, prior to step d), removing variants detected from profiling nucleic acids in the third sample from the one or more biologically-linked plasma biomarkers identified as specific to the subject.

[0012] In an aspect, a method for monitoring a subject for minimal residual disease, comprises: (a) obtaining a first sample from a subject; wherein the first sample comprises tumor tissue sample;Attorney Ref. No. GH0271WO(b) profiling nucleic acids obtained from the first sample to identify biologically-linked plasma biomarker; (c) identifying one or more biologically-linked plasma biomarkers specific to the subject; (d) determining presence / absence of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject; and (e) classifying the subject to be positive / negative for the minimal residual disease based on the presence / absence of the one or more biologically-linked plasma biomarkers in the second sample.

[0013] In some embodiments, wherein the first sample further comprises a matched normal tissue sample and / or buffy coat sample. In some embodiments, the method further comprises profiling nucleic acids obtained from the matched normal tissue sample and / or buffy coat sample. In some embodiments, the method further comprises, prior to step d), removing variants detected from profiling nucleic acids in the matched normal sample tissue and / or buffy coat sample from the one or more biologically-linked plasma biomarkers identified specific to the subject. In some embodiments, wherein the second sample is a cell-free DNA sample. In some embodiments, wherein determining the presence / absence of the one or more biologically-linked plasma biomarkers in the second sample comprises enriching nucleic acids using a custom panel specific to the subject. In some embodiments, wherein markers for profiling nucleic acids in the first sample comprises somatic variants, DNA methylation and / or RNA expression / transcriptomics. In some embodiments, wherein the biologically-linked plasma biomarkers comprises DNA methylation (for e.g., hypermethylated phenotype), cfRNA, EV-surface protein and / or circulating protein.

[0014] Accordingly, the embodiments described herein are provided, which include, but are not limited to the following:

[0015] Embodiment lis a method for monitoring a subject for minimal residual disease, comprising: a) obtaining a first sample from a subject; wherein the first sample comprises a tumor tissue sample; b) profiling nucleic acids obtained from the first sample to identify biologically-linked plasma biomarkers; c) identifying one or more biologically-linked plasma biomarkers specific to the subject; d) determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject; and e) classifying the subject to be positive or negative for minimal residual disease based on the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in the second sample.Attorney Ref. No. GH0271WO

[0016] Embodiment 2is the method of embodiment 1, further comprising obtaining a third sample from the subject, wherein the third sample is a matched normal tissue sample and / or a buffy coat sample.

[0017] Embodiment 3 is the method of the immediately preceding embodiment, wherein the third sample is a buffy coat sample.

[0018] Embodiment 4 is the method of the immediately preceding embodiment, further comprising profiling nucleic acids obtained from the third sample.

[0019] Embodiment 5 is the method of any one of embodiments 2-4, further comprising, prior to step d), removing variants detected from profiling nucleic acids in the third sample from the one or more biologically-linked plasma biomarkers identified as specific to the subject.

[0020] Embodiment 6is the method of any one of the preceding embodiments, wherein the second sample is a cell-free DNA sample.

[0021] Embodiment 7is the method of the immediately preceding embodiment, wherein the second sample is a plasma sample.

[0022] Embodiment 8is the method of any one of the preceding embodiments, wherein the profiling nucleic acids obtained from the first sample and / or the determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample further comprises a nucleic acid amplification step.

[0023] Embodiment 9is the method of any one of the preceding embodiments, wherein the profiling nucleic acids obtained from the first sample comprises sequencing nucleic acids from the first sample.

[0024] Embodiment 10 is the method of any one of the preceding embodiments, wherein the determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject comprises sequencing nucleic acids from the second sample and / or detecting one or more nucleic acids and / or proteins present in the second sample.

[0025] Embodiment 11 is the method of the immediately preceding embodiment, wherein the detecting comprises high multiplex PCR, digital PCR, quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), or sequencing.

[0026] Embodiment 12 is the method of any one of embodiments 9-11, wherein the sequencing comprises next generation sequencing.Attorney Ref. No. GH0271WO

[0027] Embodiment 13 is the method of any one of embodiments 9-11, wherein the sequencing comprises long-read sequencing.

[0028] Embodiment 14 is the method of any one of embodiments 9-11, wherein the sequencing comprises nanopore sequencing.

[0029] Embodiment 15 is the method of any one of embodiments 9-11, wherein the sequencing comprises 5-letter or 6-letter sequencing.

[0030] Embodiment 16 is the method of any one of embodiments 9-11, wherein the sequencing comprises sequencing the DNA in a manner that distinguishes the first nucleobase from the second nucleobase.

[0031] Embodiment 17 is the method of embodiment 10, wherein the detecting one or more proteins present in the second sample comprises mass spectrometry (MS).

[0032] Embodiment 18 is the method of any one of the preceding embodiments, wherein determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in the second sample comprises enriching biologically-linked plasma biomarkers in the second sample using a custom panel of capture reagents specific to the subject.

[0033] Embodiment 19 is the method of the immediately preceding embodiment, wherein the custom panel of capture reagents are configured to capture nucleic acids and / or proteins comprising the biologically-linked plasma biomarkers.

[0034] Embodiment 20 is the method of the immediately preceding embodiment, wherein the custom panel of capture reagents comprises at least 5, 7, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 capture reagents.

[0035] Embodiment 21 is the method of any one of embodiments 19-20, wherein the custom panel of capture reagents comprise a plurality of capture reagents, wherein the plurality of capture reagents comprise one or more DNA capture probes, one or more RNA capture probes, and / or one or more protein capture reagents.

[0036] Embodiment 22 is the method of the immediately preceding embodiment, wherein the one or more protein capture reagents is selected from antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp.

[0037] Embodiment 23 is the method of the immediately preceding embodiment, wherein the one or more protein capture reagents comprises an antibody.Attorney Ref. No. GH0271WO

[0038] Embodiment 24 is the method of any one of embodiments 21-23, wherein the one or more protein capture reagents comprise an oligonucleotide-capture reagent conjugate.

[0039] Embodiment 25 is the method of the immediately preceding embodiment, wherein the oligonucleotide-capture reagent conjugate comprises a molecular tag.

[0040] Embodiment 26 is the method of the immediately preceding embodiment, wherein the molecular tag is a molecular barcode.

[0041] Embodiment 27 is the method of any one of the preceding embodiments, wherein markers comprising somatic variants, DNA methylation and / or RNA expression markers are identified by profiling the nucleic acids obtained from the first sample.

[0042] Embodiment 28 is the method of any one of the preceding embodiments, wherein the biologically-linked plasma biomarkers comprise differentially methylated DNA, cell-free RNA (cfRNA), extracellular vesicle (EV)-surface protein, and / or circulating protein.

[0043] Embodiment 29 is the method of any one of the preceding embodiments, wherein markers comprising somatic variants and / or epigenetic variants are identified by profiling the nucleic acids obtained from the first sample.

[0044] Embodiment 30 is the method of the immediately preceding embodiment, wherein the biologically-linked plasma biomarkers comprise cell-free RNA (cfRNA), extracellular vesicle (EV)-surface protein, and / or circulating protein.

[0045] Embodiment 31 is the method of any one of the preceding embodiments, wherein markers comprising RNA expression markers are identified by profiling the nucleic acids obtained from the first sample.

[0046] Embodiment 32 is the method of the immediately preceding embodiment, wherein the biologically-linked plasma biomarkers comprise differentially methylated DNA, extracellular vesicle (EV)-surface protein, and / or circulating protein.

[0047] Embodiment 33 is the method of any one of embodiments 27-32, wherein the biologically-linked plasma biomarkers comprise one or more biomarkers resulting from the markers identified by profiling the nucleic acids obtained from the first sample.

[0048] Embodiment 34 is the method of any one of embodiments 27-33, wherein nucleic acid data is obtained from the first sample comprising hypomethylated DNA and the biologically-linked plasma biomarkers comprise cfRNA and / or circulating protein corresponding to one or more hypomethylated DNA regions.Attorney Ref. No. GH0271WO

[0049] Embodiment 34.1 is the method of any one of embodiments 27-34, wherein the hypomethylated DNA comprises hypomethylated LINE1 elements, the cfRNA comprises LINE1 cfRNA, and / or the circulating protein comprises LINE1 and / or ORF Ip circulating protein.

[0050] Embodiment 35 is the method of any one of embodiments 27-34.1, wherein nucleic acid data is obtained from the first sample comprising an increase in RNA expression of one or more target genes and the biologically-linked plasma biomarkers comprise one or more circulating proteins corresponding to the one or more target genes.

[0051] Embodiment 36 is the method of the immediately preceding embodiment, wherein the target genes correspond to cell-surface proteins, optionally wherein the cell-surface proteins are extracellular vesicle (EV)-proteins.

[0052] Embodiment 37 is the method of any one of embodiments 27-36, wherein nucleic acid data is obtained from the first sample comprising an increase in DNA copy number of one or more target regions and the biologically-linked plasma biomarkers comprise one or more circulating proteins corresponding to the one or more target regions.

[0053] Embodiment 38 is the method of any one of embodiments 27-37, wherein nucleic acid data is obtained from the first sample comprising one or more alternatively spliced mRNA molecules and the biologically-linked plasma markers comprise one or more circulating protein isoforms corresponding to the alternatively spliced mRNA.

[0054] Embodiment 39 is the method of any one of embodiments 27-38, wherein nucleic acid data is obtained from the first sample comprising one or more somatic DNA mutations and the biologically-linked plasma markers comprise one or more mutated circulating proteins corresponding to the one or more somatic DNA mutations.

[0055] Embodiment 40 is the method of any one of embodiments 27-39, wherein nucleic acid data is obtained from the first sample comprising one or more structural variants and the biologically-linked plasma markers comprise one or more mutated circulating proteins and / or one or more cell free RNA (cfRNA) molecules corresponding to the one or more structural variants.

[0056] Embodiment 41 is the method of any one of embodiments 27-40, wherein one or more, or each, of the biologically-linked plasma biomarkers do not comprise DNA.

[0057] Embodiment 42 is the method of any one of embodiments 27-40, wherein nucleic acid data is obtained from the first sample comprising one or more cancer associated somaticAttorney Ref. No. GH0271WOmutations associated with a gain-of-function phenotype and the biologically-linked plasma markers comprise one or more biomarkers resulting from the gain-of-function mutations.

[0058] Embodiment 43 is the method of the immediately preceding embodiment, wherein the somatic mutations associated with a gain-of-function phenotype are one or more mutations in the BRAF gene.

[0059] Embodiment 44 is the method of the immediately preceding embodiment, wherein the biologically-linked plasma markers comprise one or more hypermethylated DNA regions.

[0060] Embodiment 45 is the method of the immediately preceding embodiment, where the one or more hypermethylated DNA regions are associated with CpG island methylator phenotype (CIMP).

[0061] Embodiment 46 is the method of any one of the preceding embodiments, wherein the biologically-linked plasma biomarkers comprise biomarkers of a different type than the nucleic acids profded to identify the biologically-linked plasma biomarkers.

[0062] Embodiment 47 is the method of any one of the preceding embodiments, wherein determining the presence or absence of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject comprises determining the amounts of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject.

[0063] Embodiment 48 is the method of any one of the preceding embodiments, further comprising determining the presence or absence of minimum residual disease based on the amounts of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject.

[0064] Embodiment 49 is the method of the immediately preceding embodiment, wherein the method further comprises obtaining a third sample from the subject, wherein the third sample is a matched normal tissue sample and / or a buffy coat sample, and determining the presence or absence of minimum residual disease is not based on variants detectable in the third sample.

[0065] Embodiment 50 is the method of the immediately preceding embodiment, wherein the variants detectable in the third sample comprise clonal hematopoiesis of indeterminate potential (CHIP) variants.

[0066] Embodiment 51 is the method of any one of embodiments 49-50, wherein the one or more biologically-linked plasma biomarkers are not present in the third sample.Attorney Ref. No. GH0271WO

[0067] Embodiment 52 is the method of any one of the preceding embodiments, wherein the subject was previously diagnosed with a cancer and received one or more previous cancer treatments.

[0068] Embodiment 53 is the method of the immediately preceding embodiment, wherein the first sample is obtained at one or more preselected time points prior to the one or more previous cancer treatments.

[0069] Embodiment 54 is the method of any one of embodiments 52-53, wherein a third sample is obtained at one or more preselected time points prior to the one or more previous cancer treatments, wherein the third sample is a matched normal tissue sample and / or a buffy coat sample.

[0070] Embodiment 55 is the method of any one of embodiments 52-54, wherein the second sample is obtained at one or more preselected time points following the one or more previous cancer treatments.

[0071] Embodiment 56 is the method of any one of embodiments 52-55, further comprising determining a cancer recurrence score.

[0072] Embodiment 57 is the method of any one of embodiments 52-56, wherein the cancer recurrence status of the subject is determined to be at risk for cancer recurrence when a cancer recurrence score is determined to be at or above a predetermined threshold or the cancer recurrence status of the subject is determined to be at lower risk for cancer recurrence when the cancer recurrence score is below the predetermined threshold.

[0073] Embodiment 58 is the method of the immediately preceding embodiment, further comprising comparing the cancer recurrence score of the subject with a predetermined cancer recurrence threshold, wherein the subject is classified as a candidate for a subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold or not a candidate for a subsequent cancer treatment when the cancer recurrence score is below the cancer recurrence threshold.

[0074] Embodiment 59 is the method of any one of the preceding embodiments, further comprising partitioning at least a portion of the DNA from the first sample into at least first and second subsamples, wherein the first subsample is a hypermethylated subsample comprising methylated DNA to a greater extent than the second subsample, and the second subsample is a hypomethylated subsample.Attorney Ref. No. GH0271WO

[0075] Embodiment 60 is the method of any one of the preceding embodiments, further comprising partitioning at least a portion of the DNAfrom the second sample into at least first and second subsamples, wherein the first subsample is a hypermethylated subsample comprising methylated DNAto a greater extent than the second subsample, and the second subsample is a hypomethylated subsample.

[0076] Embodiment 61 is the method of any one of embodiments 59-60, wherein the partitioning comprises contacting the DNA with an agent that recognizes methyl cytosine in the DNA.

[0077] Embodiment 62 is the method of the immediately preceding embodiment, wherein the agent that recognizes methyl cytosine is a methyl binding reagent.

[0078] Embodiment 63 is the method of the immediately preceding embodiment, wherein the methyl binding reagent is a methyl binding domain (MBD) protein or an antibody.

[0079] Embodiment 64 is the method of any one of embodiments 59-63, wherein the methyl binding reagent specifically recognizes 5-methylcytosine.

[0080] Embodiment 65 is the method of any one of embodiments 59-64, wherein the methyl binding reagent is immobilized on a solid support.

[0081] Embodiment 66 is the method of any one of embodiments 59-65, wherein the partitioning comprises immunoprecipitation of methylated DNA.

[0082] Embodiment 67 is the method of any one of embodiments 59-66, further comprising contacting the DNAfrom the hypermethylated subsample with at least one nuclease, optionally wherein the at least one nuclease comprises at least one restriction enzyme.

[0083] Embodiment 68 is the method of the immediately preceding embodiment, wherein the at least one restriction enzyme comprises at least one methylation-sensitive restriction enzyme (MSRE).

[0084] Embodiment 69 is the method of the immediately preceding embodiment, wherein the MSRE cleaves an unmethylated CpG sequence.

[0085] Embodiment 70 is the method of any one of embodiments 68-69, wherein the MSRE comprises one or more of Aatll, AccII, Acil, Aorl3HI, Aor51HI, BspT104I, BssHII, BstUI, CfrlOI, Clal, Cpol, Eco52I, Haell, HapII, Hhal, Hin6I, Hpall, HpyCH4IV, Mlul, Nael, Notl, Nrul, Nsbl, PmaCI, Psp 14061, Pvul, SacII, Sall, Smal, and SnaBI.Attorney Ref. No. GH0271WO

[0086] Embodiment 71 is the method of any one of embodiments 59-70, further comprising contacting the DNAfrom the hypomethylated subsample with at least one nuclease, optionally wherein the at least one nuclease comprises at least one restriction enzyme.

[0087] Embodiment 72 is the method of the immediately preceding embodiment, wherein the at least one restriction enzyme comprises at least one methylation-dependent restriction enzyme (MDRE).

[0088] Embodiment 73 is the method of the immediately preceding embodiment, wherein the MDRE cleaves a methylated CpG sequence.

[0089] Embodiment 74 is the method of the immediately preceding embodiment, wherein the MDRE comprises one or more of MspJI, LpnPI, FspEI, or McrBC.

[0090] Embodiment 75 is the method of any one of embodiments 1-58, further comprising partitioning at least a portion of the DNA from the first sample into at least first and second subsamples on the basis of binding to a protein, optionally wherein the protein is a methylated protein, an acetylated protein, an unmethylated protein, or an unacetylated protein; and / or optionally wherein the protein is a histone.

[0091] Embodiment 76 is the method of the immediately preceding embodiment, wherein the partitioning comprises contacting the nucleic acids of the sample with a binding reagent which is specific for the protein and is immobilized on a solid support.

[0092] Embodiment 77 is the method of any one of the preceding embodiments, further comprising capturing a sequence-variable target region set from the first sample.

[0093] Embodiment 78 is the method of the immediately preceding embodiment, wherein the capturing is performed before the profiling step and at least a portion of the sequence-variable target region set is profiled to identify the biologically-linked plasma biomarkers.

[0094] Embodiment 79 is the method of any one of embodiments 77-78, wherein the capturing is performed after a partitioning step, if present.

[0095] Embodiment 80 is the method of the immediately preceding embodiment, wherein the sequence-variable target region set comprises at least 10, at least 20, at least 50, at least 75, at least 100, at least 200, at least 500, at least 750, at least 1,000, at least 2,000, at least 5,000, at least 7,500 or at least 10,000 target regions.

[0096] Embodiment 81 is the method of any one of embodiments 77-80, wherein the sequencevariable target region set has a footprint of at least 100 megabases.Attorney Ref. No. GH0271WO

[0097] Embodiment 82 is the method of any one of the preceding embodiments, further comprising capturing an epigenetic target region set from the first sample.

[0098] Embodiment 83 is the method of the immediately preceding embodiment, wherein the capturing is performed before the profiling step and at least a portion of the epigenetic target region set is profiled to identify the biologically-linked plasma biomarkers.

[0099] Embodiment 84 is the method of the immediately preceding embodiment, wherein the capturing is performed prior to a partitioning step, if present.

[0100] Embodiment 85 is the method of any one of embodiments 82-84, wherein the epigenetic target region set comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 target regions.

[0101] Embodiment 86 is the method of any one of embodiments 82-85, wherein the epigenetic target region set has a footprint of at least 2000 kilobases.

[0102] Embodiment 87 is the method of any one of embodiments 82-86, comprising determining a methylation level of the captured epigenetic target regions.

[0103] Embodiment 88 is the method of any one of embodiments 82-87, wherein at least one of the captured epigenetic target regions is a differentially methylated region.

[0104] Embodiment 89 is the method of any one of embodiments 82-88, wherein at least one of the captured epigenetic target regions is a fragment.

[0105] Embodiment 90 is the method of any one of embodiments 82-89, wherein at least one of the captured epigenetic target regions is a hypermethylated region, optionally wherein the hypermethylated region is a type-specific hypermethylated region.

[0106] Embodiment 91 is the method of any one of embodiments 82-90, wherein at least one of the captured epigenetic target regions is a hypomethylated region, optionally wherein the hypomethylated region is a type-specific hypomethylated region.

[0107] Embodiment 92 is the method of any one of embodiments 82-91, wherein at least one of the captured epigenetic target regions comprises a CTCF binding site, and / or a transcription start site.

[0108] Embodiment 93 is the method of any one of embodiments 82-92, wherein at least one of the captured epigenetic target regions is at least one type-specific epigenetic target region.Attorney Ref. No. GH0271WO

[0109] Embodiment 94 is the method of the immediately preceding embodiment, wherein the at least one type-specific epigenetic target region comprises type-specific differentially methylated regions and / or type specific fragments.

[0110] Embodiment 95 is the method of the immediately preceding embodiment, wherein the at least one type-specific epigenetic target region comprises type-specific hypomethylated regions and / or type-specific hypermethylated regions.[OHl] Embodiment 96 is the method of any one of embodiments 94-95, wherein the at least one type-specific epigenetic target region comprises cell-type specific, cell cluster-type specific, tissue-type specific, and / or cancer-type specific epigenetic target regions.

[0112] Embodiment 97 is the method of any one of embodiments 94-96, wherein the at least one type-specific epigenetic target region comprises type-specific epigenetic target regions that are: hypermethylated in immune cells relative to non-immune cell types present in a blood sample; differentially methylated in colon relative to other tissue types; differentially methylated in lung relative to other tissue types; differentially methylated in breast relative to other tissue types; differentially methylated in liver relative to other tissue types; differentially methylated in kidney relative to other tissue types; differentially methylated in pancreas relative to other tissue types; differentially methylated in prostate relative to other tissue types; differentially methylated in skin relative to other tissue types; or differentially methylated in bladder relative to other tissue types.

[0113] Embodiment 98 is the method of any one of embodiments 94-97, wherein the typespecific hypermethylated region or the hypermethylated regions are methylated to an extent that is at least 10%, 20%, 30%, or at least 40% greater than the average methylation of the target regions in the sample.

[0114] Embodiment 99 is the method of any one of embodiments 94-98, wherein the at least one type-specific epigenetic target region comprises target regions that are: hypomethylated in non-immune blood cells relative to the methylation level of the target regions in a different cell or tissue type in the sample; fragments specific to immune cells relative to non-immune cell types present in the sample; or fragments specific to colon, lung, breast, liver, kidney, pancreas, prostate, skin, or bladder relative to other tissue types.Attorney Ref. No. GH0271WO

[0115] Embodiment 100 is the method of any one of embodiments 94-99, comprising identifying at least one cell type or tissue type from which the at least one type-specific epigenetic target region originated.

[0116] Embodiment 101 is the method of the immediately preceding embodiment, wherein the level of the at least one type-specific epigenetic target region that originated from a cell or tissue type is determined.

[0117] Embodiment 102 is the method of the immediately preceding embodiment, wherein the level of the at least one type-specific epigenetic target regions that originated from immune cells, non-immune blood cells, colon, lung, breast, liver, kidney, prostate, skin, bladder, or pancreas are determined.

[0118] Embodiment 103 is the method of any one of the preceding embodiments, further comprising subjecting the DNA of the first sample or one or more subsamples thereof to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase, 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.

[0119] Embodiment 104 is the method of the immediately preceding embodiment, wherein the procedure that affects a first nucleobase in the DNA differently from a second nucleobase comprises a conversion procedure that changes the base pairing specificity of the base or does not change the base pairing specificity of the base, depending on the modification status of the base.

[0120] Embodiment 105 is the method of any one of embodiments 103-104, wherein the first nucleobase is an unmodified cytosine and the second nucleobase is a modified cytosine, optionally wherein the modified cytosine is 5-methylcytosine or 5-hydroxymethylcytosine.

[0121] Embodiment 106 is the method of any one of embodiments 104-105, wherein the procedure that affects a first nucleobase of the DNA differently from a second nucleobase of the DNA is methylation-sensitive conversion.

[0122] Embodiment 107 is the method of the immediately preceding embodiment, wherein the methylation-sensitive conversion is bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, APOBEC-coupled epigenetic (ACE) conversion,Attorney Ref. No. GH0271WOenzymatic methyl-seq (EM-seq) conversion, single-enzyme 5-methylcytosine sequencing (SEM-seq) conversion, or direct methylation sequencing (DM-seq).

[0123] Embodiment 108 is the method of the immediately preceding embodiment, wherein the Tet-assisted conversion further comprises a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tertbutylamine borane, or ammonia borane.

[0124] Embodiment 109 is the method of any one of embodiments 104-108, wherein the conversion procedure comprises contacting the DNA with a CpG-specific DNA methyltransferase (MTase) or a CpG-specific carboxymethyltransferase (CxMTase), a methyl donor or a carboxymethyl donor, and a cytosine deaminase.

[0125] Embodiment 110 is the method of the immediately preceding embodiment, wherein the cytosine deaminase is an APOB EC enzyme, optionally wherein the APOBEC enzyme is APOBEC3A.

[0126] Embodiment 111 is the method of any one of embodiments 104-110, wherein the procedure that affects a first nucleobase of the DNA differently from a second nucleobase of the DNA is performed before the profiling step and at least a portion of the DNA subjected to the procedure is profiled to identify the biologically-linked plasma biomarkers.

[0127] Embodiment 112 is the method of any one of the preceding embodiments, wherein the subject is a human.

[0128] Embodiment 113 is the method of any one of the preceding embodiments, further comprising determining a likelihood that the subject has precancer.

[0129] Embodiment 114 is the method of any one of embodiments 1-113, further comprising determining a likelihood that the subject has cancer.BRIEF DESCRIPTION OF THE DRAWINGS

[0130] FIG. 1 is a schematic diagram of an example of a system suitable for use with some embodiments of the disclosure.DETAILED DESCRIPTIONI. Overview

[0131] The methods disclosed herein are related to tissue-informed MRD / longitudinal monitoring assay, tracking biologically-linked biomarkers in blood. These biologically-linked biomarkers mayAttorney Ref. No. GH0271WObe gene-products (e.g. LINE! ORlFp protein) associated with tissue biomarker (e.g. LINE! DNA hypomethylation) or altered phenotypes (e.g., methylation patterns) associated with tissue detected biomarker.

[0132] The methods disclosed herein are related to a novel tissue-informed method wherein the tumor tissue nucleic acids are profded - for example, but not limited to, somatic mutation, DNA methylation, RNA expression / transcriptomics, etc.) to determine a personalized set of biomarkers, and the biologically-linked biomarkers relative to the personalized nucleic acid markers are detected in plasma (generally 'gene-products', but also related phenotypes).

[0133] Provided herein is a method for monitoring a subject for minimal residual disease, comprising: a) obtaining a first sample from a subject; wherein the first sample comprises a tumor tissue sample; b) profiling nucleic acids obtained from the first sample to identify biologically-linked plasma biomarkers; c) identifying one or more biologically-linked plasma biomarkers specific to the subject; d) determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject; and e) classifying the subject to be positive or negative for minimal residual disease based on the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in the second sample. In some embodiments, the methods provided herein further comprise obtaining a third sample from the subject, wherein the third sample is a matched normal tissue sample and / or a buffy coat sample. In some embodiments, the third sample is a buffy coat sample. In some embodiments, the methods provided herein further comprise profiling nucleic acids obtained from the third sample. In some embodiments, the methods provided herein further comprise, prior to step d), removing variants detected from profiling nucleic acids in the third sample from the one or more biologically-linked plasma biomarkers identified as specific to the subject.

[0134] In some embodiments, the second sample is a cell-free DNA sample. In some embodiments, the second sample is a plasma sample. In some embodiments, the profiling nucleic acids obtained from the first sample and / or the determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample further comprises a nucleic acid amplification step. In some embodiments, the profiling nucleic acids obtained from the first sample comprises sequencing nucleic acids from the first sample. In some embodiments, the determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject comprisesAttorney Ref. No. GH0271WOsequencing nucleic acids from the second sample and / or detecting one or more nucleic acids and / or proteins present in the second sample. In some embodiments, the detecting comprises high multiplex PCR, digital PCR, quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), or sequencing.

[0135] In some embodiments, the sequencing comprises next generation sequencing. In some embodiments, the sequencing comprises long-read sequencing. In some embodiments, the sequencing comprises nanopore sequencing. In some embodiments, the sequencing comprises 5-letter or 6-letter sequencing. In some embodiments, the sequencing comprises sequencing the DNA in a manner that distinguishes the first nucleobase from the second nucleobase. In some embodiments, the detecting one or more proteins present in the second sample comprises mass spectrometry (MS).

[0136] In some embodiments, determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in the second sample comprises enriching biologically-linked plasma biomarkers in the second sample using a custom panel of capture reagents specific to the subject. In some embodiments, the custom panel of capture reagents are configured to capture nucleic acids and / or proteins comprising the biologically-linked plasma biomarkers. In some embodiments, the custom panel of capture reagents comprises at least 5, 7, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 capture reagents. In some embodiments, the custom panel of capture reagents comprise a plurality of capture reagents, wherein the plurality of capture reagents comprise one or more DNA capture probes, one or more RNA capture probes, and / or one or more protein capture reagents. In some embodiments, the one or more protein capture reagents is selected from antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp. In some embodiments, the one or more protein capture reagents comprises an antibody. In some embodiments, the one or more protein capture reagents comprise an oligonucleotide-capture reagent conjugate. In some embodiments, the oligonucleotide-capture reagent conjugate comprises a molecular tag. In some embodiments, the molecular tag is a molecular barcode.

[0137] In some embodiments, markers comprising somatic variants, DNA methylation and / or RNA expression markers are identified by profiling the nucleic acids obtained from the first sample. In some embodiments, the biologically-linked plasma biomarkers comprise one or more biomarkers resulting from the markers identified by profiling the nucleic acids obtained from theAttorney Ref. No. GH0271WOfirst sample. A biologically-linked plasma biomarker is considered to result from a marker identified by profiling the nucleic acids obtained from the first sample when the existence, occurrence, frequency, or amount of the biologically-linked plasma biomarker depends on the marker identified by profiling the nucleic acids obtained from the first sample. For example, the marker identified by profiling the nucleic acids obtained from the first sample may be a hypomethylated DNA sequence and the biologically-linked plasma biomarker can be the level of an RNA transcribed from the hypomethylated DNA sequence, the level of a polypeptide encoded by an RNA transcribed from the hypomethylated DNA sequence, or a metabolite whose level is affected by a polypeptide encoded by an RNA transcribed from the hypomethylated DNA sequence. In another example, the marker identified by profiling the nucleic acids obtained from the first sample may be a somatic variant in a DNA sequence (“mutant sequence”) and the biologically-linked plasma biomarker can be the presence or level of an mRNA comprising the mutant sequence, the presence or level of a polypeptide encoded by the mutant sequence, or a metabolite whose presence or level is affected by a polypeptide encoded by the mutant sequence.

[0138] In some embodiments, the biologically-linked plasma biomarkers comprise differentially methylated DNA, cell-free RNA (cfRNA), extracellular vesicle (EV)-surface protein, and / or circulating protein. In some embodiments, markers comprising somatic variants and / or epigenetic variants are identified by profiling the nucleic acids obtained from the first sample. In some embodiments, the biologically-linked plasma biomarkers comprise cell-free RNA (cfRNA), extracellular vesicle (EV)-surface protein, and / or circulating protein. In some embodiments, markers comprising RNA expression markers are identified by profiling the nucleic acids obtained from the first sample. In some embodiments, the biologically-linked plasma biomarkers comprise differentially methylated DNA, extracellular vesicle (EV)-surface protein, and / or circulating protein. In some embodiments, the biologically-linked plasma biomarkers comprise one or more biomarkers resulting from the markers identified by profiling the nucleic acids obtained from the first sample. In some embodiments, nucleic acid data is obtained from the first sample comprising hypomethylated DNA and the biologically-linked plasma biomarkers comprise cfRNA and / or circulating protein corresponding to one or more hypomethylated DNA regions. In some embodiments, the hypomethylated DNA comprises hypomethylated LINE1 elements, the cfRNA comprises LINE1 cfRNA, and / or the circulating protein comprises LINE1 and / or ORF Ip circulating protein.Attorney Ref. No. GH0271WO

[0139] Tn some embodiments, nucleic acid data is obtained from the first sample comprising an increase in RNA expression of one or more target genes and the biologically-linked plasma biomarkers comprise one or more circulating proteins corresponding to the one or more target genes. In some embodiments, the target genes correspond to cell-surface proteins, optionally wherein the cell-surface proteins are extracellular vesicle (EV)-proteins. In some embodiments, nucleic acid data is obtained from the first sample comprising an increase in DNA copy number of one or more target regions and the biologically-linked plasma biomarkers comprise one or more circulating proteins corresponding to the one or more target regions. In some embodiments, nucleic acid data is obtained from the first sample comprising one or more alternatively spliced mRNA molecules and the biologically-linked plasma markers comprise one or more circulating protein isoforms corresponding to the alternatively spliced mRNA. In some embodiments, nucleic acid data is obtained from the first sample comprising one or more somatic DNA mutations and the biologically-linked plasma markers comprise one or more mutated circulating proteins corresponding to the one or more somatic DNA mutations. In some embodiments, nucleic acid data is obtained from the first sample comprising one or more structural variants and the biologically-linked plasma markers comprise one or more mutated circulating proteins and / or one or more cell free RNA (cfRNA) molecules corresponding to the one or more structural variants. In some embodiments, one or more, or each, of the biologically-linked plasma biomarkers do not comprise DNA. In some embodiments, nucleic acid data is obtained from the first sample comprising one or more cancer associated somatic mutations associated with a gain-of-function phenotype and the biologically-linked plasma markers comprise one or more biomarkers resulting from the gain-of-function mutations. In some embodiments, the somatic mutations associated with a gain-of-function phenotype are one or more mutations in the BRAF gene. In some embodiments, the biologically-linked plasma markers comprise one or more hypermethylated DNA regions. In some embodiments, the one or more hypermethylated DNA regions are associated with CpG island methylator phenotype (CIMP). In some embodiments, the biologically-linked plasma biomarkers comprise biomarkers of a different type than the nucleic acids profiled to identify the biologically-linked plasma biomarkers. Type in the context of a biomarker, e.g., a biologically-linked plasma biomarker or a biomarker detected by profiling nucleic acids such as from a tumor tissue sample, refers to the class of the molecule in which the biomarker occurs (e.g., DNA, RNA, protein, or carbohydrate).Attorney Ref. No. GH0271WO

[0140] In some embodiments, determining the presence or absence of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject comprises determining the amounts of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject. In some embodiments, the methods provided herein further comprise determining the presence or absence of minimum residual disease based on the amounts of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject. In some embodiments, the methods provided herein further comprise obtaining a third sample from the subject, wherein the third sample is a matched normal tissue sample and / or a buffy coat sample, and determining the presence or absence of minimum residual disease is not based on variants detectable in the third sample. In some embodiments, the variants detectable in the third sample comprise clonal hematopoiesis of indeterminate potential (CHIP) variants. In some embodiments, the one or more biologically-linked plasma biomarkers are not present in the third sample. In some embodiments, the subject was previously diagnosed with a cancer and received one or more previous cancer treatments. In some embodiments, the first sample is obtained at one or more preselected time points prior to the one or more previous cancer treatments. In some embodiments, a third sample is obtained at one or more preselected time points prior to the one or more previous cancer treatments, wherein the third sample is a matched normal tissue sample and / or a buffy coat sample. In some embodiments, the second sample is obtained at one or more preselected time points following the one or more previous cancer treatments. In some embodiments, the methods provided herein further comprise determining a cancer recurrence score. In some embodiments, the cancer recurrence status of the subject is determined to be at risk for cancer recurrence when a cancer recurrence score is determined to be at or above a predetermined threshold or the cancer recurrence status of the subject is determined to be at lower risk for cancer recurrence when the cancer recurrence score is below the predetermined threshold. In some embodiments, the methods provided herein further comprise comparing the cancer recurrence score of the subject with a predetermined cancer recurrence threshold, wherein the subject is classified as a candidate for a subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold or not a candidate for a subsequent cancer treatment when the cancer recurrence score is below the cancer recurrence threshold.Attorney Ref. No. GH0271WO

[0141] In some embodiments, the methods provided herein further comprise partitioning at least a portion of the DNA from the first sample into at least first and second subsamples, wherein the first subsample is a hypermethylated subsample comprising methylated DNA to a greater extent than the second subsample, and the second subsample is a hypomethylated subsample. In some embodiments, the methods provided herein further comprise partitioning at least a portion of the DNA from the second sample into at least first and second subsamples, wherein the first subsample is a hypermethylated subsample comprising methylated DNA to a greater extent than the second subsample, and the second subsample is a hypomethylated subsample. In some embodiments, the partitioning comprises contacting the DNA with an agent that recognizes methyl cytosine in the DNA. In some embodiments, the agent that recognizes methyl cytosine is a methyl binding reagent. In some embodiments, the methyl binding reagent is a methyl binding domain (MBD) protein or an antibody. In some embodiments, the methyl binding reagent specifically recognizes 5-methylcytosine. In some embodiments, the methyl binding reagent is immobilized on a solid support. In some embodiments, the partitioning comprises immunoprecipitation of methylated DNA. In some embodiments, the methods provided herein further comprise contacting the DNA from the hypermethylated subsample with at least one nuclease, optionally wherein the at least one nuclease comprises at least one restriction enzyme. In some embodiments, the at least one restriction enzyme comprises at least one methylation-sensitive restriction enzyme (MSRE). In some embodiments, the MSRE cleaves an unmethylated CpG sequence. In some embodiments, the MSRE comprises one or more of Aatll, AccII, Acil, Aorl3HI, Aor51HI, BspT104I, BssHII, BstUI, CfrlOI, Clal, Cpol, Eco52I, Haell, HapII, Hhal, Hin6I, Hpall, HpyCH4IV, Mlul, Nael, Notl, Nrul, Nsbl, PmaCI, Pspl406I, Pvul, SacII, Sall, Smal, and SnaBI.

[0142] In some embodiments, the methods provided herein further comprise contacting the DNA from the hypomethylated subsample with at least one nuclease, optionally wherein the at least one nuclease comprises at least one restriction enzyme. In some embodiments, the at least one restriction enzyme comprises at least one methylation-dependent restriction enzyme (MDRE). In some embodiments, the MDRE cleaves a methylated CpG sequence. In some embodiments, the MDRE comprises one or more of MspJI, LpnPI, FspEI, or McrBC. In some embodiments, the methods provided herein further comprise partitioning at least a portion of the DNA from the first sample into at least first and second subsamples on the basis of binding to a protein, optionally wherein the protein is a methylated protein, an acetylated protein, an unmethylated protein, or anAttorney Ref. No. GH0271WOunacetylated protein; and / or optionally wherein the protein is a histone. In some embodiments, the partitioning comprises contacting the nucleic acids of the sample with a binding reagent which is specific for the protein and is immobilized on a solid support.

[0143] In some embodiments, the methods provided herein further comprise capturing a sequencevariable target region set from the first sample. In some embodiments, the capturing is performed before the profiling step and at least a portion of the sequence-variable target region set is profiled to identify the biologically-linked plasma biomarkers. In some embodiments, the capturing is performed after a partitioning step, if present. In some embodiments, the sequence-variable target region set comprises at least 10, at least 20, at least 50, at least 75, at least 100, at least 200, at least 500, at least 750, at least 1,000, at least 2,000, at least 5,000, at least 7,500 or at least 10,000 target regions. In some embodiments, the sequence-variable target region set has a footprint of at least 100 megabases.

[0144] In some embodiments, the methods provided herein further comprise capturing an epigenetic target region set from the first sample. In some embodiments, the capturing is performed before the profiling step and at least a portion of the epigenetic target region set is profiled to identify the biologically-linked plasma biomarkers. In some embodiments, the capturing is performed prior to a partitioning step, if present. In some embodiments, the epigenetic target region set comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 target regions. In some embodiments, the epigenetic target region set has a footprint of at least 2000 kilobases.

[0145] In some embodiments, the methods provided herein further comprise determining a methylation level of the captured epigenetic target regions. In some embodiments, at least one of the captured epigenetic target regions is a differentially methylated region. In some embodiments, at least one of the captured epigenetic target regions is a fragment. In some embodiments, at least one of the captured epigenetic target regions is a hypermethylated region, optionally wherein the hypermethylated region is a type-specific hypermethylated region. In some embodiments, at least one of the captured epigenetic target regions is a hypomethylated region, optionally wherein the hypomethylated region is a type-specific hypomethylated region. In some embodiments, at least one of the captured epigenetic target regions comprises a CTCF binding site, and / or a transcription start site. In some embodiments, at least one of the captured epigenetic target regions is at least one type-specific epigenetic target region. In some embodiments, the at least one type-specific epigenetic target region comprises type-specific differentially methylated regions and / or typeAttorney Ref. No. GH0271WOspecific fragments. In some embodiments, the at least one type-specific epigenetic target region comprises type-specific hypomethylated regions and / or type-specific hypermethylated regions. In some embodiments, the at least one type-specific epigenetic target region comprises cell-type specific, cell cluster-type specific, tissue-type specific, and / or cancer-type specific epigenetic target regions.

[0146] In some embodiments, the at least one type-specific epigenetic target region comprises type-specific epigenetic target regions that are: hypermethylated in immune cells relative to non-immune cell types present in a blood sample; differentially methylated in colon relative to other tissue types; differentially methylated in lung relative to other tissue types; differentially methylated in breast relative to other tissue types; differentially methylated in liver relative to other tissue types; differentially methylated in kidney relative to other tissue types; differentially methylated in pancreas relative to other tissue types; differentially methylated in prostate relative to other tissue types; differentially methylated in skin relative to other tissue types; or differentially methylated in bladder relative to other tissue types. In some embodiments, the typespecific hypermethylated region or the hypermethylated regions are methylated to an extent that is atleast 10%, 20%, 30%, or atleast40% greater than the average methylation ofthe target regions in the sample.

[0147] In some embodiments, the at least one type-specific epigenetic target region comprises target regions that are: hypomethylated in non-immune blood cells relative to the methylation level of the target regions in a different cell or tissue type in the sample; fragments specific to immune cells relative to non-immune cell types present in the sample; or fragments specific to colon, lung, breast, liver, kidney, pancreas, prostate, skin, or bladder relative to other tissue types. In some embodiments, the methods provided herein further comprise identifying at least one cell type or tissue type from which the at least one type-specific epigenetic target region originated. In some embodiments, the level of the at least one type-specific epigenetic target region that originated from a cell or tissue type is determined. In some embodiments, the level of the at least one typespecific epigenetic target regions that originated from immune cells, non-immune blood cells, colon, lung, breast, liver, kidney, prostate, skin, bladder, or pancreas are determined.

[0148] In some embodiments, the methods provided herein further comprise subjecting the DNA of the first sample or one or more sub samples thereof to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase, wherein the first nucleobase is a modified orAttorney Ref. No. GH0271WOunmodified 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 procedure that affects a first nucleobase in the DNA differently from a second nucleobase comprises a conversion procedure that changes the base pairing specificity of the base or does not change the base pairing specificity of the base, depending on the modification status of the base. In some embodiments, the first nucleobase is an unmodified cytosine and the second nucleobase is a modified cytosine, optionally wherein the modified cytosine is 5-methylcytosine or 5-hydroxymethylcytosine. In some embodiments, the procedure that affects a first nucleobase of the DNA differently from a second nucleobase of the DNA is methylation-sensitive conversion. In some embodiments, the methylation-sensitive conversion is bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, APOBEC-coupled epigenetic (ACE) conversion, enzymatic methyl-seq (EM-seq) conversion, single-enzyme 5-methylcytosine sequencing (SEM-seq) conversion, or direct methylation sequencing (DM-seq). In some embodiments, the Tet-assisted conversion further comprises a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. In some embodiments, the conversion procedure comprises contacting the DNA with a CpG-specific DNA methyltransferase (MTase) or a CpG-specific carboxymethyltransferase (CxMTase), a methyl donor or a carboxymethyl donor, and a cytosine deaminase. In some embodiments, the cytosine deaminase is an APOBEC enzyme, optionally wherein the APOBEC enzyme is APOBEC3A. In some embodiments, the procedure that affects a first nucleobase of the DNA differently from a second nucleobase of the DNA is performed before the profiling step and at least a portion of the DNA subjected to the procedure is profiled to identify the biologically-linked plasma biomarkers.

[0149] In some embodiments, the subject is a human. In some embodiments, the methods provided herein further comprise determining a likelihood that the subject has precancer. In some embodiments, the methods provided herein further comprise determining a likelihood that the subject has cancer.

[0150] In an aspect, a method for monitoring a subject for minimal residual disease, comprises: (a) obtaining a first sample from a subject; wherein the first sample comprises tumor tissue sample; (b) profiling nucleic acids obtained from the first sample to identify biologically-linked plasma biomarker; (c) identifying one or more biologically-linked plasma biomarkers specific to theAttorney Ref. No. GH0271WOsubject; (d) determining presence / absence of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject; and (e) classifying the subject to be positive / negative for the minimal residual disease based on the presence / absence of the one or more biologically-linked plasma biomarkers in the second sample.

[0151] In some embodiments, the first sample can further include a matched normal tissue sample and / or buffy coat sample. In some embodiments, the method further comprises profiling nucleic acids obtained from the matched normal tissue sample and / or buffy coat sample. In some embodiments, the method further comprises, prior to step d), removing variants detected from profiling nucleic acids in the matched normal sample tissue and / or buffy coat sample from the one or more biologically-linked plasma biomarkers identified specific to the subject. In some embodiments, the second sample is a cell-free DNA sample. In some embodiments, the presence / absence of the one or more biologically-linked plasma biomarkers in the second sample are determined by enriching nucleic acids using a custom panel specific to the subject. In some embodiments, the markers for profiling nucleic acids in the first sample can be somatic variants, DNA methylation and / or RNA expression / transcriptomics. In some embodiments, the biologically-linked plasma biomarkers can be DNA methylation (for e.g., hypermethylated phenotype), cfRNA, EV-surface protein and / or circulating protein. Examples of tissue nucleic acid markers and corresponding biologically-linked blood biomarkers may be found in Table 1.Table 1Attorney Ref. No. GH0271WO* The gene products (biologically linked blood biomarker) are chosen such that those gene products are not present in healthy, normal plasma.

[0152] The disclosed methods can be combined with analysis of one or more additional biomarkers. In some embodiments, the disclosed methods are combined with one or more methods, such as but not limited to, methods for assessing DNA methylation patterns, DNA mutations (such as somatic mutations), nucleic acid fragmentation patterns, non-coding RNA (such as micro RNAs (miRNAs), ribosomal RNAs, transfer RNAs, small nucleolar RNAs (snow RNAs), and / or small nuclear RNAs (snRNAs)) levels, and / or cell type proportions / levels, cellular locations, and / or structural modifications of one or more proteins (such as in a sample from a subject). In some embodiments, the disclosed methods are combined with one or more analyses of genetic variations including mutations, rare mutations, indels, rearrangements, copy number variations, transversions, translocations, recombinations, inversion, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, gene fusions, chromosome fusions, gene truncations, gene amplification, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and / or abnormal changes in nucleic acid 5-methylcytosineII. Definitions

[0153] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forthAttorney Ref. No. GH0271WOthrough the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

[0154] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent upon reading this disclosure and so forth.

[0155] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In describing and claiming the methods, computer readable media, and systems, the following terminology, and grammatical variants thereof, will be used in accordance with the definitions set forth below.

[0156] About: As used herein, “about” or “approximately” as applied to one or more values or elements of interest, refers to a value or element that is similar to a stated reference value or element. In certain embodiments, the term “about” or “approximately” refers to a range of values or elements that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value or element unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value or element).

[0157] As used herein, the term “somatic biomarker” refers to one or more mutations relative to germline. Somatic mutations can occur in any cell of the body except germ cells and accordingly, are not passed on to progeny. Somatic biomarkers can comprise mutations in DNA (e.g., single nucleotide variations (SNVs), gene fusions, insertions and deletions (indels), transversions, translocations, copy number variations (CNVs), inversions, epigenetic changes (e.g., differentially methylated regions (DMRs)), including modification of cytosine (e.g., 5-methylcytosine, 5-hydroxymethylcytosine, and other more oxidized forms); in RNA (e.g., mutations in messenger RNA (mRNA), microRNA (miRNA), and other non-coding RNA); and / or in protein.

[0158] As used herein, the term “biomolecules” refers to molecules present in biologic samples obtained from a subject. Such biomolecules include but are not limited to DNA, RNA, and protein.Attorney Ref. No. GH0271WO

[0159] As used herein, the terms “structural variations”, “structural variants”, and “SVs” are used interchangeably. They refer to genomic mutations larger than a single nucleotide, and can comprise inversions, translocations, insertions, and / or deletions. In some embodiments, SVs comprise a “unique breakpoint”, which refers to a breakage in the DNA sequence relative to a reference sequence. For example, both deletions and insertions comprise unique breakpoints.

[0160] As used herein, the terms “copy number variations”, “copy number variants”, “copy number abnormalities”, “CNVs”, and “CNAs” are used interchangeably. They refer to repeats of genomic sequences present in DNA from a subject. CNVs can be detected by whole genome sequencing, or quantitative methods such as qPCR and digital PCR. As used herein, the term “multiplicity of copy number variations”, or “multiplicity of CNVs” refers to the total number of copy number variations present in the DNA from a subject. In some embodiments, the multiplicity of CNVs comprises repeats of more than one genomic region (e.g., CNVs of more than one gene). In some embodiments, the multiplicity of CNVs is an aggregate multiplicity of repeats of more than one genomic region.

[0161] As used herein, “biologically-linked plasma biomarkers” refers to biomarkers that can be detected in a plasma sample and that were selected based on analysis of nucleic acids profded from a tumor tissue sample from a subject. In some embodiments, the biologically-linked plasma biomarkers comprise differentially methylated DNA, cell-free RNA (cfRNA), extracellular vesicle (EV)-surface protein, and / or circulating protein.

[0162] As used herein, “type” in the context of a biomarker, e.g., a biologically-linked plasma biomarker or a biomarker detected by profiling nucleic acids such as from a tumor tissue sample, refers to the class of the molecule in which the biomarker occurs (e.g., DNA, RNA, protein, or carbohydrate).

[0163] Adapter: As used herein, “adapter” refers to a short nucleic acid (e.g., less than about 500 nucleotides, less than about 100 nucleotides, or less than about 50 nucleotides in length) that is typically at least partially double-stranded and is attached to either one end or both ends (i.e., two adapters are attached to both ends of the nucleic acid - one adapter at end of the nucleic acid) of a given sample nucleic acid molecule. Adapters can include nucleic acid primer binding sites to permit amplification of a nucleic acid molecule flanked by adapters at both ends, and / or a sequencing primer binding site, including primer binding sites for sequencing applications, such as various next-generation sequencing (NGS) applications. Adapters can also include binding sitesAttorney Ref. No. GH0271WOfor capture probes, such as an oli onucleotide attached to a flow cell support or the like. Adapters can also include a nucleic acid tag as described herein. Nucleic acid tags are typically positioned relative to amplification primer and sequencing primer binding sites, such that a nucleic acid tag is included in amplicons and sequence reads of a given nucleic acid molecule. Adapters of the same or different sequences can be linked to the respective ends of a nucleic acid molecule. In some embodiments, the adapters of the same sequence is linked to the respective ends of the nucleic acid molecule except that the nucleic acid tag differs. In some embodiments, the adapter is a Y-shaped adapter in which one end is blunt ended or tailed as described herein, for joining to a nucleic acid molecule, which is also blunt ended or tailed with one or more complementary nucleotides and the other end of the Y-shaped adapter comprises a non-complementary sequence which does not hybridize to form a double-strand. In still other example embodiments, an adapter is a bell-shaped adapter that includes a blunt or tailed end for joining to a nucleic acid molecule to be analyzed. Other examples of adapters include T-tailed and C-tailed adapters.

[0164] As used herein, an “asymmetric adapter” is a double stranded adapter in which the two strands are not completely complementary or are otherwise distinguishable such that synthesis of a complementary sequence of one strand of the adapter results in a sequence that is distinguishable from the sequence of the other strand of the adapter. Examples of asymmetric adapters are Y-shaped adapters and bubble adapters.

[0165] As used herein, a “Y-shaped adapter” refers to an adapter comprising two DNA strands comprising complementary and non-complementary parts, wherein the non-complementary parts form single-stranded arms. The adapter can be attached to a sample or insert DNA molecule, e.g., by ligation, such that the complementary (double-stranded) part of the adapter is proximal to the sample or insert DNA molecule. Prior to attachment, the double stranded portion of the Y-shaped adapter may have a blunt end or an overhang, e.g., of one to three nucleotides. The single stranded arms may or may not be of identical length.

[0166] As used herein, a “bubble adapter” refers to an adapter comprising two DNA strands comprising a non-complementary part flanked by complementary parts, such that the adapter has a single stranded region located between double- stranded regions. The adapter can be attached to a sample or insert DNA molecule, e.g., by ligation, such that one of the complementary (doublestranded) parts of the adapter is proximal to the sample or insert DNA molecule. Prior to attachment, the double stranded portion of the Y-shaped adapter that would be attached to the insertAttorney Ref. No. GH0271WOor sample molecule may have a blunt end or an overhang, e.g., of one to three nucleotides. The single stranded portions of the two strands may or may not be of identical length.

[0167] Amplify: As used herein, “amplify” or “amplification” in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule), where the amplification products or amplicons are generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. Amplification includes but is not limited to polymerase chain reaction (PCR).

[0168] Barcode: As used herein, “barcode” or “molecular barcode” in the context of nucleic acids refers to a nucleic acid molecule comprising a sequence that can serve as a molecular identifier. For example, individual "barcode" sequences are typically added to each DNA fragment during next-generation sequencing (NGS) library preparation so that each read can be identified and sorted before the final data analysis.

[0169] Cancer Type: As used herein, “cancer type” refers to a type or subtype of cancer defined, e.g., by histopathology. Cancer type can be defined by any conventional criterion, such as on the basis of occurrence in a given tissue (e.g., blood cancers, central nervous system (CNS), brain cancers, lung cancers (small cell and non-small cell), skin cancers, nose cancers, throat cancers, liver cancers, bone cancers, lymphomas, pancreatic cancers, bowel cancers, rectal cancers, thyroid cancers, bladder cancers, kidney cancers, mouth cancers, stomach cancers, breast cancers, prostate cancers, ovarian cancers, lung cancers, intestinal cancers, soft tissue cancers, neuroendocrine cancers, gastroesophageal cancers, head and neck cancers, gynecological cancers, colorectal cancers, urothelial cancers, solid state cancers, heterogeneous cancers, homogenous cancers), unknown primary origin and the like, and / or of the same cell lineage (e.g., carcinoma, sarcoma, lymphoma, cholangiocarcinoma, leukemia, mesothelioma, melanoma, or glioblastoma) and / or cancers exhibiting cancer markers, such as, but not limited to, Her2, CA15-3, CA19-9, CA-125, CEA, AFP, PSA, HCG, hormone receptor and NMP-22. Cancers can also be classified by stage (e g., stage 1, 2, 3, or 4) and whether of primary or secondary origin.

[0170] Captured set: As used herein, a “captured set” of nucleic acids refers to nucleic acids that have undergone capture.Attorney Ref. No. GH0271WO

[0171] Capturing: As used herein, “capturing” or “enriching” one or more target nucleic acids refers to preferentially isolating or separating the one or more target nucleic acids from non-target nucleic acids.

[0172] Cell-Free Nucleic Acid: As used herein, “cell-free nucleic acid” refers to nucleic acids not contained within or otherwise bound to a cell or, in some embodiments, nucleic acids remaining in a sample following the removal of intact cells. Cell-free nucleic acids can include, for example, all non-encapsulated nucleic acids sourced from a bodily fluid (e.g., blood, plasma, serum, urine, cerebrospinal fluid (CSF), etc.) from a subject. Cell-free nucleic acids include DNA (cfDNA), RNA (cfRNA), and hybrids thereof, including genomic DNA, mitochondrial DNA, circulating DNA, siRNA, miRNA, circulating RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi -interacting RNA (piRNA), long non-coding RNA (long ncRNA), and / or fragments of any of these. Cell-free nucleic acids can be double-stranded, single-stranded, or a hybrid thereof. A cell-free nucleic acid can be released into bodily fluid through secretion or cell death processes, e.g., cellular necrosis, apoptosis, or the like. Some cell-free nucleic acids are released into bodily fluid from cancer cells, e.g., circulating tumor DNA (ctDNA). Others are released from healthy cells. CtDNA can be non-encapsulated tumor-derived fragmented DNA. A cell-free nucleic acid can have one or more epigenetic modifications, for example, a cell-free nucleic acid can be acetylated, 5-methylated, and / or hydroxy methylated.

[0173] Cellular Nucleic Acids: As used herein, “cellular nucleic acids” means nucleic acids that are disposed within one or more cells from which the nucleic acids have originated, at least at the point a sample is taken or collected from a subject, even if those nucleic acids are subsequently removed (e.g., via cell lysis) as part of a given analytical process.

[0174] Corresponding to a target region set: As used herein, “corresponding to a target region set means that a nucleic acid, such as cfDNA, originated from a locus in the target region set or specifically binds one or more probes for the target-region set.

[0175] Coverage: As used herein, the terms “coverage”, “total molecule count”, or “total allele count” are used interchangeably. They refer to the total number of DNA molecules at a particular genomic position in a given sample.

[0176] Deoxyribonucleic Acid or Ribonucleic Acid: As used herein, “deoxyribonucleic acid” or “DNA” refers to a natural or modified nucleotide which has a hydrogen group at the 2'-position of the sugar moiety. DNA typically includes a chain of nucleotides comprising four types ofAttorney Ref. No. GH0271WOnucleotide bases; adenine (A), thymine (T), cytosine (C), and guanine (G). As used herein, “ribonucleic acid” or “RNA” refers to a natural or modified nucleotide which has a hydroxyl group at the 2'-position of the sugar moiety. RNA typically includes a chain of nucleotides comprising four types of nucleotide bases; A, uracil (U), G, and C. As used herein, the term “nucleotide” refers to a natural nucleotide or a modified nucleotide. Certain pairs of nucleotides specifically bind to one another in a complementary fashion (called complementary base pairing). In DNA, adenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine (G). In RNA, adenine (A) pairs with uracil (U) and cytosine (C) pairs with guanine (G). When a first nucleic acid strand binds to a second nucleic acid strand made up of nucleotides that are complementary to those in the first strand, the two strands bind to form a double strand. As used herein, “sequencing data,” “nucleic acid sequencing information,” “sequence information,” “nucleic acid sequence,” “nucleotide sequence”, “genomic sequence,” “sequence read” or “sequencing read” denotes any information or data that is indicative of the order and identity of the nucleotide bases (e.g., adenine, guanine, cytosine, and thymine or uracil) in a molecule (e.g., a whole genome, whole transcriptome, exome, oligonucleotide, polynucleotide, or fragment) of a nucleic acid such as DNA or RNA. It should be understood that the present teachings contemplate sequence information obtained using all available varieties of techniques, platforms or technologies, including, but not limited to: capillary electrophoresis, microarrays, ligation-based systems, polymerase-based systems, hybridization-based systems, direct or indirect nucleotide identification systems, pyrosequencing, ion- or pH-based detection systems, and electronic signature-based systems.

[0177] DNA sequence: As used herein, “DNA sequence” or “sequence” refers to “raw sequence reads” and / or “consensus sequences.” Raw sequence reads are the output of a DNA sequencer, and typically include redundant sequences of the same parent molecule, for example after amplification. “Consensus sequences” are sequences derived from redundant sequences of a parent molecule intended to represent the sequence of the original parent molecule. Consensus sequences includes the base identity at a single position. In some embodiments, consensus sequence can represent a single nucleotide base at a particular genomic position. In some embodiments, consensus sequence can represent a string of nucleotide bases at a plurality of genomic positions. Consensus sequences can be produced by voting (wherein each majority nucleotide, e.g., the most commonly observed nucleotide at a given base position, among the sequences is the consensus nucleotide) or other approaches such as comparing to a reference genome. Consensus sequencesAttorney Ref. No. GH0271WOcan be produced by tagging original parent molecules with unique or non-unique molecular tags, which allow tracking of the progeny sequences (e.g., after amplification) by tracking of the tag and / or use of sequence read internal information. Examples of tagging or barcoding, and uses of tags or barcodes, are provided in, for example, U.S. Patent Pub. Nos. 2015 / 0368708, 2015 / 0299812, 2016 / 0040229, and 2016 / 0046986, each of which is entirely incorporated herein by reference.

[0178] Genomic region: As used herein, “genomic region” refers to any region (e.g., range of base pair locations) of a genome, e.g., a chromosome, a chromosome arm, a gene, or an exon. A genomic region may be a contiguous or a non-contiguous region. A “genetic locus” (or “locus”) can be a portion or entirety of a genomic region (e.g., a gene, a portion of a gene, or a single nucleotide of a gene). In some embodiments, the size of the genomic region comprises up to a length of a chromosome / chromosome arm or a topologically associated domain (TAD). In some embodiments, the size of the genomic region can be limited to the biological activity of the region (e.g., transcriptional unit or regulatory unit).

[0179] Mutation or variation: As used herein, “mutation” or “variation” refers to a variation from a known reference sequence and includes mutations such as, for example, single nucleotide variants (SNVs), and insertions or deletions (indels). A mutation can be a germline or somatic mutation. In some embodiments, a reference sequence for purposes of comparison is a wildtype genomic sequence of the species of the subject providing a test sample, typically the human genome.

[0180] As used herein, a “double variant” refers to two or more somatic variants within 400 nucleotides of each other (e.g., within 50-400 nucleotides of each other). The somatic variants may each independently be any type of somatic variant (e.g., single nucleotide variation, or indel). The double variants are not necessarily observed in the same molecule, although this may occur. The two or more somatic variants of the double variant can be in the same molecule or different molecules. The two variants in the double variant are considered to be within 400 nucleotides of each other if any nucleotide affected by one variant is within 400 nucleotides of any nucleotide affected by another variant. A nucleotide affected by a variant refers to any nucleotide.

[0181] Neoplasm: As used herein, the terms “neoplasm” and “tumor” are used interchangeably. They refer to abnormal growth of cells in a subj ect. A neoplasm or tumor can be benign, potentially malignant, or malignant. A malignant tumor is a referred to as a cancer or a cancerous tumor.Attorney Ref. No. GH0271WO

[0182] Next-Generation Sequencing: As used herein, “next-generation sequencing” or “NGS” refers to sequencing technologies having increased throughput as compared to traditional Sanger-and capillary electrophoresis-based approaches, for example, with the ability to generate hundreds of thousands of relatively small sequence reads at a time. Some examples of next-generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. In some embodiments, next-generation sequencing includes the use of instruments capable of sequencing single molecules. Example of commercially available instruments for performing next-generation sequencing include, but are not limited to, NextSeq, HiSeq, NovaSeq, MiSeq, Ion PGM and Ion GeneStudio S5.

[0183] Nucleic Acid Tag: As used herein, “nucleic acid tag” refers to a short nucleic acid (e.g., less than about 500 nucleotides, about 100 nucleotides, about 50 nucleotides, or about 10 nucleotides in length), used to distinguish nucleic acids from different samples (e g., representing a sample index), distinguish nucleic acids from different partitions (e.g., representing a partition tag) or different nucleic acid molecules in the same sample (e.g., representing a molecular barcode), of different types, or which have undergone different processing. The nucleic acid tag comprises a predetermined, fixed, non-random, random or semi-random oligonucleotide sequence. Such nucleic acid tags may be used to label different nucleic acid molecules or different nucleic acid samples or sub-samples. Nucleic acid tags can be single-stranded, double-stranded, or at least partially double-stranded. Nucleic acid tags optionally have the same length or varied lengths. Nucleic acid tags can also include double-stranded molecules having one or more blunt-ends, include 5’ or 3’ single-stranded regions (e.g., an overhang), and / or include one or more other single-stranded regions at other locations within a given molecule. Nucleic acid tags can be attached to one end or to both ends of the other nucleic acids (e.g., sample nucleic acids to be amplified and / or sequenced). Nucleic acid tags can be decoded to reveal information such as the sample of origin, form, or processing of a given nucleic acid. For example, nucleic acid tags can also be used to enable pooling and / or parallel processing of multiple samples comprising nucleic acids bearing different molecular barcodes and / or sample indexes in which the nucleic acids are subsequently being deconvolved by detecting (e.g., reading) the nucleic acid tags. Nucleic acid tags can also be referred to as identifiers (e.g. molecular identifier, sample identifier). Additionally, or alternatively, nucleic acid tags can be used as molecular identifiers (e.g., to distinguish between different molecules or amplicons of different parent molecules in the same sample or sub-sample).Attorney Ref. No. GH0271WOThis includes, for example, uniquely tagging different nucleic acid molecules in a given sample, or non-uniquely tagging such molecules. In the case of non-unique tagging applications, a limited number of tags (i.e., molecular barcodes) may be used to tag each nucleic acid molecule such that different molecules can be distinguished based on their endogenous sequence information (for example, start and / or stop positions where they map to a selected reference genome, a subsequence of one or both ends of a sequence, and / or length of a sequence) in combination with at least one molecular barcode. Typically, a sufficient number of different molecular barcodes are used such that there is a low probability (e.g., less than about a 10%, less than about a 5%, less than about a 1%, or less than about a 0.1% chance) that any two molecules may have the same endogenous sequence information (e.g., start and / or stop positions, subsequences of one or both ends of a sequence, and / or lengths) and also have the same molecular barcode.

[0184] Polynucleotide: As used herein, “polynucleotide”, “nucleic acid”, “nucleic acid molecule”, or “oligonucleotide” refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages. Typically, a polynucleotide comprises at least three nucleosides. Oligonucleotides often range in size from a few monomeric units, e.g., 3-4, to hundreds of monomeric units. Whenever a polynucleotide is represented by a sequence of letters, such as “ATGCCTG”, the nucleotides are in3’ order from left to right, and in the case of DNA, “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes deoxythymidine, unless otherwise noted. The letters A, C, G, and T may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases.

[0185] Processing: As used herein, “processing” refers to a set of steps used to generate a library of nucleic acids that is suitable for sequencing. The set of steps can include, but are not limited to, partitioning, end repairing, addition of sequencing adapters, tagging, and / or PCR amplification of nucleic acids.

[0186] Reference Sequence: As used herein, “reference sequence” refers to a known sequence used for purposes of comparison with experimentally determined sequences. For example, a known sequence can be an entire genome, a chromosome, or any segment thereof. A reference sequence can align with a single contiguous sequence of a genome or chromosome or chromosome arm or can include non-contiguous segments that align with different regions of a genome orAttorney Ref. No. GH0271WOchromosome. Examples of reference sequences include, for example, human genomes, such as, hG19 and hG38.

[0187] Sample: As used herein, “sample” means anything capable of being analyzed by the methods and / or systems disclosed herein.

[0188] Sequencing: As used herein, “sequencing” refers to any of a number of technologies used to determine the sequence (e.g., the identity and order of monomer units) of a biomolecule, e.g., a nucleic acid such as DNA or RNA. Examples of sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy -based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co-amplification at lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, and a combination thereof. In some embodiments, sequencing can be performed by a gene analyzer such as, for example, gene analyzers commercially available from Illumina, Inc., Pacific Biosciences, Inc., or Applied Biosystems / Thermo Fisher Scientific, among many others.

[0189] Sequence Information: As used herein, “sequence information” in the context of a nucleic acid polymer means the order and identity of monomer units (e.g., nucleotides, etc.) in that polymer.

[0190] Somatic Mutation: As used herein, the terms “somatic mutation” or “somatic variation” are used interchangeably. They refer to a mutation in the genome that occurs after conception. Somatic mutations can occur in any cell of the body except germ cells and accordingly, are not passed on to progeny.

[0191] Subject: As used herein, “subject” refers to an animal, such as a mammalian species (e.g., human) or avian (e.g., bird) species, or other organism, such as a plant. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. AnimalsAttorney Ref. No. GH0271WOinclude farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companion animals (e.g., pets or support animals). A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual in need of therapy or suspected of needing therapy. The terms “individual” or “patient” are intended to be interchangeable with “subject”. For example, a subject can be an individual who has been diagnosed with having a cancer, is going to receive a cancer therapy, and / or has received at least one cancer therapy. The subject can be in remission of a cancer. As another example, the subject can be an individual who is diagnosed of having an autoimmune disease. As another example, the subject can be a female individual who is pregnant or who is planning on getting pregnant, who may have been diagnosed of or suspected of having a disease, e.g., a cancer, an auto-immune disease.

[0192] Target-region set: As used herein, “target-region set” or “set of target regions” or “target regions” or “target regions of interest” refers to a plurality of genomic loci or a plurality of genomic regions targeted for capture and / or targeted by a set of probes (e.g., through sequence compl em entari ty ) .

[0193] Tumor fraction: As used herein, “tumor fraction” refers to the proportion of cfDNA molecules that originated from tumor cells for a given sample, or sample-region pair.

[0194] The terms “or a combination thereof’ and “or combinations thereof’ as used herein refers to any and all permutations and combinations of the listed terms preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0195] As used herein, “minimum residual disease”, “minimal residual disease”, “measurable residual disease”, “molecular residual disease”, or “MRD” refers to a small number of diseased cells (e.g., cancer cells) that remain in the body after treatment (e.g., after chemotherapeutic or immunotherapeutic treatment). Detecting MRD may require highly sensitive diagnostic testing such as next generation sequencing and / or methods provided herein.Attorney Ref. No. GH0271WO

[0196] As used herein, “clonal hematopoiesis of indeterminate potential (CHIP)” is a clonally expanded hematopoietic stem cell with any one or more mutations (e.g., variants) that is detectable in a blood sample from a subject (e.g., a buffy coat or plasma sample). See e.g., Mamell et al., J. Mol Cell Cardiol (2021 doi: 10.1016 / j.yjmcc.2021.07.004. CHIP variants can be detected in the absence of disease (e.g., in a subject that does not have cancer). In some embodiments, the methods provided herein can be used to detect the presence of CHIP variants in a subject. In some embodiments, CHIP variants are not used to determine the presence or absence of cancer, precancer, orMRD.

[0197] A “reaction cleanup” refers to the removal of contaminants such as salts, enzymes, unincorporated dNTPs, primers, ethidium bromide, and other impurities that can interfere with downstream analysis. For example, when a reaction cleanup is performed between end repair and an A-tailing reaction, it removes unincorporated dNTPs such that the A-tailing reaction can be performed solely in the presence of dATP (i.e. not dCTP, dGTP and dCTP, as used in the end tailing reaction). Reaction cleanups can be performed using commercially available kits such as MinElute Reaction Cleanup Kit (Qiagen).

[0198] “Repaired regions”, also referred to as “synthesized regions” or “regions of the end-repaired DNA that were synthesized during the end repair” refer to regions of the DNA that were not present in the DNA prior to the end repair and A-tailing reactions. They are regions which have been synthesized by the polymerases used in the end repair and / or A tailing reactions, if present. In instances where the A-tailing is performed in the same tube as the end repair reaction, all four types of dNTPs will be present, and thus the polymerases used for A-tailing may generate synthesized regions, e.g. through nick translation. In instances where the A-tailing is performed separately to the end repair reaction, and these steps are separated by a reaction cleanup, only dATP will be present in the A-tailing reaction, and thus the polymerases used for A-tailing will not typically generate synthesized regions because the dNTP components are not all present in the A-tailing reaction mix.

[0199] As used herein, “base pairing specificity” refers to the standard DNA base (A, C, G, or T) for which a given base most preferentially pairs. Thus, for example, unmodified cytosine and 5-methylcytosine have the same base pairing specificity (i.e., specificity for G) whereas uracil and cytosine have different base pairing specificity because uracil has base pairing specificity for A while cytosine has base pairing specificity for G. The ability of uracil to form a wobble pair withAttorney Ref. No. GH0271WOG, for example, is irrelevant because uracil nonetheless most preferentially pairs with A among the four standard DNA bases.

[0200] A “type of dNTP” refers to a dNTP comprising a specific base, including A, T, G or C. Accordingly, wherein an end repair reaction is performed with dNTPs, wherein at least one type of dNTP comprises a modified base, the end repair reaction may be performed using dCTP comprising 5mC, and dATP, dTTP and dGTP all comprising non-modified bases.

[0201] “Capable of identifying the base modification in the at least one type of dNTP” refers to the ability of a modification-sensitive sequencing method to detect the presence or absence of the base modification in the at least one type of dNTP comprising a modified base used in the end repair. This detection of the base modification may be direct, such as in nanopore sequencing or single molecule real time sequencing, wherein the sequencing data itself indicates the presence or absence of a base modification. Alternatively, the detection of the base modification may be indirect, for example wherein the method involves a conversion procedure which alters the base pairing specificity dependent on the base modification status. It is these changes in base pairing specificity which can be detected by the sequencing method, e.g. through the comparison of the sequencing data to a reference sequence. Moreover, a modification-sensitive sequencing method is capable of identifying the base modification in the at least one type of dNTP regardless of whether it can distinguish one base modification from all other base modifications. For example, one form of modification-sensitive sequencing is sequencing after bisulfite conversion. This method is capable of distinguishing 5hmC and 5mC from unmethylated cytosine, but cannot distinguish 5hmC from 5mC.

[0202] Bases of the “same identity” refer to the same base, regardless of modification status of that base. For example, cytosine is considered to be the “same identity” as 5 -methyl cytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC), despite them having different modification statuses.

[0203] DNA is “derived from cancerous cells” if it originated from a tumor cell. Cell free DNA derived from cancerous cells includes ctDNA or circulating tumor DNA. Tumor cells are neoplastic cells that originated from a tumor, regardless of whether they remain in the tumor or become separated from the tumor (as in the cases, e.g., of metastatic cancer cells and circulating tumor cells).

[0204] The term “methylation” or “DNA methylation” refers to addition of a methyl group to a nucleotide base in a nucleic acid molecule. In some embodiments, methylation refers to additionAttorney Ref. No. GH0271WOof a methyl group to a cytosine at a CpG site (cytosine-phosphate-guanine site (i.e., a cytosine followed by a guanine in a 5’ -> 3’ direction of the nucleic acid sequence)). In some embodiments, DNA methylation refers to addition of a methyl group to adenine, such as in N6-methyladenine (6mA). In some embodiments, DNA methylation is 5-methylation (modification of the carbon in the 5th position of the cytosine ring). In some embodiments, 5-methylation refers to addition of a methyl group to the 5C position of the cytosine to create 5 -methyl cytosine (5mC). In some embodiments, methylation comprises a derivative of 5mC. Derivatives of 5mC include, but are not limited to, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-caryboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the carbon in the 3rdposition of the cytosine ring). In some embodiments, 3C methylation comprises addition of a methyl group to the 3C position of the cytosine to generate 3-methylcytosine (3mC). Methylation can also occur at non-CpG sites, for example, methylation can occur at a CpA, CpT, or CpC site. DNA methylation can change the activity of methylated DNA region. For example, when DNA in a promoter region is methylated, transcription of the gene may be repressed. DNA methylation is critical for normal development and abnormality in methylation may disrupt epigenetic regulation. The disruption, e.g., repression, in epigenetic regulation may cause diseases, such as cancer. Promoter methylation in DNA may be indicative of cancer.

[0205] The “modified nucleoside profile of DNA” means the position and identity of the nucleoside and the modification status of the nucleoside, such as methylations, within a DNA sequence. As described above, different modification sensitive sequencing methods can be used to detect such modifications. This includes methods which involve conversion followed by sequencing detect one or more different types of modified or unmodified nucleoside. For example, the TAPS method detects, but does not distinguish between, 5-methylcytosine (5mC) and 5-hydroxymethyl-cytosine (5hmC). Hence, a method for analyzing the modified nucleoside profile of DNA in a sample typically means identifying particular modifications or groups of modification, such as 5mC and / or 5hmC. Modified nucleosides are identified according to the specific method / conversion procedure being used as described above. This generally involves comparing sequence data obtained from DNA that has been subjected to a conversion procedure to a reference sequence. Typically, the method involves (i) comparing the sequence data with (A) one or more pre-determined reference sequence; or (B) sequence data obtained by sequencing a sub-sample of the DNA that was not subjected to the conversion procedure, for example a subsample that wasAttorney Ref. No. GH0271WOseparated before subjecting a separate subsample to the conversion procedure, for example as described herein; and (ii) identifying point differences between the converted DNA sequences and the reference sequence(s) (A) or non-converted DNA sequences (B) as nucleosides (in the initial sample) having a modification status that permits a change in base pairing specificity on exposure to the conversion procedure.

[0206] As used herein, a modification or other feature is present in “a greater proportion” in a first sample or population of nucleic acid than in a second sample or population when the fraction of nucleotides with the modification or other feature is higher in the first sample or population than in the second population. For example, if in a first sample, one tenth of the nucleotides are mC, and in a second sample, one twentieth of the nucleotides are mC, then the first sample comprises the cytosine modification of 5-methylation in a greater proportion than the second sample.

[0207] As used herein, “without substantially altering base-pairing specificity” of a given nucleobase means that a majority of molecules comprising that nucleobase that can be sequenced do not have alterations of the base pairing specificity of the second nucleobase relative to its base pairing specificity as it was in the originally isolated sample. In some embodiments, 75%, 90%, 95%, or 99% of molecules comprising that nucleobase that can be sequenced do not have alterations of the base pairing specificity of the second nucleobase relative to its base pairing specificity as it was in the originally isolated sample.

[0208] As used herein, “base pairing specificity” refers to the standard DNA base (A, C, G, or T) for which a given base most preferentially pairs. Thus, for example, unmodified cytosine and 5-methylcytosine have the same base pairing specificity (i.e., specificity for G) whereas uracil and cytosine have different base pairing specificity because uracil has base pairing specificity for A while cytosine has base pairing specificity for G. The ability of uracil to form a wobble pair with G is irrelevant because uracil nonetheless most preferentially pairs with A among the four standard DNA bases.

[0209] As used herein, “modified cytosine” refers to a cytosine in which at least one position of the cytosine has been substituted with a chemical moiety, such as a methyl or hydroxymethyl, that is different from the substituent at that position in unmodified cytosine. For the avoidance of doubt, “modified cytosine” does not include unmodified cytosine.

[0210] As used herein, a “combination” comprising a plurality of members refers to either of a single composition comprising the members or a set of compositions in proximity, e g., in separateAttorney Ref. No. GH0271WOcontainers or compartments within a larger container, such as a multiwell plate, tube rack, refrigerator, freezer, incubator, water bath, ice bucket, machine, or other form of storage.

[0211] The “capture yield” of a collection of probes for a given target set refers to the amount (e.g., amount relative to another target set or an absolute amount) of nucleic acid corresponding to the target set that the collection of probes captures under typical conditions. Exemplary typical capture conditions are an incubation of the sample nucleic acid and probes at 65°C for 10-18 hours in a small reaction volume (about 20 LLL containing stringent hybridization buffer. The capture yield may be expressed in absolute terms or, for a plurality of collections of probes, relative terms. When capture yields for a plurality of sets of target regions are compared, they are normalized for the footprint size of the target region set (e.g., on a per-kilobase basis). Thus, for example, if the footprint sizes of first and second target regions are 50 kb and 500 kb, respectively (giving a normalization factor of 0.1), then the DNA corresponding to the first target region set is captured with a higher yield than DNA corresponding to the second target region set when the mass per volume concentration of the captured DNA corresponding to the first target region set is more than 0.1 times the mass per volume concentration of the captured DNA corresponding to the second target region set. As a further example, using the same footprint sizes, if the captured DNA corresponding to the first target region set has a mass per volume concentration of 0.2 times the mass per volume concentration of the captured DNA corresponding to the second target region set, then the DNA corresponding to the first target region set was captured with a two-fold greater capture yield than the DNA corresponding to the second target region set.

[0212] As used herein, a “differentially methylated region” (DMR) refers to a region of DNA having a detectably different degree of methylation in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type; or having a detectably different degree of methylation in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type irrespective of the status of the biological condition of a subject; or having a detectably different degree of methylation in at least one cell or tissue type obtained from a subject having a disease or disorder relative to the degree of methylation in the same region of DNA in the same cell or tissue type obtained from a healthy subject. In some embodiments, a DMR has a detectably higher degree of methylation (e.g., a hypermethylated region) in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type orAttorney Ref. No. GH0271WOfrom the same cell or tissue type from a healthy subject. In some embodiments, a DMR has a detectably lower degree of methylation (e.g., a hypomethylated region) in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type or from the same cell or tissue type from a healthy subject.

[0213] “Specifically binds” in the context of an probe or other oligonucleotide and a target sequence means that under appropriate hybridization conditions, the oligonucleotide or probe hybridizes to its target sequence, or replicates thereof, to form a stable probe:target hybrid, while at the same time formation of stable probe :non-target hybrids is minimized. Thus, a probe hybridizes to a target sequence or replicate thereof to a sufficiently greater extent than to a nontarget sequence, to enable capture or detection of the target sequence. Appropriate hybridization conditions are well-known in the art, may be predicted based on sequence composition, or can be determined by using routine testing methods (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57, incorporated by reference herein).

[0214] “Sequence-variable target region set” refers to a set of target regions that may exhibit changes in sequence such as nucleotide substitutions (i.e., single nucleotide variations), insertions, deletions, or gene fusions or transpositions in neoplastic cells (e.g., tumor cells and cancer cells).

[0215] “Epigenetic target region set” refers to a set of target regions that may show sequenceindependent changes in neoplastic cells (e.g., tumor cells and cancer cells) or that may show sequence-independent changes in cfDNA from subjects having cancer relative to cfDNA from healthy subjects. Examples of sequence-independent changes include, but not limited to, changes in methylation (increases or decreases), nucleosome distribution, CTCF binding, transcription start sites, and regulatory protein binding regions. For present purposes, loci susceptible to neoplasia-, tumor-, or cancer-associated focal amplifications and / or gene fusions may also be included in an epigenetic target region set because detection of a change in copy number by sequencing or a fused sequence that maps to more than one locus in a reference genome tends to be more similar to detection of exemplary epigenetic changes discussed above than detection of nucleotide substitutions, insertions, or deletions, e.g., in that the focal amplifications and / or gene fusions can be detected at a relatively shallow depth of sequencing because their detection does not depend on the accuracy of base calls at one or a few individual positions.Attorney Ref. No. GH0271WO

[0216] The term “hypermethylation” refers to an increased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules within a population (e.g., sample) of nucleic acid molecules. In some embodiments, hypermethylated DNAcan include DNA molecules comprising at least 1 methylated residue, at least 2 methylated residues, at least 3 methylated residues, at least 5 methylated residues, or at least 10 methylated residues.

[0217] The term “hypomethylation” refers to a decreased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules within a population (e.g., sample) of nucleic acid molecules. In some embodiments, hypomethylated DNA includes unmethylated DNA molecules. In some embodiments, hypomethylated DNAcan include DNA molecules comprising 0 methylated residues, at most 1 methylated residue, at most 2 methylated residues, at most 3 methylated residues, at most 4 methylated residues, or at most 5 methylated residues.

[0218] As used herein, “methylation status” can refer to the presence or absence of methyl group on a DNA base (e.g. cytosine) at a particular genomic position in a nucleic acid molecule. It can also refer to the degree of methylation in a nucleic acid sequence (e.g., highly methylated, low methylated, intermediately methylated or unmethylated nucleic acid molecules). The methylation status can also refer to the number of nucleotides methylated in a particular nucleic acid molecule.

[0219] As used herein, DNA that is “not immobilized” or that is “free in solution” refers to DNA that is not bound covalently or non-covalently to a solid support, such as a bead. Such DNA may be free in solution during any step (such as all steps) of the disclosed methods.

[0220] As used herein, “partitioning” refers to physically separating or fractionating a mixture of nucleic acid molecules in a sample based on a characteristic of the nucleic acid molecules. The partitioning can be physical partitioning of molecules. Partitioning can involve separating the nucleic acid molecules into groups or sets based on the level of epigenetic feature (for e.g., methylation). For example, the nucleic acid molecules can be partitioned based on the level of methylation of the nucleic acid molecules. In some embodiments, the methods and systems used for partitioning may be found in PCT Patent Application No. PCT / US2017 / 068329, which is hereby incorporated by reference in its entirety.

[0221] As used herein, “partitioned set” or “partition” refers to a set of nucleic acid molecules partitioned into a set or group based on the differential binding affinity of the nucleic acid molecules or proteins associated with the nucleic acid molecules to a binding agent. A partitioned set may also be referred to as a subsample. The binding agent binds preferentially to the nucleicAttorney Ref. No. GH0271WOacid molecules comprising nucleotides with epigenetic modification. For example, if the epigenetic modification is methylation, the binding agent can be a methyl binding domain (MBD) protein. In some embodiments, a partitioned set can comprise nucleic acid molecules belonging to a particular level or degree of epigenetic feature (for e.g., methylation). For example, the nucleic acid molecules can be partitioned into three sets - one set for highly methylated nucleic acid molecules (first subsample, hyper partition, hyper partitioned set or hypermethylated partitioned set), a second set for low methylated nucleic acid molecules (second subsample, hypo partition, hypo partitioned set or hypomethylated partitioned set), and a third set for intermediate methylated nucleic acid molecules (third subsample, intermediate partitioned set, intermediately methylated partitioned set, residual partitioned set, or residual partition). In another example, the nucleic acid molecules can be partitioned based on the number of methylated nucleotides - one partitioned set can have nucleic acid molecules with nine methylated nucleotides, and another partitioned set can have unmethylated nucleic acid molecules (zero methylated nucleotides).

[0222] The terms “agent that recognizes a modified nucleobase in DNA,” such as an “agent that recognizes a modified cytosine in DNA” refers to a molecule or reagent that binds to or detects one or more modified nucleobases in DNA, such as methyl cytosine. A “modified nucleobase” is a nucleobase that comprises a difference in chemical structure from an unmodified nucleobase. In the case of DNA, an unmodified nucleobase is adenine, cytosine, guanine, or thymine. In some embodiments, a modified nucleobase is a modified cytosine. In some embodiments, a modified nucleobase is a methylated nucleobase. In some embodiments, a modified cytosine is a methyl cytosine, e.g., a 5-methyl cytosine. In such embodiments, the cytosine modification is a methyl. Agents that recognize a methyl cytosine in DNA include but are not limited to “methyl binding reagents,” which refer herein to reagents that bind to a methyl cytosine. Methyl binding reagents include but are not limited to methyl binding domains (MBDs) and methyl binding proteins (MBPs). In some such embodiments, the DNA may be single-stranded or double-stranded. Suitable agents include agents that recognize modified nucleotides in double-stranded DNA, singlestranded DNA, and both double-stranded and single-stranded DNA.

[0223] As used herein, “quantitative measure” refers to an absolute or relative measure. A quantitative measure can be, without limitation, a number, a statistical measurement (e.g., frequency, mean, median, standard deviation, or quantile), or a degree or a relative quantity (e.g., high, medium, and low). A quantitative measure can be a ratio of two quantitative measures. AAttorney Ref. No. GH0271WOquantitative measure can be a linear combination of quantitative measures. A quantitative measure may be a normalized measure.

[0224] A “X1 / / / / / / X2 mutation” in a specified polypeptide as used herein, where Xi and X2 are amino acids and mm is a position in an amino acid sequence, refers to a substitution in the polypeptide of amino acid Xi present at position mm of the full-length wild-type polypeptide with amino acid X2. The polypeptide is the human polypeptide unless indicated otherwise. The polypeptide comprising the X 1 / 7 / 711X2 mutation may, but does not necessarily, comprise additional differences from the wild-type sequence, including but not limited to truncations and deletions as well as other substitutions. For example, a “T1372S mutation” in TET2 refers to a substitution in a TET2 enzyme of the threonine present at position 1372 of the full-length wild-type human TET2 enzyme with a serine. Position 1372 of wild-type human TET2 aligns to position 258 and 248, respectively, of the truncated TET2 sequences disclosed as SEQ ID NOs: 23 and 24 of US Patent 10,961,525. Similarly, a “VI 900X2 mutation” where X2 is A, C, G, I, or P in TET2 refers to a substitution in a TET2 enzyme of the valine present at position 1900 of the full-length wild-type human TET2 enzyme with an alanine, cysteine, glycine, isoleucine, or proline.

[0225] “Or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise.III. Exemplary embodiments:

[0226] In some embodiments, the methods may comprise:a. Obtaining tumor tissue (and optionally buffy coat or normal tissue) from a subject in need thereof, wherein the subject has or is suspected of having cancer and / or minimal residual disease (MRD);b. Profiling Nucleic Acid from tumor tissue - for somatic DNA mutation changes that are non-silent (present in gene-product; e.g. SNV, indel, SV, CN, etc.), for DNA methylation changes known to lead to expression increase of gene-products, for RNA changes (expression level, somatic alterations, including fusions, exon skipping);c. Optionally profiling matched normal DNA (e.g., from a buffy coat sample, or a normal adjacent tissue (NAT) sample) in the same method as step b;Attorney Ref. No. GH0271WOd. From variants detected in step b, optionally fdtered by results found in step c, determining a personalized set of biomarkers to track gene-products in MRD test samples from the subject;e. Optionally generating a custom, subject-specific biomarker panel for assaying subject-specific biomarkers from step d;f. Assaying MRD test sample (e.g., a plasma sample) for the presence or absence of the personalized gene-product biomarkers to determine a the presence or absence of cancer and / or MRD; and optionally quantifying the amount of the personalized gene-product biomarkers.

[0227] In some embodiments, where the methods use extracellular vesicle (EV)-surface protein blood assays, personalized biomarker search aims to identify a or a combination of gene-products that are predicted to be displayed on surface of EVs derived from patient's tumor cells, and not in healthy normal blood / plasma. Empirical proteomics and RNA-seq data can be used to predict if protein gene-product is predicted to be expressed on cell's surface, and cancer-derived EVs have the same or a similar surfaceome as the parent cancer cell. Human proteome atlas is example of database using RNA-seq data to predict protein sub-localization in / on cell. In some embodiments, the methods described herein can result in potentially orders of magnitude higher MRD sensitivity (if gene-products have amplified signal), and potentially lower costs (if smaller number of geneproduct variants are tracked, in simpler non-NGS assays to achieve optimal sensitivity).IV. General Features of the MethodsA. Samples and Subjects

[0228] A sample can be any biological sample isolated from a subject. Samples can include body tissues, whole blood, platelets, serum, plasma, buffy coat, stool, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies (e.g., biopsies from known or suspected solid tumors), cerebrospinal fluid, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid (e.g., fluid from intercellular spaces), gingival fluid, crevicular fluid, bone marrow, pleural effusions, cerebrospinal fluid, saliva, mucous, sputum, semen, sweat, and urine. Samples may be bodily fluids, such as blood and fractions thereof, and urine. Such samples can include nucleic acids shed from tumors. The nucleic acids can include DNA and RNA, and can be in double- and single-stranded forms. A sample can be in the form originally isolated from a subject or can have been subjected to further processing to remove or add components, such asAttorney Ref. No. GH0271WOcells, enrich for one component relative to another, or convert one form of nucleic acid to another, such as RNA to DNA or single-stranded nucleic acids to double-stranded. Thus, for example, a bodily fluid for analysis can be plasma or serum containing cell-free nucleic acids, e.g., cell-free DNA (cfDNA).

[0229] In some cases, one or more DNA sample is obtained or has been obtained from a subject. In some embodiments, a first DNA sample is obtained from the subject at a first time point. In some embodiments, the first sample comprises tumor tissue. In some embodiments a second DNA sample is obtained from the subject at a second time point wherein the second timepoint is later than the first timepoint. In some embodiments, the second sample comprises cell free DNA (cfDNA). In some embodiments, the second sample comprises a plasma sample. In some embodiments, a third DNA sample is obtained from the subject at a third time point, which is identical to or different from the first time point. In some embodiments, the third sample comprises a healthy tissue sample. In some embodiments, the third sample comprises normal tissue adjacent to the tumor. In some embodiments, the third sample comprises a buffy coat sample.

[0230] The subject may be a human, a mammal, an animal, a primate, rodent (including mice and rats), or other common laboratory, domestic, companion, service or agricultural animal, for example a rabbit, dog, cat, horse, cow, sheep, goat or pig. Preferably, the subject is human. The subject may in some cases have or be suspected of having a cancer, tumor, or neoplasm. In other cases, the subject may not have cancer or a detectable cancer symptom. The subject may have been treated with one or more cancer therapy, e.g., any one or more of chemotherapies, antibodies, vaccines or biologies. The subject may be in remission, e.g. from a tumor, cancer, or neoplasia (e.g., following treatment such as chemotherapy, surgical resection, radiation, or a combination thereof). The subject may or may not be diagnosed as being susceptible to cancer or any cancer-associated genetic mutations / disorders. In some embodiments, the first sample is a diseased tissue sample. In some embodiments, the first sample is a DNA sample obtained from a tumor tissue biopsy. The cancer, tumor, or neoplasm may generally be of any type, for example a cancer tumor or neoplasm of the lung, colon, rectum (or colorectum), kidney, breast, prostate, or liver, or other type of cancer as described herein.

[0231] In some embodiments, the first, second, and third samples are obtained from a subject who was previously diagnosed with cancer and received one or more previous cancer treatments. In some such embodiments, the first and third samples are obtained at one or more preselectedAttorney Ref. No. GH0271WOtimepoints prior to the one or more previous cancer treatments. In some embodiments, the second sample is obtained from a subject in remission from a tumor, cancer, or neoplasia (e.g., following chemotherapy, surgical resection, radiation, or a combination thereof).

[0232] In any of the foregoing embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia may be of the bladder, head and neck, lung, colon, rectum, kidney, breast, prostate, skin, or liver. In some embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia is of the lung. In some embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia is of the colon or rectum. In some embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia is of the breast. In some embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia is of the prostate. In any of the foregoing embodiments, the subject may be a human subject. In some embodiments, the first, second, and / or third sample is obtained from a subject having a stage I cancer, stage II cancer, stage III cancer or stage IV cancer.

[0233] In some embodiments, the subject may have an infection, a transplant rejection, or other disease or disorder related to changes in the immune system. The subject may not have cancer or a detectable cancer symptom. The subject may have been treated with one or more cancer therapy, e.g., any one or more of chemotherapies, antibodies, vaccines or biologies. The subject may be in remission. The subject may or may not be diagnosed as being susceptible to cancer or any cancer-associated genetic mutations / disorders.

[0234] In some embodiments, the sample volume of bodily fluid taken from a subject depends on the desired read depth for sequenced regions. Examples of volumes are about 0.4-40 milliliters (mb), about 5-20 mL, about 10-20 mb. For example, the volume can be about 0.5 mb, about 1 mb, about 5 mL, about 10 mL, about 20 mL, about 30 mL, about 40 mL, or more milliliters. A volume of sampled plasma is typically between about 5 mL to about 20 mL.

[0235] The sample can comprise various amounts of nucleic acid. Typically, the amount of nucleic acid in a given sample is equates with multiple genome equivalents. For example, a sample of about 30 nanograms (ng) DNAcan contain about 10,000 (104) haploid human genome equivalents and, in the case of cfDNA, about 200 billion (2 x 1011) individual polynucleotide molecules. Similarly, a sample of about 100 ng of DNA can contain about 30,000 haploid human genome equivalents and, in the case of cfDNA, about 600 billion individual molecules.Attorney Ref. No. GH0271WO

[0236] In some embodiments, a sample comprises nucleic acids from different sources, e.g., from cells and from cell-free sources (e.g., blood samples, etc.). Typically, a sample includes nucleic acids carrying mutations. For example, a sample optionally comprises DNA carrying germline mutations and / or somatic mutations. Typically, a sample comprises DNA carrying cancer-associated mutations (e.g., cancer-associated somatic mutations). A sample can comprise an epigenetic variant, wherein the epigenetic variant associated with the presence of a genetic variant such as a cancer-associated mutation. In some embodiments, the sample comprises an epigenetic variant associated with the presence of a genetic variant, wherein the sample does not comprise the genetic variant.

[0237] In some embodiments, a population of nucleic acids is obtained from a serum, plasma or blood sample from a subject having or suspected of having neoplasia, a tumor, precancer, or cancer or previously diagnosed with neoplasia, a tumor, precancer, or cancer. The population includes nucleic acids having varying levels of sequence variation and / or epigenetic variation, such as posttranslation modifications (PTMs) of chromatin and / or nucleobase modifications, e.g., modifications of cytosine, particularly at the 5-position of the nucleobase, e.g., 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine.

[0238] The first, second, and / or third DNA sample may be or comprise cell free nucleic acids or cfDNA. The cfDNA may be obtained from a test subject, for example as described above. For example, the sample for analysis may be plasma or serum containing cell-free nucleic acids. “Cell-free DNA” “cfDNA molecules,” or “cfDNA”, for example, include DNA molecules that naturally occur in a subject in extracellular form (e.g., in blood, serum, plasma, or other bodily fluids such as lymph, cerebrospinal fluid, urine, or sputum). While the cfDNA originally existed in a cell or cells in a large complex biological organism, e.g., a mammal, it has undergone release from the cell(s) in vivo into a fluid found in the organism, and may be obtained by obtaining a sample of the fluid without the need to perform an in vitro cell lysis step. In other words, cell-free nucleic acids or cfDNA are nucleic acids or DNA not contained within or otherwise bound to a cell, or the nucleic acids or DNA remaining in a sample after removing intact cells. Cell-free nucleic acids include DNA, RNA, and hybrids thereof, including genomic DNA, mitochondrial DNA, siRNA, miRNA, circulating 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 can be double-stranded, single-stranded, or a hybrid thereof. A cell-freeAttorney Ref. No. GH0271WOnucleic acid can be released into bodily fluid through secretion or cell death processes, e.g., cellular necrosis and apoptosis. Some cell-free nucleic acids are released into bodily fluid from cancer cells e.g., circulating tumor DNA, (ctDNA). 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 by a mixture of tumor cells and non-tumor cells.

[0239] Example amounts of cell-free nucleic acids in a sample before amplification typically range from about 1 femtogram (fg) to about 1 microgram (pg), e.g., about 1 picogram (pg) to about 200 nanograms (ng), about 1 ng to about 100 ng, about 10 ng to about 1000 ng. In some embodiments, a sample includes 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. Optionally, the amount is at least about 1 fg, at least about 10 fg, at least about 100 fg, at least about 1 pg, at least about 10 pg, at least about 100 pg, at least about 1 ng, at least about 10 ng, at least about 100 ng, at least about 150 ng, or at least about 200 ng of cell-free nucleic acid molecules. In some embodiments, the amount is up to about 1 fg, about 10 fg, about 100 fg, about 1 pg, about 10 pg, about 100 pg, about 1 ng, about 10 ng, about 100 ng, about 150 ng, or about 200 ng of cell-free nucleic acid molecules. In some embodiments, methods include obtaining between about 1 fg to about 200 ng cell-free nucleic acid molecules from samples.

[0240] Cell-free nucleic acids typically have a size distribution of between about 100 nucleotides in length and about 500 nucleotides in length, with molecules of about 110 nucleotides in length to about 230 nucleotides in length representing about 90% of molecules in the sample, with a mode of about 168 nucleotides length (in samples from human subjects) and a second minor peak in a range between about 240 nucleotides to about 440 nucleotides in length. In some embodiments, cell-free nucleic acids are from about 160 nucleotides to about 180 nucleotides in length, or from about 320 nucleotides to about 360 nucleotides in length, or from about 440 nucleotides to about 480 nucleotides in length.

[0241] In some embodiments, cell-free nucleic acids are isolated from bodily fluids through a partitioning step in which cell-free nucleic acids, as found in solution, are separated from intact cells and other non-soluble components of the bodily fluid. In some embodiments, partitioning includes techniques such as centrifugation or fdtration. Alternatively, cells in bodily fluids may be lysed, and cell-free and cellular nucleic acids may be processed together. Generally, afterAttorney Ref. No. GH0271WOaddition of buffers and wash steps, cell-free nucleic acids may be precipitated with, for example, an alcohol. In some embodiments, additional clean-up steps are used, such as silica-based columns to remove contaminants or salts. Non-specific bulk carrier nucleic acids, for example, are optionally added throughout the reaction to optimize aspects of the example procedure, such as yield. After such processing, samples typically include various forms of nucleic acids including double-stranded DNA, single-stranded DNA and / or single-stranded RNA. Optionally, singlestranded DNA and / or single-stranded RNA are converted to double-stranded forms so that they are included in subsequent processing and analysis steps.

[0242] A sample can be isolated or obtained from a subject and transported to a site of sample analysis. The sample may be preserved and shipped at a desirable temperature, e.g., room temperature, 4°C, -20°C, and / or -80°C. A sample can be isolated or obtained from a subject at the site of the sample analysis.

[0243] In some embodiments, the DNA from each of the first, second, and third samples comprises cell-free DNA. In some embodiments, the DNA from the second sample comprises cell free DNA. In some embodiments the DNA sample from each of the first, second, and third samples is a DNA sample from a formalin fixed paraffin embedded (FFPE) sample. In some embodiments, the first sample is a tumor tissue sample. In some embodiments, the first sample is a FFPE tumor tissue sample.

[0244] In some embodiments, the DNA from the second sample is from plasma. The volume of plasma used to obtain the DNA sample can depend on the desired read depth for sequenced regions. Exemplary volumes are 0.4-40 ml, 5-20 ml, 10-20 ml. For example, the volume can be 0.5 m , 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, or 40 mL. A volume of sampled plasma may be 5 to 20 mL. In some embodiments, the sample volume is 3-5 mL of plasma, such as 4 mL of plasma, per 10 mL whole blood.

[0245] In some embodiments, the sample comprises whole blood. Exemplary volumes of sampled whole blood are 0.4-40 mL, 5-20 mL, 10-20 mL, 1-6 mL, 1-3 mL, and 3-5 mL. For example, the volume can be 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, or 40 mL. A volume of sampled whole blood may be 5 to 20 mL. In some embodiments, the sample volume is 1-5 mL of whole blood, such as 2.5 mL of whole blood.

[0246] In some embodiments, the third sample comprises buffy coat separated from whole blood. Exemplary volumes of sampled buffy coat are 0.1-20 mL, 1-10 mL, 1-5 mL, 0.2-0.6 mL, and 0.3-Attorney Ref. No. GH0271WO0.5 mL. For example, the volume can be 0.1 mL, 0.2 mL, 0.3 L, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mb, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL 10 mL, or 20 mL. A volume of sampled buffy coat may be 1 to 10 mL. In some embodiments, the sample volume is 0.1-0.5 mL of buffy coat, such as 0.3 mL of buffy coat, per 10 mL whole blood.

[0247] In some embodiments, the third sample comprises PBMCs separated from whole blood. Exemplary volumes of sampled PBMCs are 0.1-20 mL, 1-10 mL, 1-5 mL, 0.2-0.6 mL, and 0.3-0.5 mL. For example, the volume can be 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL 10 mL, or 20 mL. A volume of sampled PBMCs may be 1 to 10 mL. In some embodiments, the sample volume is 0.1-0.5 mL of PBMCs, such as 0.3 mL of PBMCs, per 10 mL whole blood.

[0248] In some embodiments, the third sample comprises leukocytes separated from subject blood using leukapheresis. Exemplary volumes of sampled leukocytes from leukapheresis are 0.1-20 mL, 1-10 mL, 1-5 mL, 0.2-0.6 mL, and 0.3-0.5 mL. For example, the volume can be 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, or 20 mL. A volume of sampled leukocytes from leukapheresis may be 1 to 10 mL. In some embodiments, the sample volume is 0.1 -0.6 mL of leukocytes from leukapheresis, such as 0.4 mL of leukocytes, per 10 mL whole blood.

[0249] A sample can comprise various amounts of DNA that contain genome equivalents. For example, a sample of about 30 ng DNA can contain about 10,000 (104) haploid human genome equivalents and, in the case of cell free DNA (cfDNA), about 200 billion (2xlOn) individual polynucleotide molecules. Similarly, a sample of about 100 ng of DNA can contain about 30,000 haploid human genome equivalents and, in the case of cfDNA, about 600 billion individual molecules.

[0250] After such processing, samples can include various forms of nucleic acid including double stranded DNA, single stranded DNA and single stranded RNA. In some embodiments, single stranded DNA and RNA can be converted to double stranded forms so they are included in subsequent processing and analysis steps.

[0251] The methods disclosed herein are also particularly suited for the analysis of DNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. While the formalin fixation process adequately preserves the ultrastructure of the tissues, it results in various types of damage to the DNA within the tissues, such as nicks in the DNA. As explained elsewhere herein, these nicks canAttorney Ref. No. GH0271WOlead to synthesis of regions of the DNA molecule in the end repair process. The methods disclosed herein allow for these regions to be identified and the sequence data to be interpreted accordingly.

[0252] Reference or control molecules can be added to or spiked into a sample as a control or normalization standard. For example, a certain amount of modified DNA from a species other than the species of the subject from which the sample was obtained or synthetic nucleic acids comprising certain modifications may be added to the sample. In some embodiments, the reference or control molecules are distinguishable from the molecules originally present in the sample. In some embodiments, the detected DNA sequences are normalized to the reference or control molecules.B. End repair and A-tailing

[0253] In some embodiments, the disclosed methods comprise subjecting DNA from the first, second, and / or third samples or a subsample thereof (e g., a hypomethylated and / or hypermethylated subsample thereof) to end repair to generate end-repaired DNA molecules. In some embodiments, end repair is performed prior to a sequencing step. In some embodiments, end repair is performed prior to a capturing step. In some embodiments, end repair is performed after a capturing step and prior to a sequencing step. In some embodiments, the end repair is performed before separating at least a portion of probe-target complexes from other nucleic acids in the sample. In some embodiments, the end repair is performed after separating at least a portion of probe-target complexes from other nucleic acids in the sample. In some embodiments, the end repair is performed before two or more, or each, of the steps mentioned above. In some embodiments, the end repair is performed using deoxynucleotide triphosphates (dNTPs). In some embodiments, at least one type of dNTP comprises a modified base, and the at least one dNTP comprising a modified base is incorporated into repaired regions of the end-repaired DNA molecules at one or more locations.

[0254] End repair includes methods for repairing DNA by the conversion of non-blunt ended DNA into blunt ended DNA. Sequencing workflows typically use end repair to make ends of DNA molecules compatible with adapters, which are subsequently ligated onto the DNA. Fragmented and / or damaged DNA (e.g., cfDNA or DNA from FFPE samples) often contain non-blunt ends, which contain 3 ’ overhangs and / or 5 ’ overhangs. A 3 ’ overhang refers to the 3 ’ end of a DNA strand which extends beyond the 5 ’end of the paired strand, resulting in one or more unpaired nucleotides at the 3 ’end of the DNA strand. Conversely, a 5 ’overhang refers to the 5’ end of a DNA strandAttorney Ref. No. GH0271WOwhich extends beyond the 3’end of the paired strand, resulting in one or more unpaired nucleotides at the 5 ’end of the DNA strand.

[0255] The process of end repair involves the conversion of double-stranded DNA with 3’overhangs and / or 5’overhangs to double-stranded DNA without overhangs. This can be done using one or more enzymes such as T4 DNA polymerase and / or Klenow fragment. The 3’ to 5’ exonuclease activity of these enzymes removes the 3 ’ends at 3’overhangs and the 5’ to 3’ polymerase activity of these enzymes extends the 3’ ends at 5’ overhangs to remove the 5’ overhang, thereby generating a blunt-ended DNA molecule. In order to fill in these 5’ overhangs, end repair is conducted in the presence of dATP, dCTP, dGTP and dTTP End repair can also include a second step, which involves the addition of a phosphate group to the 5' ends of DNA, by an enzyme such as polynucleotide kinase. This makes the 5’ends of the end-repaired DNA molecules compatible with the subsequent action of DNA polymerases and DNA ligases.

[0256] As used herein, the term “A-tailing” refers to the addition of a single deoxyadenosine residue to the end of a blunt-ended double-stranded DNA fragment to form a 3' deoxyadenosine single-base overhang. Such A tailing reactions are conducted with polymerases which have the ability to add a non-templated A to the 3' end of a blunt, double-stranded DNA molecule. Polymerases capable of A-tailing typically do not possess 3’-5’ exonuclease activity. When A-tailing is performed as a separate reaction to end repair, it is typically conducted in the presence of dATP, but the absence of dCTP, dTTP and dGTP. A-tailed fragments are not compatible for selfligation (i.e., self-circularizatian and concatenation of the DNA), but they are compatible with 3' T-overhangs, which can be used on adapters. Methods comprising end repair, A-tailing and ligation to adapters with 3' T-overhangs can result in higher efficiency ligation, compared to blunt ended ligation, as blunt ligation can lead to self-ligation of the adapters and / or DNA molecules.

[0257] In some embodiments, the methods disclosed herein comprise end repair of the DNA molecules followed by blunt end ligation of adapters. In other embodiments, the methods disclosed herein comprise end repair of the DNA molecules followed by A-tailing and sticky-end ligation of T-tailed adapters. When the methods disclosed herein comprise an A-tailing step, it may be performed separately from the end repair with an intervening reaction clean-up step or it may be performed in the same reaction as the end repair (e.g. using NEBNext® Ultra™ II End Repair / dA-Tailing Module (E7546)). In some embodiments, the reaction clean-up step removes unincorporated dNTPs. In instances wherein the A-tailing reaction is performed in the sameAttorney Ref. No. GH0271WOreaction as end repair, the sticky-end ligation may be performed with a mixture of T-tailed adapters and C-tailed adapters.

[0258] End repair and A tailing reactions can have varying impacts on the composition of the DNA molecule, dependent on the exact workflow and reaction components used. These reactions can lead to the synthesis of regions at the 3 ’ends of DNA strands, but also the synthesis of internal regions through nick translation and through gap filling followed by ligation.

[0259] In some embodiments, end repair can lead to 3 ’fill in with unmethylated cytosines, which may not reflect the true methylation status of that position in the DNA molecule prior to the generation of the 5 ’overhang. In nicked DNA, polymerases which contain 5’ to 3’ exonuclease activity and / or strand displacement activity can lead to the synthesis of internal regions of the DNA molecule through nick translation. If the end repair reaction is conducted with non-methylated deoxycytidine triphosphate (dCTP), the synthesized regions will incorporate the non-methylated dCTP, potentially at positions which initially comprised methylated cytosines. In gapped DNA, both the DNA polymerases used in end repair and A tailing can lead to the generation of synthesized regions. The gaps can be fdled in with DNA polymerases used in the end repair reaction, regardless of whether they possess 5’ to 3’ exonuclease activity or strand displacement activity. After this gap filling, a nick will still exist between the synthesized region and the region of the original DNA molecule 3’ of the gap. The A-tailing enzymes may then introduce further synthesized regions through nick translation, as described for the nicked DNA. This synthesized region may extend to the 3 ’end of the DNA molecule.

[0260] In some embodiments, the end-repair and the A-tailing reactions are performed in a single tube. In such cases, the A tailing reaction can be performed at a higher temperature than the end repair. Optionally, end repair is performed at ambient temperature (e.g. 15-35°C) and A tailing is performed at a temperature over 60°C, including e.g., about 60°C-75°C. The A tailing reaction can be performed using a thermostable polymerase (e.g. Taq DNA polymerase, Tfl DNA polymerase, Bst DNA Polymerase, Large Fragment or Tth DNA polymerase) and the method further comprises increasing temperature of the sample after the end repair to inactivate the polymerase used in end repair (e.g. T4 DNA polymerase or Klenow fragment). In some embodiments the A-tailing is performed using a DNA polymerase that: (i) does not possess 5 ’-3’ exonuclease activity; and / or (ii) is not a strand displacing DNA polymerase. These properties reduce the ability of the DNA polymerase to extend from nick. This reduces the level of synthesis which may occur during theAttorney Ref. No. GH0271WOend repair and A-tailing reactions thus reducing the proportion of sequencing data that may be filtered out as potentially containing artifactual data. Accordingly, in some embodiments, the A-tailing is performed using a DNA polymerase that cannot extend from a nick in the DNA such as HemoKlen Taq. In other embodiments, the A-tailing is performed using Taq DNA polymerase. In other embodiments, the A-tailing is performed using Tfl polymerase, Bst DNA Polymerase, Large Fragment or Tth polymerase.

[0261] In order to reduce the level of synthesized regions, the end repair reaction can be performed using DNA polymerases can be used which lack 5 ’to 3’ exonuclease activity and / or strand displacement activity (e.g. T4 DNA polymerase or Klenow fragment).

[0262] In nicked DNA and gapped DNA, nick translation is reduced in end repair through the use of polymerases which lack 5 ’to 3’ exonuclease activity and / or strand displacement activity. The separation of the end repair and A tailing reaction by a reaction clean-up means that only dATP (not dCTP, dTTP or dGTP) is present in the A tailing reaction. This means that efficient nick translation cannot occur in the A tailing reaction because the three of the four nucleotide components are not present in the reaction mixture. In gapped DNA, the gaps can be filled in with DNA polymerases used in the end repair reaction, regardless of whether they possess 5’ to 3’ exonuclease activity or strand displacement activity. These filled gaps thereby generate synthesized regions.

[0263] In some embodiments, the end-repair is performed with a polymerase which lacks 5 ’to 3’ exonuclease activity and / or strand displacement activity. In some cases, the polymerase used in the end repair reaction may be Q5® High-Fidelity DNA Polymerase, Q5U® Hot Start High-Fidelity DNAPolymerase, Phusion® High-Fidelity DNAPolymerase, Hemo Klen 7c / r / , phi29DNA Polymerase, T7 DNA Polymerase, DNA Polymerase I (E. co / i), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”) or T4 DNA Polymerase. In some embodiments, the polymerase used in the end repair is T4 DNA Polymerase or Klenow fragment. In some embodiments, the end repair is performed with a DNA polymerase which has 5 ’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

[0264] In some embodiments, the methods disclosed herein comprise an A tailing reaction after the end repair and before the ligation reaction, wherein the end repair and A tailing reactions are separated by a reaction cleanup. The A tailing reaction is typically performed in the presence ofAttorney Ref. No. GH0271WOdATP, but in the absence of dCTP, dTTP and dGTP In some embodiments, the A tailing reaction is performed using Klenow Fragment lacking 3'-5' exonuclease activity.

[0265] In some embodiments, a dNTP that comprises a modified base is used in end repair, which may be any modified base wherein the presence or the absence of the modification can be detected by a type of sequencing. In some embodiments, a dNTP comprising a modified base can be used in a combined end repair and A-tailing reaction. In some embodiments, the modified base is incorporated in the synthesized regions at both CpG sites and CpH sites (i.e. CpA, CpC and CpT sites). While methylation of cytosines in non-CpG contexts has been described, it is thought to comprise 0.02% of total methyl-cytosine in differentiated somatic cells (Jang et al. Genes (Basel).2017 Jun; 8(6): 148). The presence of methylated cytosine in non-CpG contexts in the end-repaired DNA can therefore be interpreted as being introduced during the end repair and / or A-tailing reactions. Using a dNTP comprising a modified base can therefore be used to effectively label the synthesized regions of the end repaired DNA.

[0266] In some embodiments, a dNTP that comprises a modified base may comprise any modified base wherein the presence or the absence of the modification can be detected by a type of sequencing. The modified base may be 5-caryboxylcytosine (5-caC), 4-methylcytosine (4mC), 5-methylcytosine (5mC), 5-hydroxymethyl-cytosine (5hmC), N6-methyladenosine (6mA), bromodeoxyuridine (BrdU), 5-fluorodeoxyuridine (FldU), 5-iododeoxyuridine (IdU), 5-ethynyldeoxyuridine (EdU) and / or 8-oxoguanine (8oxoG).

[0267] When a dNTP comprising a modified base is used, it may be used in place of the equivalent unmodified base in the end repair reaction. For instance, if a dCTP comprising 5mC is used in the end repair reaction, there may be no dCTP comprising an unmodified cytosine. This would ensure that dCTPs incorporated into the DNA molecule during the end repair reaction contain 5mC. In some embodiments, multiple types of dNTP comprising a modified base are used in the end repair. For example, dATP comprising 6mA and dCTP comprising 5mC can be used in the end repair reaction in place of dATP comprising unmodified adenine and dCTP comprising unmodified cytosine. The use of multiple types of dNTP comprising a modified base is advantageous because it provides increased resolution in defining the regions of the end-repaired DNA molecule which have been synthesized during the end repair reaction. This is because, in this example, the end of a synthesized region can be defined as the first unmodified adenine or unmodified cytosine after aAttorney Ref. No. GH0271WOstretch of containing 6mAs and / or 5mCs, rather than relying on the detection of solely an unmodified adenine or solely an unmodified cytosine.

[0268] The sequencing method used will depend on the type of modified base used in the endrepair reaction such that the specific modification can be detected. Exemplary conversion-based methods are described above alongside the base modification which they can detect. Moreover, nanopore-based sequencing can be used to detect 5-caC, 4mC, 5mC, 5hmC, 6mA, BrdU, FldU, IdU, and EdU, and single-molecule real time (SMRT) sequencing from Pacific Biosciences can be used to detect 5-caC, 4mC, 5mC, 5hmC, 6mA, and 8oxoG.

[0269] The disclosed methods use at least one type of dNTP which comprises a modified base (e.g. a methylated deoxycytidine triphosphate, such as deoxycytidine triphosphate comprising 5-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC)) in the end repair reaction. In particular embodiments, the dNTP is 5mC. During end repair reactions, the methylated deoxycytidine triphosphate will be incorporated into the synthesized regions regardless of the sequence context. This will result in methylated cytosines in non-CpG positions (i.e., methylated cytosines in a CpH context), which are very rare in nature. These methylated non-CpG cytosines can therefore be used as labels for identifying synthesized regions in the end repaired DNA molecule. Similarly, other types of dNTP which comprise a modified base uncommon or absent in nature can be used. The identification of such modified bases can be performed using sequencing, and regions comprising these modifications can be interpreted as defining regions which were synthesized in the end repair reaction.

[0270] In other embodiments of the disclosed methods, the modified base is a methylated cytosine, such as 5mC or 5hmC. In some embodiments, the methylated cytosine is 5mCpH (where H = A, T, or C). In other embodiments, the modified base is other than 5mC or 5hmC. In some embodiments wherein the modified base is other than 5mC or 5hmC, a repaired region is defined as (i) the sequence between two non-modified bases spanning a modified base, wherein the bases are of the same identity to the modified bases present in the at least one type of dNTP; and / or (ii) the sequence between a non-modified base and the end of a sequence read, wherein there is no additional non-modified bases between the non-modified base and the end of the sequence read, where the non-modified bases are the same identity as the modified base present in the at least one type of dNTP. In some embodiments wherein the modified base is a methylated cytosine, such as 5mC or 5hmC, a repaired region is defined as (i) the sequence between two non-methylatedAttorney Ref. No. GH0271WOcytosines which span one or more methylated CpH cytosines; and / or (ii) the sequence between a methylated CpH cytosine and an end of a sequence read, wherein the methylated CpH cytosine is the CpH cytosine most distant from the end of the sequence read, or a subsequence thereof comprising one or more methylated CpH cytosines.

[0271] In some embodiments, a CpG-binding protein binds mCpG / mCpG dsDNA dyads. In some embodiments, in which the end repair reaction is conducted using an unmethylated deoxy cytidine triphosphate (dCTP), synthesized regions in the end-repaired molecules are called as unmethylated cytosines (on both strands) at these positions. In some embodiments, in which the end repair reaction is conducted using a methylated deoxycytidine triphosphate (dmCTP), synthesized regions (on both strands) in the end-repaired molecules are called as methylated cytosines at these positions if the opposing, template strand comprises an mCpG.C. Ligation to Adapters

[0272] In some embodiments, the methods comprise ligating adapters to DNA from the first, second, and / or third samples or a subsample thereof (e.g., a hypomethylated and / or hypermethylated subsample thereof). In some embodiments DNA molecules can be subjected to blunt-end ligation with blunt-ended adapters. In some embodiments, nucleic acids, e.g., DNA molecules can be subjected to sticky-end ligation with sticky-ended adapters. In some embodiments, once the nucleic acids, e.g., DNA has been end-repaired it can be subjected to blunt-end ligation with blunt-ended adapters, in cases where A-tailing is not performed, or sticky end ligation with T-tailed adapters, when A tailing is performed. Nucleic acids, e g., DNA molecules can be ligated to adapters at either one end or both ends. Nucleic acids, e.g., DNA molecules can be ligated with at least partially double stranded adapter (e.g., a Y shaped or bell-shaped adapter). In some embodiments, the ligation is performed prior to a sequencing step. In some embodiments, the ligation is performed prior to a capturing step. In some embodiments, the ligation is performed after a capturing step and prior to a sequencing step. When a conversion step is performed, in some embodiments, the ligation step can take place before or after the conversion step. In general, “conversion step” or “conversion procedure” refers to any step or procedure that changes the base pairing specificity of one or more nucleotides. In some embodiments, the ligation step occurs after an end-repair. In some embodiments, the ligation step occurs before contacting the DNA (e.g., DNA in the sample or subsample thereof) with a CpG-binding protein. In some embodiments, the ligation step occurs before contacting the DNA with a methyl-sensitive deaminase. In someAttorney Ref. No. GH0271WOembodiments, the ligation step occurs before contacting the DNA with a methyl-sensitive deaminase. In some embodiments, adapters are ligated to end-repaired DNA molecules or the adapters are ligated to the DNA molecule or a plurality of DNA molecules. In some such embodiments, the ligation reaction also seals nicks present in the end-repaired DNA.

[0273] In some embodiments, DNA ligase and adapters are added to ligate DNA molecules in the sample with an adapter on one or both ends, i.e. to form adapted DNA. As used herein, “adapter” refers to short nucleic acids (e.g., less than about 500, less than about 100 or less than about 50 nucleotides in length, or be 20-30, 20-40, 30-50, 30-60, 40-60, 40-70, 50-60, 50-70, 20-500, or 30-100 bases from end to end) that are typically at least partially double-stranded and can be ligated to the end of a given sample DNA molecule. In some instances, two adapters can be ligated to a single sample DNA molecule, with one adapter ligated to each end of the sample nucleic acid molecule.

[0274] In some embodiments, the ligase used in ligation reactions can act on both single strand DNA nicks and double stranded DNA ends. In some cases, the ligase is T4 DNA ligase or T3 DNA ligase. Adapters can include nucleic acid primer binding sites to permit amplification of a sample DNA molecule flanked by adapters at both ends, and / or a sequencing primer binding site, including primer binding sites for sequencing applications, such as various next generation sequencing (NGS) applications. Adapters can include a sequence for hybridizing to a solid support, e.g., a flow cell sequence. Adapters can also include binding sites for capture probes, such as an oligonucleotide attached to a flow cell support or the like. Adapters can also include sample indexes and / or molecular barcodes. These are typically positioned relative to amplification primer and sequencing primer binding sites, such that the sample index and / or molecular barcode is included in amplicons and sequencing reads of a given DNA molecule. Adapters of the same or different sequence can be linked to the respective ends of a sample DNA molecule. In some cases, adapters of the same or different sequence are linked to the respective ends of the DNA molecule except that the sample index and / or molecular barcode differs in its sequence. In some embodiments, the adapter is a Y-shaped adapter in which one end is blunt ended or tailed as described herein, for joining to a nucleic acid molecule, which is also blunt ended or tailed with one or more complementary nucleotides to those in the tail of the adapter. In another exemplary embodiment, an adapter is a bell-shaped adapter that includes a blunt or tailed end for joining to a DNA molecule to be analyzed. Other exemplary adapters include T-tailed, C-tailed or hairpinAttorney Ref. No. GH0271WOshaped adapters. For example, a hairpin shaped adapter can comprise a complementary double stranded portion and a loop portion, where the double stranded portion can be attached (e g. ligated) to a double-stranded polynucleotide. Hairpin shaped sequencing adapters can be attached to both ends of a polynucleotide fragment to generate a circular molecule, which can be sequenced multiple times. The adapters used in the methods of the present disclosure comprise one or more known modified nucleosides, such as methylated nucleosides. In some embodiments, the modified nucleosides comprise modification resistant cytosines. In some embodiments, each cytosine in each adapter is a modification resistant cytosine. In some embodiments, the modification resistant cytosine is a deamination resistant cytosine. In some embodiments, the deamination resistant cytosine comprises 5-propynylC (5pyC), 5-pyrrolo-dC (5pyrC), 5-hydroxymethylcytosine (5hmC), glucosylated5-hydroxymethylcytosine (5ghmC), cytosine 5-methylenesulfonate (CMS), or N4-modified cytosine. In some embodiments, the adapters are resistant to digestion by a methylation resistant restriction enzyme (MSRE). In some embodiments, the MSRE digestionresistant adapters comprise one or more methylated nucleotides, comprise one or more nucleotide analogs resistant to methylation sensitive restriction enzymes, or do not comprise a nucleotide sequence recognized by the MSRE. In some embodiments, the one or more methylated nucleotides in the MSRE digestion-resistant adapters comprise 5-methylcytosine and / or 5-hydroxymethylcytosine. In some embodiments, the adapters are resistant to digestion by a methylation dependent restriction enzyme (MDRE). In some embodiments, the MDRE digestionresistant adapters comprise one or more unmethylated nucleotides, comprise one or more nucleotide analogs resistant to methylation dependent restriction enzymes, or do not comprise a nucleotide sequence recognized by the MDRE.

[0275] In instances where two adapters are ligated to a sample nucleic acid (one at each end), either or both of the adapters may comprise one or more known modified nucleosides. Typically, the primer binding site(s), sequencing primer binding site(s), sample index(es) and / or molecular barcode(s), if present, do not comprise the known modified nucleosides that change base pairing specificity as a result of the conversion procedure.

[0276] In some embodiments, adapters may be added to the DNA from each of the first, second, and / or third samples or a subsample thereof. Adapters can be ligated to DNA at any point in the methods herein. In some embodiments, adapters are ligated to the DNA in a sample, to end-repaired DNA molecules, to DNA after contacting the DNA with a CpG-binding protein, to DNAAttorney Ref. No. GH0271WOafter separating CpG protein-bound DNA from unbound DNA, to DNA after removing the CpG-binding protein from the DNA, to DNA in a converted sample, or to separated CpG-dense DNA. In some embodiments, adapters are ligated to the DNA in the sample prior to contacting the CpG-dense DNA with a methyl-sensitive deaminase. In some embodiments, adapters are ligated to the CpG protein-bound DNA prior to removing the CpG-binding protein from the DNA. In some embodiments, adapters are ligated to the DNA in the sample prior to contacting the DNA in the sample with a CpG-binding protein. In some embodiments, adapters are ligated to DNA eluted from a converted sample. In some embodiments, the eluted DNA is single-stranded DNA. In some embodiments, the eluted DNA is CpG-dense DNA. In some embodiments, DNA in the sample may be contacted with a methylation sensitive restriction enzyme (MSRE). In some embodiments, DNA in the sample may be contacted with a methylation dependent restriction enzyme (MDRE). In some embodiments, CpG-dense DNAis partitioned (i.e., separated) from DNAthat is not CpG-dense using CpG-binding proteins. In some embodiments, CpG-dense DNAmay be contacted with an MSRE. In some embodiments, DNAthat is not CpG-dense may be contacted with an MDRE. In some embodiments, DNA in the sample is contacted with an MSRE before or after ligating adapters to end-repaired DNA molecules, after contacting CpG-rich DNA with a methyl-sensitive deaminase, after removing the CpG-binding protein from the CpG protein-bound DNA, and / or after contacting DNA in the sample with a CpG-binding protein. In some embodiments, DNA in the sample is contacted with an MDRE before or after ligating adapters to end-repaired DNA molecules, after contacting DNA in the sample with a CpG-binding protein, after separating the CpG protein-bound DNA from unbound DNA, after removing the CpG-binding protein from the CpG protein-bound DNA, and / or after contacting CpG-dense DNA with a methyl-sensitive deaminase. In some embodiments, adapters are ligated to the DNA of a sample or subsample thereof prior to annealing primers to the DNA for capture probe generation. In some such embodiments, the adapter-ligated DNA is amplified prior to annealing primers to the DNA for capture probe generation. In some embodiments, adapters are ligated to the DNA of a sample or subsample thereof before the DNA is contacted with the capture probes. In some embodiments, the DNA to which the adapters are ligated is in the same sample or subsample as the DNA used as a template to generate capture probes. In some embodiments, the DNA to which the adapters are ligated is in a different sample or subsample, e.g., a second sample or a second subsample of a firstAttorney Ref. No. GH0271WOsample, than the DNA used as a template to generate capture probes. In some embodiments, the adapters ligated to DNA captured by the capture probes.

[0277] In some embodiments, the primers used to generate capture probes are not complementary to adapters, and the resulting capture probes therefore do not comprise adapters. Adapter-ligated DNA can therefore be selectively amplified in the presence of capture probes that do not comprise adapters. Similarly, adapter-ligated DNA can be separated from DNA that does not comprise adapters.

[0278] In some embodiments, the disclosed methods comprise analyzing DNA in each of the first, second, and / or third samples. In such methods, adapters may be added to the DNA. This may be done concurrently with an amplification procedure, e.g., by providing the adapters in a 5’ portion of a primer (where PCR is used, this can be referred to as library prep-PCR or LP-PCR), before, or after an amplification step. In some embodiments, adapters are added by other approaches, such as ligation. In some such methods, first adapters are added to the 3’ ends of the nucleic acids by ligation, which may include ligation to single-stranded DNA. In some embodiments, prior to any partitioning or capturing steps, first adapters are added to the nucleic acids by ligation, which may include ligation to single-stranded DNA (e.g., to the 3’ ends thereof). In some embodiments, the capture probes can be isolated after partitioning and ligation. For example, the hypomethylated partition can be ligated with adapters and a portion of the ligated hypomethylated partition can then be used to generate the capture probes for rearrangements. The adapter can be used as a priming site for second-strand synthesis, e.g., using a universal primer and a DNA polymerase. A second adapter can then be ligated to at least the 3’ end of the second strand of the now doublestranded molecule. In some embodiments, the first adapter comprises an affinity tag, such as biotin, and nucleic acid ligated to the first adapter is bound to a solid support (e.g., bead), which may comprise a binding partner for the affinity tag such as streptavidin. For further discussion of a related procedure, see Gansauge et al., Nature Protocols 8:737-748 (2013). Commercial kits for sequencing library preparation compatible with single-stranded nucleic acids are available, e.g., the Accel-NGS® Methyl-Seq DNA Library Kit from Swift Biosciences. In some embodiments, after adapter ligation, nucleic acids are amplified.

[0279] In some embodiments, the single-stranded DNA library preparation is performed in a one-step combined phosphorylation / ligation reaction, e.g., as described in Troll et al., BMC Genomics, 20:1023 (2019), available at https: / / doi.org / 10.1186 / sl2864-019-6355-0. This method, calledAttorney Ref. No. GH0271WOSingle Reaction Single-stranded LibrarY (“SRSLY,”) can be performed without end-polishing. SRSLY may be useful for converting short and fragmented DNA molecules, e.g., cfDNA fragments, into sequencing libraries while retaining native lengths and ends. The SRSLY method can create sequencing libraries (e.g., Illumina sequencing libraries) from fragmented or degraded template (input) DNA. In particular embodiments, template DNA is first heat denatured and then immediately cold shocked to render the template DNA molecules single-stranded. The DNA can be maintained as single-stranded throughout the ligation reaction by the inclusion of a thermostable single-stranded binding protein (SSB). Next, the template DNA, which at this point can be singlestranded and coated with SSB, is placed in a phosphorylation / ligation dual reaction with directional dsDNA NGS adapters that contain single-stranded overhangs. Both the forward and reverse sequencing adapters can share similar structures but differ in which termini is unblocked in order to facilitate proper ligations. Both sequencing adapters can comprise a dsDNA portion and a single-stranded splint overhang of random nucleotides that occurs on the 3-prime terminus of the bottom strand of the forward adapter and the 5-prime terminus of the bottom strand of the reverse adapter. In this way, the forward adapter (e.g., (P5) Illumina adapter) can be delivered to the 5-prime end of template molecules and the reverse adapter (e.g., (P7) Illumina adapter) is delivered to the 3-prime end of template molecules. Thus, the native polarity of input DNA molecules can be retained.

[0280] During the dual phosphorylation / ligation reaction, T4 Polynucleotide Kinase (PNK) can be used to prepare template DNA termini for ligation by phosphorylating 5-prime termini and dephosphorylating 3-prime termini. T4 PNK works on both ssDNAand dsDNA molecules and has no activity on the phosphorylation state of proteins. Simultaneously, the random nucleotides of the splint adapter can be annealed to the single-stranded template molecule. This creates a short, localized dsDNA molecule, enabling ligation of template to adapter with a ligase such as T4 DNA ligase, which has high ligation efficiency on dsDNA templates but low efficiency on ssDNA. After the single phosphorylation / ligation reaction is complete, the library DNA can be, e.g., purified and placed directly into standard NGS indexing PCR, compatible with both traditional single or dual index primers.

[0281] In some embodiments, the adapters include different tags of sufficient numbers that the number of combinations of tags results in a low probability e g., 95, 99 or 99.9% of two nucleic acids with the same start and stop points receiving the same combination of tags. Adapters, whetherAttorney Ref. No. GH0271WObearing the same or different tags, can include the same or different primer binding sites, but preferably adapters include the same primer binding site.

[0282] In some embodiments, following attachment of adapters, the nucleic acids are subject to amplification. The amplification can use, e.g., universal primers that recognize primer binding sites in the adapters.

[0283] In some embodiments, following attachment of adapters, the DNA or a subsample or portion of the DNA is partitioned, comprising contacting the DNA with an agent that preferentially binds to nucleic acids bearing an epigenetic modification. The nucleic acids are partitioned into at least two partitioned subsamples differing in the extent to which the nucleic acids bear the modification from binding to the agents. For example, if the agent has affinity for nucleic acids bearing the modification, nucleic acids overrepresented in the modification (compared with median representation in the population) preferentially bind to the agent, whereas nucleic acids underrepresented for the modification do not bind or are more easily eluted from the agent. The nucleic acids can then be amplified from primers binding to the primer binding sites within the adapters. Partitioning may be performed instead before adapter attachment, in which case the adapters may comprise differential tags that include a component that identifies which partition a molecule occurred in.

[0284] In some embodiments, the nucleic acids are linked at both ends to Y-shaped adapters including primer binding sites and tags. In some embodiments, the molecules are amplified.D. Molecular Tagging

[0285] In some embodiments, the nucleic acid molecules (from the first, second, and / or third sample or a subsample thereof) may be tagged with sample indexes and / or molecular barcodes (referred to generally as “tags”). In some embodiments, the nucleic acids from the first, second, and / or third sample or a subsample thereof comprise barcodes.

[0286] Tags can be molecules, such as nucleic acids, containing information that indicates a feature of the molecule with which the tag is associated. For example, nucleic acid (e.g., DNA) molecules can bear a sample tag or sample index (which distinguishes molecules in one sample from those in a different sample), a partition tag (which distinguishes molecules in one partition from those in a different partition) and / or a molecular tag / molecular barcode (which distinguishes different molecules from one another (in both unique and non-unique tagging scenarios)).Attorney Ref. No. GH0271WO

[0287] Tagging strategies can be divided into unique tagging and non-unique tagging strategies. In unique tagging, all or substantially all of the molecules in a sample bear a different tag, so that reads can be assigned to original molecules based on tag information alone. Tags used in such methods are sometimes referred to as “unique tags”. In non-unique tagging, different molecules in the same sample can bear the same tag, so that other information in addition to tag information is used to assign a sequence read to an original molecule. Such information may include start and stop coordinate, coordinate to which the molecule maps, start or stop coordinate alone, etc. Tags used in such methods are sometimes referred to as “non-unique tags”. Accordingly, it is not necessary to uniquely tag every molecule in a sample. It suffices to uniquely tag molecules falling within an identifiable class within a sample. Thus, molecules in different identifiable families can bear the same tag without loss of information about the identity of the tagged molecule.

[0288] In certain embodiments, a tag can comprise one or a combination of barcodes. As used herein, the term “barcode” refers to a nucleic acid molecule having a particular nucleotide sequence, or to the nucleotide sequence, itself, depending on context. A barcode can have, for example, between 10 and 100 nucleotides. A collection of barcodes can have degenerate sequences or can have sequences having a certain Hamming distance, as desired for the specific purpose. So, for example, a molecular barcode can be comprised of one barcode or a combination of two barcodes, each attached to different ends of a molecule. Additionally or alternatively, for different partitions and / or samples, different sets of molecular barcodes, molecular tags, or molecular indexes can be used such that the barcodes serve as a molecular tag through their individual sequences and also serve to identify the partition and / or sample to which they correspond based the set of which they are a member. For example, barcodes can be used to allow the origin of DNA (e.g., the subject, biological sample (e.g., samples collected at various time points), an enriched DNA sample (e.g., enriched DNA comprising an epigenetic target region set or enriched DNA comprising a sequence-variable target region set), partition, or similar) to be identified, e.g., following pooling of a plurality of samples for parallel sequencing. Tags comprising barcodes can be incorporated into or otherwise joined to adapters. Tags can be incorporated by ligation, overlap extension PCR among other methods. Tags can be used to label the individual polynucleotide population partitions so as to correlate the tag (or tags) with a specific partition. Alternatively, tags can be used in embodiments of the disclosure that do not employ a partitioning step. In some embodiments, a single tag can be used to label a specific partition. In some embodiments, multipleAttorney Ref. No. GH0271WOdifferent tags can be used to label a specific partition. In embodiments employing multiple different tags to label a specific partition, the set of tags used to label one partition can be readily differentiated for the set of tags used to label other partitions. In some embodiments, the tags may have additional functions, for example the tags can be used to index sample sources or used as unique molecular identifiers (which can be used to improve the quality of sequencing data by differentiating sequencing errors from mutations, for example as in Kinde et al., Proc Nat’l Acad Sci USA 108: 9530-9535 (2011), Kou et al., PLoS 0NE,W. e0146638 (2016)) or used as nonunique molecule identifiers, for example as described in US Pat. No. 9,598,731. Similarly, in some embodiments, the tags may have additional functions, for example the tags can be used to index sample sources or used as non-unique molecular identifiers (which can be used to improve the quality of sequencing data by differentiating sequencing errors from mutations).

[0289] Tags may be incorporated into or otherwise joined to adapters by chemical synthesis, ligation (e.g., as described above, e.g., by blunt-end ligation or sticky-end ligation), or overlap extension polymerase chain reaction (PCR), among other methods. Such adapters are ultimately joined to the target nucleic acid molecule. In other embodiments, one or more rounds of amplification cycles (e.g., PCR amplification) may be applied to introduce sample indexes to a nucleic acid molecule using conventional nucleic acid amplification methods. The amplifications may be conducted in one or more reaction mixtures (e.g., a plurality of microwells in an array). Molecular barcodes and / or sample indexes may be introduced simultaneously, or in any sequential order. In some embodiments, molecular barcodes and / or sample indexes are introduced prior to and / or after any conversion procedure. In the case of molecular barcodes and / or sample indexes being introduced through amplification processes, the conversion step will occur before the molecular barcodes and / or sample indexes are introduced. In some embodiments, molecular barcodes and / or sample indexes are introduced prior to and / or after sequence capturing steps, if present, are performed. In some embodiments, only the molecular barcodes are introduced prior to probe capturing and the sample indexes are introduced after sequence capturing steps are performed. In some embodiments, both the molecular barcodes and the sample indexes are introduced prior to performing probe-based capturing steps, if present. In some embodiments, the sample indexes are introduced after sequence capturing steps are performed. In some embodiments, sample indexes are incorporated to the nucleic acid molecules through overlap extension polymerase chain reaction (PCR).Attorney Ref. No. GH0271WO

[0290] In some embodiments, the tags may be located at one end or at both ends of the sample nucleic acid molecule. In some embodiments, tags are predetermined or random or semi-random sequence oligonucleotides. In some embodiments, the tag(s) may be less than about 500, 200, 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides in length. Typically tags are about 5 to 20 or 6 to 15 nucleotides in length. The tags may be linked to sample nucleic acids randomly or non-randomly.

[0291] In some embodiments, each sample or subsample thereof is uniquely tagged with a sample index or a combination of sample indexes. In some embodiments, each nucleic acid molecule of a sample or subsample is uniquely tagged with a molecular barcode or a combination of molecular barcodes. In other embodiments, a plurality of molecular barcodes may be used such that molecular barcodes are not necessarily unique to one another in the plurality (e g., non-unique molecular barcodes). In these embodiments, molecular barcodes are generally attached (e.g., by ligation as part of an adapter) to individual molecules such that the combination of the molecular barcode and the sequence it may be attached to creates a unique sequence that may be individually tracked. Detection of non-unique molecular barcodes in combination with endogenous sequence information (e.g., the beginning (start) and / or end (stop) genomic location / position corresponding to the sequence of the original nucleic acid molecule in the sample, start and stop genomic positions corresponding to the sequence of the original nucleic acid molecule in the sample, the beginning (start) and / or end (stop) genomic location / position of the sequence read that is mapped to the reference sequence, start and stop genomic positions of the sequence read that is mapped to the reference sequence, sub-sequences of sequence reads at one or both ends, length of sequence reads, and / or length of the original nucleic acid molecule in the sample) typically allows for the assignment of a unique identity to a particular molecule. In some embodiments, beginning region comprises the first 1, first 2, the first 5, the first 10, the first 15, the first 20, the first 25, the first 30 or at least the first 30 base positions at the 5' end of the sequencing read that align to the reference sequence. In some embodiments, the end region comprises the last 1, last 2, the last 5, the last 10, the last 15, the last 20, the last 25, the last 30 or at least the last 30 base positions at the 3' end of the sequencing read that align to the reference sequence. The length, or number of base pairs, of an individual sequence read are also optionally used to assign a unique identity to a given molecule. As described herein, fragments from a single strand of nucleic acid having beenAttorney Ref. No. GH0271WOassigned a unique identity, may thereby permit subsequent identification of fragments from the parent strand, and / or a complementary strand.

[0292] In certain embodiments of non-unique tagging, the number of different tags used can be sufficient that there is a very high likelihood (e.g., at least 99%, at least 99.9%, at least 99.99% or at least 99.999% that all DNA molecules of a particular group bear a different tag. It is to be noted that when barcodes are used as tags, and when barcodes are attached, e.g., randomly, to both ends of a molecule, the combination of barcodes, together, can constitute a tag. This number, in term, is a function of the number of molecules falling into the calls. For example, the class may be all molecules mapping to the same start-stop position on a reference genome. The class may be all molecules mapping across a particular genetic locus, e.g., a particular base or a particular region (e.g., up to 100 bases or a gene or an exon of a gene).

[0293] In certain embodiments, the number of different tags used to uniquely identify a number of molecules, z, in a class can be between any of 2*z, 3*z, 4*z, 5*z, 6*z, 7*z, 8*z, 9*z, 10*z, 11 *z, 12*z, 13*z, 14*z, 15*z, 16*z, 17*z, 18*z, I9*z, 20*z or 100*z (e.g., lower limit) and any of 100,000*z, 10,000*z, 1000*z or 100*z (e.g., upper limit). In some embodiments, molecular barcodes are introduced at an expected ratio of a set of identifiers (e.g., a combination of unique or non-unique molecular barcodes) to molecules in a sample. One example format uses from about 2 to about 1,000,000 different molecular barcode sequences, or from about 5 to about 150 different molecular barcode sequences, or from about 20 to about 50 different molecular barcode sequences, ligated to both ends of a target molecule. Alternatively, from 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 the 20-50 different molecular barcode sequences can be attached to each end of the target molecule) can be used. Such numbers of identifiers are typically sufficient for different molecules having the same start and stop points to 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 molecules have the same combinations of molecular barcodes. For example, in a sample of about 5 ng to 30 ng of cell free DNA, one expects around 3000 molecules to map to a particular nucleotide coordinate, and between about 3 and 10 molecules having any start coordinate to share the same stop coordinate. Accordingly, about 50 to about 50,000 different tags (e.g., between about 6 and 220 barcode combinations) can suffice to uniquely tag all such molecules. To uniquely tag all 3000 moleculesAttorney Ref. No. GH0271WOmapping across a nucleotide coordinate, about 1 million to about 20 million different tags would be required.

[0294] In some embodiments, the assignment of unique or non-unique molecular barcodes in reactions is performed using methods and systems described in, for example, U.S. Patent Application Nos. 20010053519, 20030152490, and 20110160078, and U.S. PatentNos. 6,582,908, 7,537,898, 9,598,731, and 9,902,992, each of which is hereby incorporated by reference in its entirety.

[0295] In some embodiments, the assignment of unique molecular barcodes in reactions is performed using methods and systems described in Lim et al., Communications Biology. (2025)8:1098, e.g., SPIDER-seq. In some such embodiments, amplicons are tagged with a pair of two unique molecular barcodes using primers that contain a barcode. Successive daughter strands synthesized through each round of PCR amplification are grouped into clusters (e.g., peer-to peer networks, as illustrated in Fig. 1c of Lim et al.) based on a chain of common unique barcodes between immediate parent and daughter strands. That is, strand synthesis events (with a synthesized strand as a template) involve copying one barcode from the template and include one new barcode from the primer, so each daughter strand shares a unique barcode with its parent. By clustering strands in this way, a consensus can be generated that reduces errors.

[0296] Alternatively, in some embodiments, different nucleic acid molecules of a sample may be identified using only endogenous sequence information (e g., start and / or stop positions, subsequences of one or both ends of a sequence, and / or lengths).

[0297] After sequencing, analysis of reads to detect genetic variants can be performed. Tags are used to sort reads from different molecules. Analysis can include in silico analysis to determine genetic and epigenetic variation (one or more of methylation, chromatin structure, etc.) using sequence information, genomic coordinates length, coverage and / or copy number. In some embodiments, higher coverage can correlate with higher nucleosome occupancy in genomic region while lower coverage can correlate with lower nucleosome occupancy or a nucleosome depleted region (NDR).

[0298] In some embodiments, the tagged nucleic acids are sequenced after loading into a microwell plate. The microwell plate can have 96, 384, or 1536 microwells. In some cases, they are introduced at an expected ratio of unique tags to microwells. For example, the unique tags may be loaded so that more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000,Attorney Ref. No. GH0271WO50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000 or 1,000,000,000 unique tags are loaded per genome sample. In some cases, the unique tags may be loaded so that less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000 or 1,000,000,000 unique tags are loaded per genome sample. In some cases, the average number of unique tags loaded per sample genome is less than, or greater than, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000 or 1,000,000,000 unique tags per genome sample.

[0299] In some embodiments a format uses 20-50 different tags (e.g., barcodes) ligated to both ends of target nucleic acids. For example, 35 different tags (e.g., barcodes) ligated to both ends of target molecules creating 35 x 35 permutations, which equals 1225 for 35 tags. Such numbers of tags are sufficient so that different molecules having the same start and stop points have a high probability (e.g., at least 94%, 99.5%, 99.99%, 99.999%) of receiving different combinations of tags. Other barcode combinations include any number between 10 and 500, e.g., about 15x15, about 35x35, about 75x75, about 100x100, about 250x250, about 500x500.

[0300] In some cases, unique tags may be predetermined or random or semi-random sequence oligonucleotides. In other cases, a plurality of barcodes may be used such that barcodes are not necessarily unique to one another in the plurality. In this example, barcodes may be ligated to individual molecules such that the combination of the barcode and the sequence it may be ligated to creates a unique sequence that may be individually tracked. As described herein, detection of non-unique barcodes in combination with sequence data of beginning (start) and end (stop) portions of sequence reads may allow assignment of a unique identity to a particular molecule. The length or number of base pairs, of an individual sequence read may also be used to assign a unique identity to such a molecule. As described herein, fragments from a single strand of nucleic acid having been assigned a unique identity, may thereby permit subsequent identification of fragments from the parent strand.

[0301] In some embodiments, the method includes adding one or more internal control DNAs and forward and reverse primers for amplifying the internal control DNAs. The internal control DNAs may be added before amplification using the primers that anneal upstream and downstream of the rearrangement breakpoints. The forward and reverse primers for amplifying the internal control DNAs may be included with, or added at the same time as, the primers that anneal upstream and downstream of the rearrangement breakpoints. The internal control DNAs may comprise or consistAttorney Ref. No. GH0271WOof sequences that do not occur in the genome of the subject, or that do not occur in the genome of the species of which the subject is a member (e.g., the human genome). The forward and / or reverse primers for amplifying the internal control DNAs may comprise sequences that are not complementary to any sequence in the genome of the subject, e.g., the human genome. The internal control DNAs may be used to ensure that the amplification process proceeded as designed. As such, the method may comprise detecting (e.g., sequencing) molecules amplified from and / or captured by the one or more internal control DNAs. The method can comprise comparing an amount of internal control DNAs (e.g., number of molecules or reads detected that correspond to an internal control DNA sequence) to a predetermined threshold, and either rejecting sequencing results if the predetermined threshold is not met or accepting sequencing results if the predetermined threshold is met. The predetermined threshold may be established, e.g., based on historical data or by testing the method on samples of DNA from test subjects, such as healthy volunteers. For example, amplification and detection of the one or more internal control DNAs provides confirmation that the amplification process proceeded properly, thus reducing the likelihood of a false negative.

[0302] In some embodiments, the DNA is tagged before a step of contacting the DNA in the sample with a CpG-binding protein, and tagging may also occur after an end-repair step. In some embodiments, the DNA is tagged after a step of contacting the DNA in the sample with a CpG-binding protein. In some embodiments, the DNA is tagged before a step of separating the CpG protein-bound DNA from unbound DNA. In some embodiments, the DNA is tagged after a step of separating the CpG protein-bound DNA from unbound DNA. In some embodiments, the DNA is tagged before a step of contacting CpG-dense DNA with a methyl-sensitive deaminase. In some embodiments, the DNA is tagged after a step of contacting CpG-dense DNA with a methylsensitive deaminase.

[0303] In some embodiments, the DNA is tagged after a step of contacting the DNA in the sample with a CpG-binding protein and before a step of step of separating the CpG protein-bound DNA from unbound DNA. In some embodiments, the DNA is tagged after a step of contacting the DNA in the sample with a CpG-binding protein and before a step of contacting CpG-dense DNA with a methyl-sensitive deaminase. In some embodiments, the DNA is tagged after a step of separating the CpG protein-bound DNA from unbound DNA and before a step of contacting CpG-dense DNA with a methyl-sensitive deaminase.Attorney Ref. No. GH0271WO

[0304] In some embodiments, wherein the separating the CpG protein-bound DNA from unbound DNA comprises partitioning the DNA in the sample into a plurality of partitioned subsamples, a first partitioned subsample is differentially tagged relative to a second partitioned subsample. Thus, in some embodiments, the method disclosed herein comprises differentially tagging the first partitioned subsample and the second partitioned subsample. In some embodiments, the first partitioned subsample is tagged after the partitioning. In some embodiments, the second partitioned subsample is tagged after the partitioning. In some embodiments, the first partitioned subsample comprises methylated DNA in a greater proportion than the second partitioned subsample, and is differentially tagged relative to the second partitioned subsample.

[0305] In some embodiments, the DNA in the first, second, and / or third sample or a subsample thereof (e.g., a hypomethylated and / or hypermethylated subsample thereof) comprises barcodes. In some embodiments, the disclosed method comprises ligating one or more adapters to the DNA after a separating step, wherein the one or more adapters comprise molecular barcodes. In some embodiments, wherein the separating the CpG protein-bound DNA from unbound DNA comprises partitioning the DNA in the sample into a plurality of partitioned subsamples, the DNA of a first partitioned subsample is contacted with an MSRE after ligating adapters comprising barcodes to the DNA (e.g., end-repaired DNA) and / or before contacting the DNA in the first partition with a methyl-sensitive deaminase. In some embodiments, wherein the separating the CpG protein-bound DNA from unbound DNA comprises partitioning the DNA in the sample into a plurality of partitioned subsamples, DNA in the second partition is contacted with an MDRE after ligating adapters comprising barcodes to the DNA (e.g., end-repaired DNA) and / or before contacting the DNA in the second partition with a methyl-sensitive deaminase. In some embodiments, the methods disclosed herein comprise ligating one or more adapters comprising barcodes to the DNA after the separating step and / or before contacting the CpG-dense DNA with a methyl-sensitive deaminase.E. Conversion; Contacting the DNA with a Deaminase

[0306] The methods disclosed herein can comprise converting DNA, e.g., contacting DNA from the first, second and / or third samples or a subsample thereof (e.g., a hypomethylated and / or hypermethylated subsample thereof) with a deaminase, such as a methyl-sensitive deaminase, thereby providing a converted sample in which unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, conversion is performed prior to a sequencing step. In someAttorney Ref. No. GH0271WOembodiments, conversion is performed prior to a capturing step. In some embodiments, conversion is performed after a capturing step and prior to sequencing step. In some embodiments, this step occurs after one or more of contacting DNAin a sample with a CpG-binding protein, eluting CpG-dense DNA from the CpG-binding protein, an end-repair step, and / or a tagging (e.g., adapter ligation step) and may also occur before one or more of an amplification step, and a sequencing step. In some embodiments, the methyl-sensitive deaminase is a dsDNA deaminase and / or a ssDNA deaminase. In some embodiments, the dsDNA deaminase has methyl-sensitive deaminase activity on ssDNA. The step of contacting the DNA with a methyl-sensitive deaminase can be referred to as, or be included in, a conversion procedure, such as any of the conversion procedures described elsewhere herein. For an exemplary description of conversion using a methyl-sensitive deaminase, see, e.g., Vaisvila et al., Mol Cell. 2024 Mar 7;84(5):854-866.e7. DNAin the converted sample is then amplified, separated, partitioned, and / or enriched. In some embodiments, the converted DNA is sequenced, and a level of methylation at one or more differentially methylated regions of the DNA is quantified. Such embodiments may also comprise a step of end-repair prior to the sequencing, prior to contacting the DNA with a methyl-sensitive deaminase, and / or prior to contacting the DNAin the sample with a CpG-binding protein.

[0307] In some embodiments, the contacting the DNA with a methyl-sensitive deaminase provides a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, all unmethylated CpGs in the sample can be converted to UpGs using the methyl-sensitive deaminase. In some embodiments, at least a portion of unmethylated CpGs are not converted to UpGs using the methyl-sensitive deaminase. In some embodiments, at least a portion of methylated CpGs can be converted to UpGs using the methyl-sensitive deaminase.

[0308] As outlined below, there are various methods of detecting and / or identifying modified nucleosides that rely on a conversion procedure that changes the base-pairing specificity of a nucleoside, based on the modification status of the nucleosides. These changes of base-pairing specificity can then be detected, and thus the modification status of the nucleoside inferred, by sequencing.

[0309] In some embodiments, the conversion procedure used in the methods of the disclosure is one that changes the base pairing specificity of an unmodified nucleoside (e.g. cytosine), but does not change the base pairing specificity of the corresponding modified nucleoside (e g. methylatedAttorney Ref. No. GH0271WOcytosine, such as 5hmC and / or 5mC) or does not change the base pairing specificity of any modified nucleoside (e.g. modified cytosine, adenosine, guanosine and thymidine (or uracil)). In methods that require denaturation for conversion, failure to denature a DNA molecule will result in non-conversion of all bases in the DNA molecule. As biological changes in methylation are predominantly concerted to a localized regions of interest, these non-random (localized) nonconversion events can appear as false negatives (non-methylated regions). Random nonconversion methods can maximally affect a low percent of bases within a region, and thus the specificity of methylation change detection can be maximized (reduce false positives) by placing a threshold on percentage of bases within a region that are methylated / non-methylated.

[0310] Hence, in some cases, a conversion procedure that does not involve denaturation is preferred. The skilled person can select a suitable method according to their needs, including which nucleoside modifications are to be detected and / or identified and which type of modified base is used in the end repair reaction.

[0311] The methods described herein could in principle use any suitable methyl-sensitive enzymatic conversion procedure that changes the base-pairing specificity of the unmodified cytosine and thereby allows the modified base to be distinguished from the corresponding unmodified cytosine and / or other types of modification when sequenced. For example, any enzymatic conversion procedure could be used allowing 5-methylcytosine (m5c or 5-mC or 5mC) to be distinguished from unmodified cytosine. In particular embodiments, the conversion procedure converts unmodified (e g., unmethylated) cytosines to uracils using a methyl-sensitive deaminase.

[0312] In some embodiments, the conversion procedure comprises enzymatic conversion of a nucleobase using a non-specific, modification-sensitive double-stranded DNA deaminase, e.g., as in SEM-seq. See, e.g., Vaisvila et al. Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA. Mol Cell. 2024 Mar 7;84(5):854-866.e7. SEM-Seq employs a non-specific, modification-sensitive double-stranded DNA deaminase (e.g., modification-sensitive DNA deaminase A (MsddA)) in a nondestructive single-enzyme 5-methylctyosine sequencing (SEM-seq) method that deaminates unmodified cytosines. Accordingly, SEM-seq does not require the TET2 and T4-PGT or 5-hydroxymethylcytosine carbamoyltransferase protection and denaturing steps that are of use, e.g.,Attorney Ref. No. GH0271WOin AP0EC3A-based protocols. Additionally, MsddA does not deaminate 5-formylated cytosines (5fC) or 5-carboxylated cytosines (5-caC). In SEM-seq, unmodified cytosines in the DNA are deaminated to uracil and is read as “T” during sequencing. Modified cytosines (e.g., 5mC) are not converted and are read as “C” during sequencing. Cytosines that are read as thymines are identified as unmodified (e.g., unmethylated) cytosines or as thymines in the DNA. Performing SEM-seq conversion thus facilitates identifying positions containing 5mC using the sequence reads obtained. In some embodiments, the conversion procedure comprises enzymatic conversion of unmodified cytosine using MsddA or a modification-sensitive DNA deaminase A (MsddA)-like deaminase. For an exemplary description of MsddA and MsddA-like deaminases, see, e.g., Vaisvila et al. Mol Cell. 2024 Mar 7;84(5):854-866.e7, which illustrates in Fig. 2A-C that MsddA-like deaminases have reduced activity on each of 5mC, 5hmC, and 5gmC relative to unmodified cytosine in dsDNA, e.g., a reduction of about 75%, 80%, or more on each of 5mC, 5hmC, and 5gmC relative to unmodified cytosine (e.g., using assay conditions as described in Vaisvila et al., such as analysis of deamination of C in E. coli or lambda dem- DNA, deamination of 5mC in XP12 phage DNA, deamination of 5hmC in a C-hydroxymethylated adenovirus PCR fragment or fully C-hydroxymethylated T4147 phage DNA, and deamination of 5gmC in alpha-glucosyltransferase knockout (AGT-) T4 phage DNA. Deamination can be performed by contacting substrate DNA with deaminase and analyzed using NGS as follows: 50 ng of unmodified E. coli C2566 genomic DNA can be combined with the control DNAs (about 1 ng of Lambda, XP12, and T4147, and 0.1 ng of the 5hmC Adenovirus PCR fragment), sheared to about 300 bp and ligated to pyrrolo-dC adapters with 1 uL of in vitro synthesized deaminase (e.g., synthesized using the PURExpress In Vitro Protein Synthesis kit (NEB, Ipswich, MA) following manufacturer’s recommendations with 100-400 ng of PCR fragment template DNA containing codon-optimized deaminase coding sequence and T7 promoter and terminator). Exemplary deamination reaction conditions are 50 mM Bis-Tris pH 6.0, 0.1% Triton X-100 for 1 hour at 37 degrees C. After the deamination reaction, 1 uL of Thermolabile Proteinase K (NEB, Ipswich, MA) can be added and incubated for 30 min at 37 degrees C and then the Proteinase K can be heat inactivated at 60 degrees C for 10 minutes. The deaminated product can then be used for library amplification using the NEBNext Q5U Master Mix (New England Biolabs, Ipswich, MA, USA) with 5mMof NEBNext Unique Dual Index Primers. The resulting library can be purified using IX NEBNext Sample Purification Beads according to the manufacturer’s instructions and the purified library can be analyzed and quantifiedAttorney Ref. No. GH0271WOby an Agilent Bioanalyzer 2100 DNA Highsensitivity chip. The libraries can be sequenced using the Illumina NextSeq andNovaSeq platforms. Paired-end sequencing of 75 cycles (2 x 75 bp) can be performed for all the sequencing runs. Base calling and demultiplexing can be carried out with the standard Illumina pipeline.

[0313] In some embodiments, a methyl-sensitive deaminase used in a method described herein is a dsDNA deaminase and / or a ssDNA deaminase. In some embodiments, the dsDNA deaminase has methyl-sensitive deaminase activity on ssDNA. The step of contacting the DNA with a methylsensitive deaminase can be referred to as, or be included in, a conversion procedure, such as any of the conversion procedures described elsewhere herein. For an exemplary description of conversion using a methyl-sensitive deaminase, see, e.g., Vaisvila et al., Mol Cell. 2024 Mar 7;84(5):854-866.e7. DNAin the converted sample is then amplified, separated, partitioned, and / or enriched. In some embodiments, the converted DNA is sequenced, and a level of methylation at one or more differentially methylated regions of the DNA is quantified. Such embodiments may also comprise a step of end-repair prior to the sequencing, prior to contacting the DNA with a methyl-sensitive deaminase, and / or prior to contacting the DNAin the sample with a CpG-binding protein.

[0314] In some embodiments, the contacting the DNA with a methyl-sensitive deaminase provides a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, all unmethylated CpGs in the sample can be converted to UpGs using the methyl-sensitive deaminase. In some embodiments, at least a portion of unmethylated CpGs are not converted to UpGs using the methyl-sensitive deaminase. In some embodiments, at least a portion of methylated CpGs can be converted to UpGs using the methyl-sensitive deaminase.

[0315] Optionally, the conversion procedure further includes enzymatic protection of 5hmCs, such as by glucosylation of the 5hmCs (e.g., using PGT) or by carbamoylation of the 5hmCs (e.g., using 5-hydroxymethylcytosine carbamoyltransferase), in the DNA prior to the deamination of unprotected modified cytosines. In this method, 5hmC can be protected from conversion, for example through glucosylation using P-glucosyl transferase (PGT), forming (5-glucosylhydroxymethylcytosine) 5ghmC, or through carbamoylation using 5-hydroxymethylcytosine carbamoyltransferase, forming 5cmC. This is described, for example, in Yu et al., Cell 2012; 149: 1368-80, and in Yang et al., Bio-protocol, 2023; 12(17): e4496.Attorney Ref. No. GH0271WOGlucosylation or carbamoylation of 5hmC can reduce or eliminate deamination of 5hmC by a deaminase such as MsddA or an MsddA-like deaminase.

[0316] In some embodiments, a TET protein can be used to convert 5mC and optionally 5hmC (but not unmodified C) into substrates (e.g., 5caC) that cannot be deaminated by a deaminase, and then a deaminase (e.g., MsddA or an MsddA-like deaminase) can be used to deaminate unmodified cytosines, converting them to uracils. Various TET enzymes may be used in the disclosed methods as appropriate. In some embodiments, the one or more TET enzymes comprise TETv. TETv is described in US Patent 10,260,088 and its sequence is SEQ ID NO: 1 therein. In some embodiments, the one or more TET enzymes comprise TETcd. TETcd is described in US Patent 10,260,088 and its sequence is SEQ ID NO: 3 therein. In some embodiments, the one or more TET enzymes comprise TET1. In some embodiments, the one or more TET enzymes comprise TET2. TET2 may be expressed and used as a fragment comprising TET2 residues 1129-1480 joined to TET2 residues 1844-1936 by a linker as described, e.g., in US Patent 10,961,525. In some embodiments, the one or more TET enzymes comprise TET1 and TET2. In some embodiments, the one or more TET enzymes comprise a T1372 TET mutant, such as T1372S. In some embodiments, the one or more TET enzymes comprise a V1900 TET mutant, such as a V1900A, V1900C, V1900G, VI 9001, or V1900P TET mutant. In some embodiments, the one or more TET enzymes comprise a VI 900 TET2 mutant, such as a VI 900 A, V1900C, V1900G, VI 9001, or V1900P TET2 mutant. It can be beneficial to use a TET enzyme that maximizes formation of 5-carboxylcytosine (5-caC) relative to less oxidized modified cytosines, particularly 5-formylcytosine, because 5-caC is not a substrate for enzymatic deamination, e.g., by methylsensitive deaminase enzymes such as MsddA or an MsddA-like deaminase. Maximizing formation of 5-caC thus reduces the risk of false calls in which a base is identified as unmethylated because it underwent deamination even though it was methylated (or hydroxymethylated) in the original sample. Accordingly, in some embodiments, the TET enzyme comprises a mutation that increases formation of 5-caC. Exemplary mutations are set forth above. “A mutation that increases formation of 5-caC” means that the TET enzyme having the mutation produces more 5-caC than a TET enzyme that lacks the mutation but is otherwise identical. 5-caC production can be measured as described, e.g., in Liu et al., Nat Chem Biol 13:181-187 (2017) (see Online Methods section, TET reactions in vitro subsection, “driving” conditions). Any variants and / or mutants described in Liu et al. (2017) can be used in the disclosed methods as appropriate.Attorney Ref. No. GH0271WO

[0317] In some embodiments, the one ormoreTET enzymes compri se a TET2 enzyme comprising a T1372S mutation, such as TET2-CS-T1372S and TET2-CD-T1372S. A TET2 comprising a T1372S mutation is described in US Patent 10,961,525 and may be expressed and used as a fragment comprising TET2 residues 1129-1480 joined to TET2 residues 1844-1936 by a linker. Position 1372 of TET2 corresponds to position 258 of SEQ ID NO: 21 (wild type TET2 catalytic domain) of US Patent 10,961,525. Thus, the sequence of a T1372S TET2 catalytic domain may be obtained by changing the threonine at position 258 of SEQ ID NO: 21 of US Patent 10,961,525 to serine. TET2 comprising a T1372S mutation is also described in Liu et al., Nat Chem Biol. 2017 February; 13(2): 181-187. As demonstrated in Liu et al., TET2 comprising a T1372S mutation can more efficiently oxidize 5mC to produce 5-carboxylcytosine (5-caC) than other versions of TET2 such as TET2 lacking a T1372S mutation. In some embodiments, the TET2 enzyme is a human TET2 enzyme comprising a T1372S mutation.

[0318] In some embodiments, the methyl-sensitive deaminase is thermally inactivated after contacting the mCpG-dense DNA with the methyl-sensitive deaminase. In some embodiments, the thermal inactivation comprises heating or cooling of the methyl-sensitive deaminase to a temperature at which the methyl-sensitive deaminase has reduced or inhibited activity relative to a methyl -sensitive deaminase that has not been subjected to heating or cooling. In some embodiments, the thermal inactivation completely inhibits the activity of the methyl-sensitive deaminase or reduces the activity of the methyl-sensitive deaminase by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 90%, about 95%, about 98%, about 99%, or 100% relative to a methyl-sensitive deaminase that has not been subjected to heating or cooling.F. Sequence-specifically degrading a plurality of nucleic acid sequences

[0319] In some embodiments, methods disclosed herein comprise a step of sequence-specifically degrading a plurality of nucleic acid sequences comprising a modification, e.g., in the first, second, and / or third samples or a subsample thereof (e.g., a hypomethylated and / or hypermethylated subsample thereof). In some embodiments, the sequence-specific degrading comprises contacting the nucleic acids in a sample or subsample thereof with a sequence-specific nuclease. The modified sequences that are degraded are prevalent in nucleic acid sequences obtained from healthy subjects. In some embodiments, the nucleic acid sequences are cfDNA sequences. In someAttorney Ref. No. GH0271WOembodiments, the modification is methylation. In some embodiments, the methylation comprises or consists of cytosine methylation. In some embodiments, methylated sequences that are degraded comprise a CpG site that is commonly methylated in cfDNA obtained from healthy subjects. In some embodiments, sequences that comprise a CpG site that is not commonly methylated is not degraded by a sequence-specific nuclease. In some embodiments, methods herein comprise additional elements or steps to deplete unmodified sequences and sequences comprising modifications other than methylation or the modification of interest that are prevalent in cfDNA obtained from healthy subjects, such as additional elements or steps disclosed herein. The combination of depleting sequences that are modified in cfDNA from healthy subjects and depleting sequences that are not modified or contain other modifications in cfDNA from healthy subjects can provide a treated sample enriched for DNA molecules that are prevalent only in subjects that are not healthy. In some embodiments, sequences remaining uncleaved after the sequence-specific degrading step are detected. Thus, some methods herein provide detection of the presence or absence of aberrantly modified nucleic acid sequences by specifically degrading normally modified nucleic acid sequences prevalent in samples obtained from healthy subjects. In some embodiments, the treated sample is enriched for modified sequences that are associated with cancer. In general, the methods described herein can facilitate more efficient usage of capacity in downstream analytical steps (e.g., detection of sequences of interest, such as by sequencing). This is achieved by degrading non-informative sequences, e.g., which bear a modification such as methylation in all, essentially all, or a majority of samples, so that such uninformative sequences do not participate in and / or consume capacity during the downstream analytical steps. In some embodiments, the non-informative sequences comprise repetitive DNA elements (e.g., transposable elements, SINEs, LINEs, and / or Alu elements) and / or DNA that is methylated in healthy cells in the erythroid lineage.

[0320] In some embodiments, the sequence-specific degrading comprises cleavage of both strands of double-stranded DNA, resulting in formation of a double-strand end. In some embodiments, the double-strand end is a blunt end. In some embodiments, the double-strand end comprises an overhang of one or more nucleosides.

[0321] In some embodiments, the sequence-specific degrading is performed after partitioning the DNA based on methylation status. In some such embodiments, the methods comprise contacting the DNA with a methyl binding domain (MBD) specific for methyl cytosine. In someAttorney Ref. No. GH0271WOembodiments, the sequence-specific degrading is performed on DNA of a hypermethylated partition but not performed on DNA of a hypomethylated partition. In some embodiments, the sequence specific degrading occurs before or after the adapters containing barcodes are ligated to DNA. In a particular embodiment, the sequence- specific degrading occurs after the adapters containing barcodes are ligated to DNA. In some embodiments, the sequence specific degrading occurs after the ligating barcode-containing adapters to the DNA. In some embodiments, the sequence-specific degrading occurs prior to a sequencing step. In some embodiments, the sequence-specific degrading occurs prior to a capture step, if present. In some embodiments, the sequence-specific degrading occurs prior to a sequencing step and after a capture step, if present.

[0322] In some embodiments, the sequence-specific degrading is performed after partitioning the DNA based on methylated status, ligating adapters containing barcodes to the DNA, and degrading DNA of a hypermethylated partition with a MSRE. In some such embodiments, the DNA remaining after the sequence-specific degrading is amplified by PCR. In some embodiments, adapters containing barcodes are ligated to DNA of a hypomethylated partition, the DNA is then amplified by PCR and enriched using DNA or RNA probes of target region sets. In some such embodiments, DNA of the hypomethylated partition is not sequence-specifically degraded. In some embodiments, enriched DNA of the hypermethylated partition is combined with DNA remaining in the hypermethylated partition before sequencing and analysis.

[0323] In some embodiments, the sequence-specific degrading comprises using a kit for depletion of uninformative sequences. Such kits are known in the art and described herein. Examples of kits for sequence specific degradation include but are not limited to: DepleteX™ (Jumpcode Genomics), CRISPRclean1M(Jumpcode Genomics), and AnyDeplete1M(Tecan).1. Sequence-specific nucleases

[0324] In some embodiments, the sequence-specific degrading is performed by contacting nucleic acids, such as DNA, with a sequence-specific nuclease. In some embodiments, the sequencespecific nuclease is a modification-independent sequence-specific nuclease. Examples of modification-independent sequence-specific nucleases include but are not limited to CRISPR nucleases, TALENS, zinc fingers, and Argonaute nucleases.

[0325] In some embodiments, the modification-independent sequence-specific nuclease is a CRISPR nuclease. Exemplary CRISPR nucleases include Type II and Type V Cas nucleases,Attorney Ref. No. GH0271WOincluding Cas9, such as a Streptococcus pyogenes Cas9 nuclease or a variant thereof, a Staphylococcus aureus Cas9, or a variant thereof; Cast 2, such as a Cast 2a or Cast 2b nuclease, or a variant thereof; and CasX nucleases or variants thereof. In some embodiments, a Cas nuclease is a multi -turnover Cas nuclease or a high-fidelity variant. Some exemplary CRISPR nucleases are further described in, e.g., Yourik et al. Staphylococcus aureus Cas9 is a multiple-turnover enzyme. RNA25:35-44 (2019) and Kleinstiver et al. High-fidelity CRISPR-Cas9 variantswith undetectable genome-wide off-targets. Nature 529: 490-495 (2016), which are hereby incorporated by reference in their entirety.2. Guide RNAs and specifically degraded sequences

[0326] In some embodiments, the sequence-specifically degrading comprises contacting DNA with one or more guide RNAs that guide a nuclease to the specific sequence or sequences to be degraded. Appropriate guide RNA sequences are chosen based on the specific sequences to be degraded and on the nuclease to be used. For example, if a CRISPR nuclease is used, one or more appropriate guide RNAs that are recognized by the CRISPR nuclease are used. In some embodiments, single guide RNAs (“sgRNAs”) or other types of fused or truncated CRISPR guide RNAs are used with the appropriate CRISPR nuclease. Exemplary guide RNAs are described in, e.g., Fu et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32:279-284 (2014), which is hereby incorporated by reference in its entirety.

[0327] In some embodiments, the one or more guide RNAs comprise one or more modifications. In some such embodiments, the guide RNAs comprise a modified internucleoside linkage. In some embodiments, the modified internucleoside linkage is a phosphorothioate intemucleoside linkage. In some embodiments, the guide RNAs comprise a modified sugar. In some embodiments, the modified sugar comprises a 2’- substitution. In some embodiments, the 2’ -substitution is a 2’-fluoro, 2’-O-methoxyethyl, 2’-O-alkyl substitution, or a 2’-hydroxy substitution. In the context of a guide RNA, a sugar comprising a 2’-hydroxy substitution, such as a 2’-deoxyribosyl sugar, is a modified sugar. In some embodiments, the 2’-O-alkyl substitution is a 2’-O-methyl substitution. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the bicyclic sugar is a LNA, cEt, or ENA sugar. In some embodiments, the modified sugar is a linear sugar. In some embodiments, the linear sugar is a UNA sugar.

[0328] A guide RNA that is configured to bind to a certain sequence comprises a portion that specifically binds to the certain sequence. In some embodiments, a portion of each guide RNAAttorney Ref. No. GH0271WOspecifically binds to a target region or a portion of a target region of DNA. In some embodiments, a portion of each guide RNA specifically binds to a sequence of DNA comprising a modification, wherein the modified version of the sequence is prevalent in cfDNA obtained from healthy subjects.

[0329] In some embodiments, the sequence-specific degrading comprises contacting DNA with a plurality of guide RNAs. In some embodiments, each unique guide RNA specifically binds to a different member of a plurality of specific sequences, if present, in a sample or subsample thereof. In some embodiments, the plurality of guide RNAs comprises guide RNAs that specifically bind to sequences modified in cfDNA obtained from healthy subjects. In some embodiments, the plurality of guide RNAs comprises guide RNAs that specifically bind to sequences comprising a CpG motif that is methylated in cfDNA obtained from a healthy subject. In some embodiments, the plurality of sequences degraded is 1-100 million sequences. In some embodiments, the plurality of sequences degraded is 20-250,000; 20-100,000; 20-10,000; 20-1,000; or 20-100 sequences. In some embodiments, the plurality of sequences degraded is 50-100,000; 50-10,000; 50-1,000; or 50-100 sequences. In some embodiments, the plurality of sequences degraded is 100-100,000; 100-10,000; or 100-1,000 sequences. In some embodiments, each of the plurality of sequences degraded is a sequence present in a different gene or genetic locus. In some embodiments, sequences that are degraded comprise, or are selected from, repetitive elements (e.g., transposable elements; LINEs such as LINE1; Alu elements; SINEs, centromeric repeats, telomeric repeats, and / or minisatellite repeats). In some embodiments, sequences that are degraded comprise sequences known to be modified in cfDNA obtained from healthy subjects. In some embodiments, sequences that are degraded comprise sequences determined to be modified in cfDNA obtained from a healthy subject using existing methods of detecting sites of modifications in DNA sequences.

[0330] In some embodiments, methods herein comprise an element or step to deplete unmodified or unmethylated sequences prevalent in cfDNA obtained from healthy subjects. In some such embodiments, the sequence-specific degrading comprises degrading sequences lacking motifs that may be modified or methylated. In some such embodiments, the sequences that are degraded comprise sequences lacking CpG motifs. In some embodiments, the sequence-specific degrading comprises contacting DNA with a plurality of guide RNAs, wherein the plurality of guide RNAs comprises guide RNAs that specifically bind to sequences that are unmethylated in cfDNAAttorney Ref. No. GH0271WOobtained from healthy subjects. Tn some embodiments, each of the plurality of guide RNAs specifically binds to a sequence prevalent in a form comprising a modification in cfDNA from a healthy subject or to a sequence prevalent in a form lacking the modification in cfDNA from a healthy subject.G. Separating and Partitioning

[0331] In some embodiments, a heterogeneous DNA sample from the first, second, and / or third samples is separated.

[0332] In some embodiments, separating comprises partitioning. In some instances, a heterogeneous nucleic acid sample is partitioned into two or more partitions (sub-samples). In some instances, a heterogeneous nucleic acid sample is partitioned into two or more partitions (sub-samples) using a CpG-binding protein or mCpG-binding protein. In some embodiments, each partition is differentially tagged. Tagged partitions can then be pooled together for collective sample prep and / or sequencing. The partitioning-tagging-pooling steps can occur more than once, with each round of partitioning occurring based on a different characteristics, and tagged using differential tags that are distinguished from other partitions and partitioning means. In some embodiments, the separating comprises partitioning the DNA in the sample into a plurality of partitioned subsamples. In some embodiments, the plurality of partitioned subsamples comprises a first partitioned subsample and a second partitioned subsample. In some embodiments, the first partitioned subsample comprises methylated DNA and / or CpG-dense DNA in a greater proportion than the second partitioned subsample. The partitioning step can occur before or after contacting the DNA (e.g., CpG-dense DNA) with a methyl-sensitive deaminase. In some embodiments, the partitioning is performed prior to the sequencing the DNA from the first sample and DNA from the second sample. In some embodiments, the partitioning is performed prior to a capture step, if present. In some embodiments, the partitioning is performed after a capture step, if present. The partitioning step can occur after contacting the DNA (e.g., DNA in the sample) with a CpG-binding protein. In some embodiments, the partitioning step occurs before contacting the DNA with a methyl-sensitive deaminase. In some embodiments, the partitioning step occurs before a conversion step.

[0333] Examples of characteristics that can be used for partitioning include sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and / or proteins that bind to DNA. Resulting partitions can include one or more of the following nucleic acid forms:Attorney Ref. No. GH0271WOsingle-stranded DNA (ssDNA), double-stranded DNA (dsDNA), shorter DNA fragments and longer DNA fragments. In some embodiments, partitioning based on a cytosine modification (e.g., cytosine methylation) or methylation generally is performed and is optionally combined with at least one additional partitioning step, which may be based on any of the foregoing characteristics or forms of DNA. In some embodiments, a heterogeneous population of DNA from each of the first, second, and / or third sample is partitioned into DNA with one or more base modifications and without the one or more base modifications. Examples of base modifications are described elsewhere herein. Alternatively or additionally, a heterogeneous population of DNA can be partitioned into nucleic acid molecules associated with nucleosomes and nucleic acid molecules devoid of nucleosomes. Alternatively or additionally, a heterogeneous population of DNA may be partitioned into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Alternatively, or additionally, a heterogeneous population of DNA may be partitioned based on nucleic acid length (e.g., molecules of up to 160 bp and molecules having a length of greater than 160 bp).

[0334] In some cases, different procedures are applied to different partitions to determine different characteristics of the initial sample. In some embodiments, the DNA of at least one partition is subjected to an end repair and sequencing procedure described herein. In some embodiments at least one partition is not subjected to the end repair and sequencing procedure described herein. In cases where the method comprises a conversion procedure, corresponding sequences from the converted and non-converted partitions can be compared to identify single nucleotides that have undergone conversion and therefore identify corresponding modified nucleosides in the initial sample.

[0335] In some embodiments, partition tagging comprises tagging molecules in each partition with a partition tag. After re-combining partitions (e.g., to reduce the number of sequencing runs needed and avoid unnecessary cost) and sequencing molecules, the partition tags identify the source partition. In another embodiment, different partitions are tagged with different sets of molecular tags, e.g., comprised of a pair of barcodes. In this way, each molecular barcode indicates the source partition as well as being useful to distinguish molecules within a partition. For example, a first set of 35 barcodes can be used to tag molecules in a first partition, while a second set of 35 barcodes can be used tag molecules in a second partition.

[0336] In some embodiments, after partitioning and tagging with partition tags, the molecules may be pooled for sequencing in a single run. In some embodiments, a sample tag is added to theAttorney Ref. No. GH0271WOmolecules, e.g., in a step subsequent to addition of partition tags and pooling. Sample tags can facilitate pooling material generated from multiple samples for sequencing in a single sequencing run.

[0337] Alternatively, in some embodiments, partition tags may be correlated to the sample as well as the partition. As a simple example, a first tag can indicate a first partition of a first sample; a second tag can indicate a second partition of the first sample; a third tag can indicate a first partition of a second sample; and a fourth tag can indicate a second partition of the second sample.

[0338] While tags may be attached to molecules already partitioned based on one or more characteristics, the final tagged molecules in the library may no longer possess that characteristic. For example, while single stranded DNA molecules may be partitioned and tagged, the final tagged molecules in the library are likely to be double stranded. Similarly, while DNA may be subject to partition based on different levels of methylation, in the final library, tagged molecules derived from these molecules are likely to be unmethylated. Accordingly, the tag attached to a molecule in the library typically indicates the characteristic of the “parent molecule” from which the ultimate tagged molecule is derived, not necessarily to characteristic of the tagged molecule, itself.

[0339] As an example, barcodes 1, 2, 3, 4, etc. are used to tag and label molecules in the first partition; barcodes A, B, C, D, etc. are used to tag and label molecules in the second partition; and barcodes a, b, c, d, etc. are used to tag and label molecules in the third partition. Differentially tagged partitions can be pooled prior to sequencing. Differentially tagged partitions can be separately sequenced or sequenced together concurrently, e.g., in the same flow cell of an Illumina sequencer.

[0340] After sequencing, analysis of reads can be performed on a partition-by-partition level, as well as a whole DNA population level. Tags are used to sort reads from different partitions. Analysis can include in silico analysis to determine genetic and epigenetic variation (one or more of methylation, chromatin structure, etc.) using sequence information, genomic coordinates length, coverage, and / or copy number. In some embodiments, higher coverage can correlate with higher nucleosome occupancy in genomic region while lower coverage can correlate with lower nucleosome occupancy or a nucleosome depleted region (NDR).

[0341] Disclosed methods herein comprise analyzing biomolecules (e.g., DNA) in a first, second, and third sample from a subject. In some embodiments described herein, the disclosed methods comprise partitioning DNA. In such methods, different forms of DNA (e.g., hypermethylated andAttorney Ref. No. GH0271WOhypomethylated DNA) can be physically partitioned based on one or more characteristics of the DNA. This approach can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated. In some embodiments, a first subsample or aliquot of a sample is subjected to steps for making capture probes as described elsewhere herein and a second subsample or aliquot of a sample is subjected to partitioning. In some embodiments, a sample or subsample or aliquot thereof is subjected to partitioning and differential tagging, followed by a capture step using capture probes for rearranged sequences and optionally additional capture probes, e.g., for sequence-variable and / or epigenetic target regions.

[0342] Methylation profiling can involve determining methylation patterns across different regions of the genome. For example, after partitioning molecules based on extent of methylation (e.g., relative number of methylated nucleobases per molecule) and sequencing, the sequences of molecules in the different partitions can be mapped to a reference genome. This can show regions of the genome that, compared with other regions, are more highly methylated or are less highly methylated. In this way, genomic regions, in contrast to individual molecules, may differ in their extent of methylation.

[0343] In some embodiments, the partitioning comprises contacting the DNA with an agent that recognizes a modification associated with (e.g., in) the DNA. In some embodiments, the agent that recognizes the modification is an antibody or a CpG-binding protein. In some embodiments, the agent is immobilized on a solid support. In some embodiments, the solid support comprises a bead. In some embodiments, the partitioning comprises immunoprecipitation, e.g., using the antibody agent, such as an antibody or a CpG-binding protein, immobilized on solid support.

[0344] In some embodiments, the separating comprises precipitating the CpG protein-bound DNA. In some embodiments, the separating comprises precipitating the CpG protein-bound DNA to separate it from the unbound DNA. In some embodiments, the partitioning comprises precipitating the CpG protein-bound DNA. In some embodiments, the partitioning comprises precipitating the CpG protein-bound DNA to separate it from the unbound DNA. In some embodiments, the precipitating the CpG protein-bound DNA can be performed using any pair of binding partners. In some embodiments, one of the binding partners may be linked to the CpG protein, and the other binding partner may be linked to a solid support. In some embodiments, the binding partner comprises biotin and streptavidin. In some embodiments, the biotin may be linked to the CpG-binding protein, and the streptavidin may be linked to a solid support. In someAttorney Ref. No. GH0271WOembodiments, the CpG-binding protein is linked to a solid support, optionally using any pair of binding partners. In some embodiments, the separating comprises immunoprecipitating the CpG protein-bound DNA. In some embodiments, the separating comprises immunoprecipitating the CpG protein-bound DNA separately from the unbound DNA. In some embodiments, the partitioning comprises immunoprecipitating the CpG protein-bound DNA. In some embodiments, the partitioning comprises immunoprecipitating the CpG protein-bound DNA separately from the unbound DNA.

[0345] In some embodiments, the CpG-binding protein domain binds to methylated CpG dinucleotides and unmethylated CpG dinucleotides with about equal affinity, e.g., mCpG-binding domain 3 (MBD3). In some embodiments, the mCpG-binding protein has an affinity (e.g., Kd) for a methylated CpG dinucleotide within a factor of about 10, about 5, about 2, or about 1.5 of its affinity for an unmethylated CpG dinucleotide. In some embodiments, the CpG-binding protein domain does not preferentially bind to methylated CpG dinucleotides relative to unmethylated CpG dinucleotides, e.g., mCpG-binding domain 3 (MBD3). In some embodiments, the CpG-binding protein domain does not preferentially bind to unmethylated CpG dinucleotides relative to methylated CpG dinucleotides, e.g., mCpG-binding domain 3 (MBD3).

[0346] In some embodiments, the CpG-binding protein domain preferentially binds to methylated CpG dinucleotides relative to unmethylated CpG dinucleotides (i.e., an mCpG-binding protein). In some embodiments, the mCpG-binding protein has a greater affinity for a methylated CpG dinucleotide relative to an unmethylated CpG dinucleotide. In some embodiments, the mCpG-binding protein has an affinity (e.g., Kd) for a methylated CpG dinucleotide that is stronger than the affinity for an unmethylated CpG dinucleotide by a factor of about 10, about 5, about 2, or about 1.5. In some embodiments, the mCpG-binding protein comprises mCpG-binding domain 4 (MBD4). In some embodiments, the mCpG-binding protein comprises mCpG-binding domain 2 (MBD2). In some embodiments, the mCpG-binding protein comprises methyl CpG binding protein 2 (MeCP2). In some embodiments, the mCpG-binding protein comprises mCpG-binding domain 1 (MBD1). In some embodiments, the MBD1 contains three internal CXXC zinc finger domains: CXXC-1, CXXC-2, and CXXC-3. In some embodiments, MBD1 contains two internal CXXC zinc finger domains: CXXC-1 and CXXC-2 In some embodiments, MBD1 contains internal CXXC zinc finger domains CXXC-1 and CXXC-2 and does not contain CXXC-3Attorney Ref. No. GH0271WO

[0347] In some embodiments, the modification is methylation, and in some such embodiments, the partitioning comprises partitioning on the basis of methylation level. In some such embodiments, the agent is a methyl binding reagent. In some embodiments, the methyl binding reagent specifically recognizes 5-methylcytosine. In some such embodiments, the agent is a hydroxymethyl binding reagent. In some embodiments, the methyl binding reagent specifically recognizes 5-hydroxymethylcytosine, biotinylated 5-hydroxymethylcytosine, glucosylated 5-hydroxymethylcytosine, or sulfonylated 5-hydroxymethylcytosine. In some embodiments, the partitioning comprises partitioning on the basis of binding to a protein comprising contacting the sample comprising the DNA with a binding reagent specific for the protein. In some such embodiments, binding reagent specifically binds a methylated protein, an acetylated protein, such as a methylated or acetylated histone. In some embodiments, the binding reagent specifically binds an unmethylated or unacetylated protein epitope.

[0348] In some embodiments, the modification is hydroxymethylation, and in some such embodiments, the partitioning comprises partitioning on the basis of hydroxymethylation level. In some such embodiments, the agent is a hydroxymethyl binding reagent, such as an antibody. In some embodiments, the hydroxymethyl binding reagent (e.g., antibody) specifically recognizes 5-hydroxymethylcytosine (5-hmC). In some embodiments, a modification such as hydroxymethylation is labeled (e.g., biotinylated, glucosylated, or sulfonated) before being contacted with an agent that recognizes the labeled form of the modification. For example, 5-hmC can be enzymatically glucosylated and then partitioned based on binding to J-binding protein 1. Exemplary methods of labeling and / or partitioning 5-hmC are provided, e.g., in Song et al., Nat. Biotech. 29:68-72 (2010); Ko et al., Nature 468:839-843 (2010); and Robertson et al., Nucleic Acids Res. 39:e55 (2011).

[0349] Where immunoprecipitation is used and involves an antibody that recognizes singlestranded DNA, the DNA may be converted to double- stranded form by complementary strand synthesis before a subsequent step. Such synthesis may use an adapter as a primer binding site, or can use random priming.

[0350] Partitioning nucleic acid molecules in a sample can increase a rare signal, e.g., by enriching rare nucleic acid molecules that are more prevalent in one partition of the sample. For example, a genetic variation present in hypermethylated DNA but less (or not) present in hypomethylated DNA can be more easily detected by partitioning a sample into hypermethylated andAttorney Ref. No. GH0271WOhypomethylated nucleic acid molecules. By analyzing multiple partitions of a sample, a multidimensional analysis of a single molecule can be performed and hence, greater sensitivity can be achieved. Partitioning may include physically partitioning nucleic acid molecules into partitions or subsamples based on the presence or absence of one or more methylated nucleobases. A sample may be partitioned into partitions or subsamples based on a characteristic that is indicative of differential gene expression or a disease state. A sample may be partitioned based on a characteristic, or combination thereof that provides a difference in signal between a normal and diseased state during analysis of nucleic acids, e.g., cell free DNA (cfDNA), non-cfDNA, tumor DNA, circulating tumor DNA (ctDNA) and cell free nucleic acids (cfNA).

[0351] In some embodiments, hypermethylation and / or hypomethylation variable epigenetic target regions are analyzed to determine whether they show differential methylation characteristic of tumor cells or cells of a type that does not normally contribute to the DNA sample being analyzed (such as cfDNA), and / or particular immune cell types.

[0352] In some instances, heterogeneous DNA in a sample is partitioned into two or more partitions (e.g., at least 3, 4, 5, 6 or 7 partitions). In some embodiments, each partition is differentially tagged. Tagged partitions can then be pooled together for collective sample prep and / or sequencing. The partitioning-tagging-pooling steps can occur more than once, with each round of partitioning occurring based on a different characteristic (examples provided herein), and tagged using differential tags that are distinguished from other partitions and partitioning means. In other instances, the differentially tagged partitions are separately sequenced.

[0353] In some embodiments, sequence reads from differentially tagged and pooled DNA are obtained and analyzed in silico. After sequencing, analysis of reads can be performed on a partition-by-partition level, as well as a whole DNA population level. Tags are used to sort reads from different partitions. Analysis to detect genetic variants can be performed on a partition-by-partition level, as well as whole nucleic acid population level. For example, analysis can include in silico analysis to determine genetic variants, such as copy number variations (CNVs), single nucleotide variations (SNVs), insertions / deletions (indels), and / or fusions in nucleic acids in each partition. In some instances, in silico analysis can include analysis to determine epigenetic variation (one or more of methylation, chromatin structure, etc.). Analysis can include in silico using sequence information, genomic coordinates length, coverage, and / or copy number. For example, coverage of sequence reads can be used to determine nucleosome positioning inAttorney Ref. No. GH0271WOchromatin. Tags are used to sort reads from different partitions. Higher coverage can correlate with higher nucleosome occupancy in genomic region while lower coverage can correlate with lower nucleosome occupancy or nucleosome depleted region (NDR).

[0354] The agents used to partition populations of nucleic acids within a sample can be affinity agents, such as antibodies with the desired specificity, natural binding partners or variants thereof (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68-72 (2011)), or artificial peptides selected e.g., by phage display to have specificity to a given target. In some embodiments, the agent used in the partitioning is an agent that recognizes a modified nucleobase. In some embodiments, the modified nucleobase recognized by the agent is a modified cytosine, such as a methylcytosine (e.g., 5-methylcytosine). In some embodiments, the modified nucleobase recognized by the agent is a product of a procedure that affects the first nucleobase in the DNA differently from the second nucleobase in the DNA of the sample. In some embodiments, the modified nucleobase may be a “converted nucleobase,” meaning that its base pairing specificity was changed by a procedure. For example, certain procedures convert unmethylated or unmodified cytosine to dihydrouracil, or more generally, at least one modified or unmodified form of cytosine undergoes deamination, resulting in uracil (considered a modified nucleobase in the context of DNA) or a further modified form of uracil. Examples of partitioning agents include antibodies, such as antibodies that recognize a modified nucleobase, which may be a modified cytosine, such as a methylcytosine (e.g., 5-methylcytosine). In some embodiments, the partitioning agent is an antibody that recognizes a modified cytosine other than 5-methylcytosine, such as 5-carboxylcytosine (5-caC). Alternative partitioning agents include methyl binding domain (MBDs) and methyl binding proteins (MBPs) as described herein, including proteins such as MeCP2, MBD4, MBD2, MBD1, and antibodies preferentially binding to 5-methylcytosine. Where an antibody is used to immunoprecipitate methylated DNA, the methylated DNA may be recovered in single-stranded form. In such embodiments, a second strand can be synthesized. Hypermethylated (and optionally intermediately methylated) subsamples may then be contacted with a methylation sensitive nuclease that does not cleave hemi-methylated DNA, such as Hpall, BstUI, or Hin6i. Alternatively or in addition, hypomethylated (and optionally intermediately methylated) subsamples may then be contacted with a methylation dependent nuclease that cleaves hemi-methylated DNA.Attorney Ref. No. GH0271WO

[0355] Additional, non-limiting examples of partitioning agents are histone binding proteins which can separate nucleic acids bound to histones 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.

[0356] In some embodiments, partitioning can comprise both binary partitioning and partitioning based on degree / level of modifications. For example, methylated fragments can be partitioned by methylated DNA immunoprecipitation (MeDIP), or all methylated fragments can be partitioned from unmethylated fragments using methyl binding domain proteins (e.g., MethylMinder Methylated DNA Enrichment Kit (ThermoFisher Scientific). Subsequently, additional partitioning may involve eluting fragments having different levels of methylation by adjusting the salt concentration in a solution with the methyl binding domain and bound fragments. As salt concentration increases, fragments having greater methylation levels are eluted.

[0357] Analyzing DNA may comprise detecting or quantifying DNA of interest. Analyzing DNA can comprise detecting genetic variants and / or epigenetic features (e.g., DNA methylation and / or DNA fragmentation). In some embodiments, the DNA of interest is one or more differentially methylated regions of the DNA. In some embodiments, the detecting or quantifying the DNA of interest comprises quantifying and / or detecting a level of methylation at one or more differentially methylated regions of the DNA. In some embodiments, quantifying and / or detecting the level of methylation at one or more differentially methylated regions of the DNA comprises sequencing at least a portion of the amplified DNA or quantitative PCR (qPCR).

[0358] In some embodiments, methylation levels can be determined using partitioning, modification-sensitive conversion such as direct detection during sequencing, methylationsensitive restriction enzyme digestion, methylation-dependent restriction enzyme digestion, or any other suitable approach. For example, different forms of DNA (e g., hypermethylated and hypomethylated DNA) can be physically partitioned based on one or more characteristics of the DNA. For example, a methylated DNA binding protein (e.g., an MBD such as MBD1, MBD2, MBD4, or MeCP2) or an antibody specific for 5-methylcytosine (as in MeDIP) can be used to partition the DNA. This approach can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated. In some embodiments, a DNA fragmentation pattern can be determined based on endpoints and / or centerpoints of DNA molecules, such as cfDNA molecules.Attorney Ref. No. GH0271WO

[0359] In some instances, the final partitions are enriched in nucleic acids having different extents of modifications (overrepresentative or underrepresentative of modifications). Overrepresentation and underrepresentation can be defined by the number of modifications born by a nucleic acid relative to the median number of modifications per strand in a population. For example, if the median number of 5-methylcytosine residues in nucleic acid in a sample is 2, a nucleic acid including more than two 5-methylcytosine residues is overrepresented in this modification and a nucleic acid with 1 or zero 5-methylcytosine residues is underrepresented. The effect of affinity separation is to enrich for nucleic acids overrepresented in a modification in a bound phase and for nucleic acids underrepresented in a modification in an unbound phase (i.e. in solution). The nucleic acids in the bound phase can be eluted before subsequent processing.

[0360] When using MeDIP or MethylMiner®Methylated DNA Enrichment Kit (ThermoFisher Scientific) various levels of methylation can be partitioned using sequential elutions. For example, a hypomethylated partition (no methylation) can be separated from a methylated partition by contacting the nucleic acid population with the MBD from the kit, which is attached to magnetic beads. The beads are used to separate out the methylated nucleic acids from the non- methylated nucleic acids. Subsequently, one or more elution steps are performed sequentially to elute nucleic acids having 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, e.g., at least 150 mM, at least 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or 2000 mM. After such methylated nucleic acids are eluted, magnetic separation is once again used to separate higher level of methylated nucleic acids from those with lower level of methylation. The elution and magnetic separation steps can be repeated to create various partitions such as a hypomethylated partition (enriched in nucleic acids comprising no methylation), a methylated partition (enriched in nucleic acids comprising low levels of methylation), and a hyper methylated partition (enriched in nucleic acids comprising high levels of methylation).

[0361] In some methods, nucleic acids bound to an agent used for affinity separation based partitioning are subjected to a wash step. The wash step washes off nucleic acids weakly bound to the affinity agent. Such nucleic acids can be enriched in nucleic acids having the modification to an extent close to the mean or median (i.e., intermediate between nucleic acids remaining bound to the solid phase and nucleic acids not binding to the solid phase on initial contacting of the sample with the agent).Attorney Ref. No. GH0271WO

[0362] The affinity separation results in at least two, and sometimes three or more partitions of nucleic acids with different extents of a modification. While the partitions are still separate, the nucleic acids of at least one partition, and usually two or three (or more) partitions are linked to nucleic acid tags, usually provided as components of adapters, with the nucleic acids in different partitions receiving different tags that distinguish members of one partition from another. The tags linked to nucleic acid molecules of the same partition can be the same or different from one another. But if different from one another, the tags may have part of their code in common so as to identify the molecules to which they are attached as being of a particular partition.

[0363] For further details regarding portioning nucleic acid samples based on characteristics such as methylation, see WO20f8 / f 19452, which is incorporated herein by reference.

[0364] In some embodiments, nucleic acid molecules can be partitioned into different partitions by methods disclosed elsewhere herein, including e.g., by sequence-specifically degrading a plurality of nucleic acid sequences comprising a modification, e.g., in the sample or a subsample thereof.

[0365] In some embodiments, the nucleic acid molecules can be partitioned into different partitions based on the nucleic acid molecules that are bound to a specific protein or a fragment thereof and those that are not bound to that specific protein or fragment thereof.

[0366] Nucleic acid molecules can be partitioned based on DNA-protein binding. Protein-DNA complexes can be partitioned based on a specific property of a protein. Examples of such properties include various epitopes, modifications (e g., histone methylation or acetylation) or enzymatic activity. Examples of proteins which may bind to DNA and serve as a basis for fractionation may include, but are not limited to, protein A and protein G. Any suitable method can be used to partition the nucleic acid molecules based on protein bound regions. Examples of methods used to partition nucleic acid molecules based on protein bound regions include, but are not limited to, SDS-PAGE, chromatin-immuno-precipitation (ChIP), heparin chromatography, and asymmetrical field flow fractionation (AF4).

[0367] In some embodiments, the partitioning comprises contacting the DNA with a methylation sensitive restriction enzyme (MSRE) and / or a methylation dependent restriction enzyme (MDRE). Following the treatment of the DNA with a MSRE or a MDRE, the DNA may be partitioned based on size to generate hypermethylated (longest DNA molecules following MSRE treatment and shortest DNA fragments following MDRE treatment), intermediate (intermediate length DNAAttorney Ref. No. GH0271WOmolecules following MSRE or MDRE treatment), and hypom ethylated (shortest DNA molecules following MSRE treatment and longest DNA fragments following MDRE treatment) subsamples.

[0368] In some embodiments, the partitioning is performed by contacting the nucleic acids with a methyl binding domain (“MBD”) of a methyl binding protein (“MBP”). In some such embodiments, the nucleic acids are contacted with an entire MBP. In some embodiments, an MBD binds to 5-methylcytosine (5mC), and an MBP comprises an MBD and is referred to interchangeably herein as a methyl binding protein or a methyl binding domain protein. In some embodiments, MBD is coupled to paramagnetic beads, such as Dynabeads® M-280 Streptavidin via a biotin linker. Partitioning into fractions with different extents of methylation can be performed by eluting fractions by increasing the NaCl concentration.

[0369] In some embodiments, bound DNA is eluted by contacting the antibody or MBD with a protease, such as proteinase K. This may be performed instead of or in addition to elution steps using NaCl as discussed above.

[0370] Examples of agents that recognize a modified nucleobase contemplated herein include, but are not limited to:

[0371] (a) MeCP2 is a protein that preferentially binds to 5-methyl-cytosine over unmodified cytosine.

[0372] (b) RPL26, PRP8 and the DNA mismatch repair protein MHS6 preferentially bind to 5-hydroxymethyl-cytosine over unmodified cytosine.

[0373] (c) FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3 preferably bind to 5-formyl-cytosine over unmodified cytosine (lurlaro et al., Genome Biol. 14: R119 (2013)).

[0374] (d) Antibodies specific to one or more methylated or modified nucleobases or conversion products thereof, such as 5mC, 5-caC, or DHU.

[0375] In general, elution is a function of the number of modifications, such as the number of methylated sites per molecule, with molecules having more methylation eluting under increased salt concentrations. To elute the DNA into distinct populations based on the extent of methylation, one can use a series of elution buffers of increasing NaCl concentration. Salt concentration can range from about 100 nm to about 2500 mM NaCl. In one embodiment, the process results in three (3) partitions. Molecules are contacted with a solution at a first salt concentration and comprising a molecule comprising an agent that recognizes a modified nucleobase, which molecule can be attached to a capture moiety, such as streptavidin. At the first salt concentration a population ofAttorney Ref. No. GH0271WOmolecules will bind to the agent and a population will remain unbound. The unbound population can be separated as a “hypomethylated” population. For example, a first partition enriched in hypomethylated form of DNAis that which remains unbound at a low salt concentration, e.g., 100 mM or 160 mM. A second partition enriched in intermediate methylated DNA is eluted using an intermediate salt concentration, e.g., between 100 mM and 2000 mM concentration. This is also separated from the sample. A third partition enriched in hypermethylated form of DNAis eluted using a high salt concentration, e.g., at least about 2000 mM.

[0376] In some embodiments, the eluted DNAis single-stranded DNA. In some embodiments, one or more adapters are ligated to the eluted DNA. In some embodiments, the eluted DNA comprises CpG-dense DNA. In some embodiments, the CpG-binding protein is removed from the eluted DNA comprising CpG-dense DNA. In some embodiments, CpG protein-bound DNA is eluted from the CpG-binding protein.

[0377] In some embodiments, a monoclonal antibody raised against 5-methylcytidine (5mC) is used to purify methylated DNA. DNA is denatured, e.g., at 95°C in order to yield single-stranded DNA fragments. Protein G coupled to standard or magnetic beads as well as washes following incubation with the anti-5mC antibody are used to immunoprecipitate DNA bound to the antibody. Such DNA may then be eluted. Partitions may comprise unprecipitated DNA and one or more partitions eluted from the beads.

[0378] In some embodiments, the partitions of DNA are desalted and concentrated in preparation for enzymatic steps of library preparation.

[0379] Sequences that comprise aberrantly high copy numbers may tend to be hypermethylated. Accordingly, in some embodiments, the DNA contacted with capture probes specific for members of an epigenetic target region set comprising a plurality of target regions that are both type-specific differentially methylated regions and copy number variants comprises at least a portion of a hypermethylated partition. The DNA from or comprising at least a portion of the hypermethylated partition may or may not be combined with DNA from or comprising at least a portion of one or more other partitions, such as an intermediate partition or a hypomethylated partition.

[0380] In some cases, different procedures are applied to different partitions to determine different characteristics of the initial sample. In some embodiments, the DNA of at least one partition is subjected to an end repair and sequencing procedure described herein. In some embodiments at least one partition is not subjected to the end repair and sequencing procedure according to theAttorney Ref. No. GH0271WOmethods of the disclosure described herein. In cases where the sequencing procedure comprises a conversion procedure, corresponding sequences from the converted and non-converted partitions can be compared to identify single nucleotides that have undergone conversion and therefore identify corresponding modified nucleosides in the initial sample.

[0381] Disclosed methods herein can comprise analyzing DNA in a sample. In some embodiments described herein, the disclosed methods comprise partitioning DNA. In such methods, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically partitioned based on one or more characteristics of the DNA. This approach can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated. In some embodiments, a first subsample or aliquot of a sample is subjected to steps for making capture probes as described elsewhere herein and a second subsample or aliquot of a sample is subjected to partitioning. In some embodiments, a sample or subsample or aliquot thereof is subjected to partitioning and differential tagging, followed by a capture step using capture probes for rearranged sequences and optionally additional capture probes, e.g., for sequence-variable and / or epigenetic target regions.

[0382] Methylation profiling can involve determining methylation patterns across different regions of the genome. For example, after partitioning molecules based on extent of methylation (e.g., relative number of methylated nucleobases per molecule) and sequencing, the sequences of molecules in the different partitions can be mapped to a reference genome. This can show regions of the genome that, compared with other regions, are more highly methylated or are less highly methylated. In this way, genomic regions, in contrast to individual molecules, may differ in their extent of methylation.H. Amplification

[0383] In some embodiments, DNA from each of the first, second, and / or third samples or a subsample thereof (e.g., a hypomethylated and / or hypermethylated subsample thereof) is amplified. In some embodiments, the DNA from each of the first, second, and / or third sub samples or a subsample thereof (e.g., a hypomethylated and / or hypermethylated subsample thereof) can be subjected to a plurality of distinct amplification reactions. For example, DNA in a converted sample, as described herein, can be amplified.Attorney Ref. No. GH0271WO

[0384] In some embodiments, the amplification of the DNA in the converted sample comprises using a DNA polymerase. In some embodiments, the DNA polymerase is a uracil-tolerant DNA polymerase.

[0385] In some embodiments, the uracil-tolerant polymerase may be Q5U® Hot Start High-Fidelity DNA Polymerase, On Taq DNA Polymerase, Taq DNA Polymerase, LongAmp® Taq DNA Polymerase, Hemo KlenTaty, Epimark® Hot Start Taq DNA Polymerase, Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, Bst 2.0 DNA Polymerase, Bst 3.0 DNA Polymerase, Bsu DNA Polymerase, Large Fragment, phi29 DNA Polymerase, phi29-XT DNA Polymerase, Therminator™ DNA Polymerase, DNA Polymerase I (E. coli), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”), Klenow Fragment (3'— >5' exo-), or any combination thereof.

[0386] In some embodiments, DNA can be amplified by methylation-preserving amplification. In some embodiments, the methylation-preserving amplification can occur before the contacting the DNA in a sample with a CpG-binding protein.

[0387] Amplification methods of use herein, including methylation-preserving amplification, can include any suitable methods, such as known to those of ordinary skill in the art. Sample nucleic acids may be flanked by adapters and amplified by PCR and other amplification methods using nucleic acid primers binding to primer binding sites in adapters flanking a DNA molecule to be amplified. In some embodiments, amplification methods involve cycles of extension, denaturation, and annealing resulting from thermocycling, or can be isothermal such as in linear amplification methods, transcription-mediated amplification, recombinase polymerase amplification (RPA), helices dependent amplification (HDA), loop-mediated isothermal amplification (LAMP) (Notomi et al., Nuc. Acids Res., 28, e63, 2000), rolling-circle amplification (RCA) (Blanco et al., J. Biol. Chem., 264, 8935-8940, 1989), or hyperbranched rolling circle amplification (Lizard et al., Nat. Genetics, 19, 225-232, 1998). . Other examples of amplification methods that may be optionally utilized include the ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustained sequence-based replication. In some embodiments, the methylation-preserving amplification comprises linear amplification with thermocycling.

[0388] Typically, the amplification reactions generate a plurality of non-uniquely or uniquely tagged nucleic acid amplicons with molecular barcodes and sample indexes at size ranging fromAttorney Ref. No. GH0271WOabout 150 nucleotides (nt), to about 700 nt, from 250 nt to about 350 nt, or from about 320 nt to about 550 nt. In some embodiments, the amplicons have a size of about 180 nt. In some embodiments, the amplicons have a size of about 200 nt.

[0389] In some embodiments, methylation-preserving amplification comprises amplification performed in the presence of a methyltransferase. Methylating agents of use in methylationpreserving amplification methods described herein are known to those of ordinary skill in the art, and can include, for example, any suitable methyltransferase. In some embodiments, the methylating agent is DNMT1. DNMT1 is the most abundant DNA methyltransferase in mammalian cells and predominantly methylates hemimethylated CpG di-nucleotides in the mammalian genome. For example, DNA molecules replicated using PCR amplification with DNMT1 incubation will maintain their methylation status post-amplification, for use in further analyses, such as those described herein (such as an epigenetic base conversion step and / or an enrichment step).

[0390] Additional methylating agents useful herein include the mammalian methyltransferases, DNMT3a and DNMT3b, the plant methyltransferases, MET1, and CMT3. In some embodiments, DNMT1 or another suitable methyltransferase is used with a methyl donor and may be used with or without cofactors known to those of ordinary skill in the art. DNMT1 works in vitro at 95% efficiency without a cofactor; however, DNMT1 may be used with a cofactor such as NP95(Uhrfl), such as described in Bashtrykov PI, et al. “The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism.” J Biol Chem. 2014. In some embodiments, DNMT1 is used at a concentration of about 50-10000 U / mL, such as about 50-2000, about 50-5000, about 2500-7500, or about 5000-10000 U / mL. In some embodiments, DNMT1 is used at a concentration of about 100-500, about 500-1000, about 100-1000, about 1000-1500, about 500-1500, about 600-1400, about 700-1300, about 800-1200, about 900-1100, or about 950-1050 U / mL. In some embodiments, DNMT1 is used at a concentration of about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, or about 2000 U / mL. In some embodiments, DNMT1 is used at a concentration of about 1,000 U / ml.

[0391] In some embodiments, enriching methylated DNA in a sample comprises amplification, such as embodiments comprising quantitative PCR (qPCR) or digital PCR. In some embodiments,Attorney Ref. No. GH0271WOthe copy number of a nucleic acid region (e.g., a DNA region encoding a target gene or an RNA transcript) is quantified using qPCR. Some such embodiments comprising targeted detection of DNA sequences using qPCR or digital PCR do not comprise standard DNA library preparation steps, such as adapter ligation or tagging. In some embodiments, the sample comprises biomolecules comprising RNA (e.g., mRNA) . In some such embodiments, the RNA can be reverse transcribed into DNA using reverse-transcription PCR (RT-PCR).

[0392] In some embodiments, DNA in a converted sample, as described herein, can be amplified. In some embodiments, the amplification of the DNA in the converted sample comprises using a DNA polymerase. In some embodiments, the DNA polymerase is a uracil -tolerant DNA polymerase. In some embodiments, the uracil-tolerant polymerase may be Q5U® Hot Start High-Fidelity DNA Polymerase, OneTaq® DNA Polymerase, Taq DNA Polymerase, LongAmp® Taq DNA Polymerase, Hemo KlenT f , Epimark® Hot Start Taq DNA Polymerase, Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, Bst 2.0 DNA Polymerase, Bst 3.0 DNA Polymerase, Bsu DNA Polymerase, Large Fragment, phi29 DNA Polymerase, phi29-XT DNA Polymerase, Therminator™ DNA Polymerase, DNA Polymerase I (E. coli), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”), Klenow Fragment (3'— 5' exo-), or any combination thereof.

[0393] In some embodiments, amplification is performed prior to a sequencing step. In some embodiments, amplification performed prior to a capturing step. In some embodiments, amplification is performed after a capturing step, and prior a sequencing step.

[0394] In some embodiments, the present methods perform dsDNA ligations with T-tailed and C-tailed adapters. The addition of C-tailed adapters can increase ligation efficiency because the A-tailing reaction can also add G-tails to a small portion of the DNA molecules, when the A tailing is performed in the presence of dGTP, such as when the A-tailing is performed in the same reaction as the end repair. The use of T-tailed and C-tailed adapters can result in amplification of at least 50, 60, 70 or 80% of double stranded nucleic acids. The present methods can increase the amount or number of amplified molecules relative to control methods performed with T-tailed adapters alone by at least 10, 15, or 20%.

[0395] In some embodiments, an amplification of the DNA in a sample, such as a converted sample, comprises amplifying rolling-circle amplification (RCA). In some embodiments, RCA comprises circularizing a DNA template (e.g., DNA in the converted sample). In someAttorney Ref. No. GH0271WOembodiments, RCA comprises copying the circularized DNA template using a rolling circle polymerase to generate a plurality of circularized DNA templates. In some embodiments, the rolling circle polymerase is a phi29 DNA polymerase. Exemplary methods of RCA are provided, e.g., in Lou et al., Proc. Natl. Acad. Sci. 110 (49) 19872-19877 (2013). In some embodiments, the RCA occurs prior to a step of sequencing the DNA.

[0396] In some embodiments, adapted DNA is amplified before sequencing. This may be an additional amplification step subsequent to an earlier amplification step. In some embodiments, amplification of adapted DNA comprises RCA, e.g., as described above. In some embodiments, RCA comprises copying the circularized DNA template using a rolling circle polymerase to generate a plurality of circularized DNA templates. In some embodiments, the rolling circle polymerase is a phi29 DNA polymerase. Amplification may in some cases be before one or more capture steps. In some embodiments, the ligation step occurs after the conversion step. In some embodiments, the ligation occurs before or simultaneously with amplification.

[0397] In some embodiments, sequencing DNA that was amplified using RCA provides sequence reads comprising multiple copies of the sequence of an original sample molecule or converted molecule and the copies are used to determine a consensus sequence of the original sample molecule or converted molecule.1. Custom Primer Sets

[0398] In some embodiments, the methods provided herein further comprise obtaining a panel of custom primers configured to amplify DNA and / or RNA comprising at least a portion of the one or more biologically-linked plasma biomarkers. In some embodiments, the panel of custom primers comprises at least 5, 7, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 custom primers. In some embodiments, the methods provided herein further comprise amplifying DNA and / or RNA from the second sample by contacting the second sample with the panel of custom primers prior to a sequencing step. In some embodiments, the determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject comprises sequencing at least a portion of the amplified DNA from the second sample. In some embodiments, the presence or absence of at least 5, 7, 10, 15, 20, 30, 4050, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 of the biologically-linked plasma biomarkers in the second sample are determined.Attorney Ref. No. GH0271WO

[0399] In some embodiments, the custom panel of PCR primers comprises sequences that hybridize to a portion of the DNA or RNA comprising at least a portion of the one or more biologically-linked plasma biomarkers. For example, if the nucleic acid data obtained from the first (e.g., tumor) sample comprises structural variations (e.g., structural variations comprising a unique breakpoint), the custom PCR primers can be designed to hybridize to a region of DNA or RNA spanning the breakpoint. If the one or more biologically-linked plasma biomarkers are copy number variations, the custom PCR primers can be designed to hybridize to one or more target gene loci and / or one or more target gene mRNA for quantitative PCR (qPCR) or reverse transcription quantitative PCR (RT-qPCR) to detect the number of copies of one or more target gene against one or more control or reference genes.I. Hybridization, Isolating, Enriching, Capturing, and Using Capture Reagents

[0400] In some embodiments, DNA in a sample or a subsample thereof (e.g., a hypomethylated and / or hypermethylated subsample thereof) can be contacted with a plurality of capture reagents (which can be “capture,” “enriching,” or “enrichment”), in which molecules having target nucleic acids and / or proteins are captured for subsequent analysis or depleted from a sample.

[0401] In some embodiments, sequences are enriched prior to sequencing the nucleic acids. Enrichment optionally performed for specific target regions or nonspecifically (“target sequences”). In some embodiments, targeted regions of interest may be enriched / captured with nucleic acid capture probes ("baits") selected for one or more bait set panels using a differential tiling and capture scheme. A differential tiling and capture scheme generally uses bait sets of different relative concentrations to differentially tile (e.g., at different "resolutions") across genomic regions associated with the baits, subject to a set of constraints (e.g., sequencer constraints such as sequencing load, utility of each bait, etc.), and capture the targeted nucleic acids at a desired level for downstream sequencing. These targeted genomic regions of interest optionally include natural or synthetic nucleotide sequences of the nucleic acid construct. In some embodiments, biotin-labeled beads with probes to one or more regions of interest can be used to capture target sequences, and optionally followed by amplification of those regions, to enrich for the regions of interest.

[0402] Sequence capture typically involves the use of oligonucleotide probes that hybridize to the target nucleic acid sequence. In some embodiments, a probe set strategy involves tiling the probes across a region of interest. Such probes can be, for example, from about 60 to about 120Attorney Ref. No. GH0271WOnucleotides in length. The set can have a depth (e g., depth of coverage) of about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 50X, or more than 50X. The effectiveness of sequence capture generally depends, in part, on the length of the sequence in the target molecule that is complementary (or nearly complementary) to the sequence of the probe.

[0403] In some embodiments, contacting is performed prior to a step of amplifying DNA and prior to a step of sequencing the DNA, and optionally after contacting the DNA with a deaminase.

[0404] In some embodiments, the contacting step is performed prior to a step of amplifying DNA, prior to a step of sequencing the DNA, after contacting the CpG-dense DNA with the methylsensitive deaminase, and after separating the CpG protein-bound DNA from unbound DNA. In some embodiments, the capture step is performed prior to a step of amplifying methylation-separated DNA, prior to a step of sequencing the DNA, after contacting the CpG-dense DNA with the methyl-sensitive deaminase, and after partitioning the DNA in the sample into a plurality of subsamples. In some embodiments, the capture step is performed prior to a step of amplifying methylation-separated DNA, prior to a step of sequencing the DNA, after separating the CpG protein-bound DNA from unbound DNA, and after partitioning the DNA in the sample into a plurality of subsamples. In some embodiments, the capture step is performed prior to a step of amplifying methylation-separated DNA, after contacting the CpG-dense DNA with the methylsensitive deaminase, after separating the CpG protein-bound DNA from unbound DNA, and after partitioning the DNA in the sample into a plurality of subsamples. In some embodiments, the capture step is performed prior to a step of sequencing the DNA, after contacting the CpG-dense DNA with the methyl-sensitive deaminase, after separating the CpG protein-bound DNA from unbound DNA, and after partitioning the DNA in the sample into a plurality of subsamples.

[0405] In some embodiments, methods disclosed herein comprise capturing (i.e., enriching) one or more sets of target regions of DNA, such as cfDNA. In some embodiments, capture is performed prior to a step of amplifying DNA, prior to a step of sequencing the DNA, after contacting the DNA with a deaminase, and / or after partitioning the DNA in the sample into a plurality of sub samples.

[0406] Capture may be performed using any suitable approach using a plurality of capture reagents (e.g., RNA and / or DNA capture probes). In some embodiments, the capture probes comprise a target-hybridizing sequence of about 80 to about 140 nucleotides in length. In some embodiments, the capture probes comprise a target-hybridizing sequence of about 90 to about 130 nucleotides inAttorney Ref. No. GH0271WOlength. In some embodiments, the capture probes comprise a target-hybridizing sequence of about 100 to about 120 nucleotides in length. In some embodiments the capture probes comprise a targethybridizing sequence of 100 nucleotides in length. In some embodiments, the capture probes comprise a target-hybridizing sequence of 120 nucleotides in length. In some embodiments, the capture probes are about 80 to about 140 nucleotides in length. In some embodiments, the capture probes are about 90 to about 130 nucleotides in length. In some embodiments, the capture probes are about 100 to about 120 nucleotides in length. In some embodiments the capture probes are 100 nucleotides in length. In some embodiments, the capture probes are 120 nucleotides in length. In some embodiments, the capture probes are more than 120 nucleotides in length. In some embodiments, the capture probes are less than 120 nucleotides in length. Target capture can involve use of a bait set comprising oligonucleotide baits (a type of probe useful herein) labeled with a capture moiety, such as biotin or the other examples noted below. The probes can have sequences selected to tile across a panel of regions, such as genes. Such bait sets are combined with a sample under conditions that allow hybridization of the target molecules with the baits. Then, captured molecules are isolated using the capture moiety. For example, a biotin capture moiety by bead-based streptavidin. Such methods are further described in, for example, U.S. patent 9,850,523, issuing December 26, 2017, which is incorporated herein by reference.

[0407] Capture moieties include, without limitation, biotin, avidin, streptavidin, a nucleic acid comprising a particular nucleotide sequence, digoxygenin, a histidine tag, an affinity tag, an immunoglobulin constant domain, a hapten recognized by an antibody, and magnetically attractable particles. In some embodiments, the immunoglobulin constant domain may be bound using protein A, protein G, or a secondary antibody. In some embodiments, the secondary antibody comprises an anti-mouse secondary antibody. In some embodiments, the anti-mouse secondary antibody is a goat anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, or a donkey anti-mouse secondary antibody. In some embodiments, a CpG-binding protein comprises a capture moiety. The extraction moiety can be a member of a binding pair, such as biotin / streptavidin or hapten / antibody. In some embodiments, a capture moiety that is attached to an analyte is captured by its binding pair which is attached to an isolatable moiety, such as a magnetically attractable particle or a large particle that can be sedimented through 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 captureAttorney Ref. No. GH0271WOmoieties are biotin which allows affinity separation by binding to streptavidin linked or linkable to a solid phase or an oligonucleotide, which allows affinity separation through binding to a complementary oligonucleotide linked or linkable to a solid phase.

[0408] A panel of regions targeted for enrichment can be selected such that they do not contain regions known to include the base modification used in the end repair reaction. When the end repair is performed with dNTPs comprising 5mC or 5hmC, a panel of regions targeted for enrichment may be selected such that they do not contain CpH dinucleotides which are known to be naturally methylated in the subject (e.g. humans). Such CpH dinucleotides can be identified through the use of publicly available resources (e.g. MethBankS.0: a database ofDNA methylomes across a variety’ of species Nucleic Acids Res 2018). Such an approach has the advantage that any detected methylated CpH dinucleotides can unambiguously be attributed to regions synthesized in the end repair.

[0409] In some embodiments, capturing comprises contacting the DNA to be captured with a set of target-specific probes. The set of target-specific probes may have any of the features described herein for sets of target-specific probes, including but not limited to in the embodiments set forth above and the sections relating to probes below. Capturing may be performed on one or more subsamples (e.g., one or more hypomethylated and / or hypermethylated subsamples) of each of the first, second, and / or third samples prepared during methods disclosed herein.

[0410] The capturing step may be performed using conditions suitable for specific nucleic acid hybridization, which generally depend to some extent on features of the probes such as length, base composition, etc. Those skilled in the art will be familiar with appropriate conditions given general knowledge in the art regarding nucleic acid hybridization. In some embodiments, complexes of target-specific probes and DNA are formed.

[0411] In some embodiments, a method described herein comprises capturing DNA obtained from a subject (e.g., DNA from the first, second, and / or third samples or a subsample thereof (e.g., a hypomethylated and / or hypermethylated subsample thereof)) for a plurality of sets of target regions. In some embodiments, the DNA obtained from a subject is cell free DNA (cfDNA).

[0412] In some embodiments, the target regions comprise at least a portion of the biologically-linked plasma biomarkers identified by comparing the nucleic acid data from the first (e.g., tumor) sample to the nucleic acid data from the third (e.g., buffy) sample and / or a reference sample. InAttorney Ref. No. GH0271WOsome such embodiments, the target regions comprise biologically-linked plasma biomarker target regions.

[0413] In some embodiments, the target regions comprise epigenetic target regions, which may show differences in methylation levels and / or fragmentation patterns depending on whether they originated from a tumor or from healthy cells. In some embodiments, the target regions comprise sequence-variable target regions, which may show differences in sequence depending on whether they originated from a tumor or from healthy cells. The capturing step produces a captured set of DNA molecules, and the DNA molecules corresponding to the sequence-variable target region set are captured at a greater capture yield in the captured set of DNA molecules than DNA molecules corresponding to the epigenetic target region set. For additional discussion of capturing steps, capture yields, and related aspects, see W02020 / 160414, which is incorporated herein by reference for all purposes.

[0414] In some embodiments, a method described herein comprises contacting DNA (e.g., cfDNA) obtained from a subject with a set of target-specific probes, wherein the set of targetspecific probes is configured to capture DNA corresponding to the sequence-variable target region set at a greater capture yield than DNA corresponding to the epigenetic target region set.

[0415] It can be beneficial to capture DNA (e.g., cfDNA) corresponding to the sequence-variable target region set at a greater capture yield than DNA corresponding to the epigenetic target region set because a greater depth of sequencing may be necessary to analyze the sequence-variable target regions with sufficient confidence or accuracy than may be necessary to analyze the epigenetic target regions. The volume of data needed to determine fragmentation patterns (e.g., to test for perturbation of transcription start sites or CTCF binding sites) or fragment abundance (e.g., in hypermethylated and hypomethylated partitions) is generally less than the volume of data needed to determine the presence or absence of cancer-related sequence mutations. Capturing the target region sets at different yields can facilitate sequencing the target regions to different depths of sequencing in the same sequencing run (e.g., using a pooled mixture and / or in the same sequencing cell).

[0416] In various embodiments, the methods further comprise sequencing the captured DNA e.g., to different degrees of sequencing depth for the epigenetic and sequence-variable target region sets, consistent with the discussion herein.Attorney Ref. No. GH0271WO

[0417] In some embodiments, complexes of target-specific probes and DNA are separated from DNA not bound to target-specific probes. For example, where target-specific probes are bound covalently or noncovalently to a solid support, a washing or aspiration step can be used to separate unbound material. Alternatively, where the complexes have chromatographic properties distinct from unbound material (e.g., where the probes comprise a ligand that binds a chromatographic resin), chromatography can be used.

[0418] In some embodiments, complexes of target-specific probes and DNA are separated from DNA not bound to target-specific probes. For example, where target-specific probes are bound covalently or noncovalently to a solid support, a washing or aspiration step can be used to separate unbound material. Alternatively, where the complexes have chromatographic properties distinct from unbound material (e.g., where the probes comprise a ligand that binds a chromatographic resin), chromatography can be used.

[0419] As discussed in detail elsewhere herein, the set of target-specific probes may comprise a plurality of sets such as probes for a sequence-variable target region set and probes for an epigenetic target region set. In some such embodiments, the capturing step is performed with the probes for the sequence-variable target region set and the probes for the epigenetic target region set in the same vessel at the same time, e.g., the probes for the sequence-variable and epigenetic target region sets are in the same composition. This approach provides a relatively streamlined workflow. In some embodiments, the concentration of the probes for the sequence- variable target region set is greater than the concentration of the probes for the epigenetic target region set.

[0420] Alternatively, the capturing step is performed with the sequence-variable target region probe set in a first vessel and with the epigenetic target region probe set in a second vessel, or the contacting step is performed with the sequence-variable target region probe set at a first time and a first vessel and the epigenetic target region probe set at a second time before or after the first time. This approach allows for preparation of separate first and second compositions comprising captured DNA corresponding to the sequence-variable target region set and captured DNA corresponding to the epigenetic target region set. The compositions can be processed separately as desired (e.g., to fractionate based on methylation as described elsewhere herein) and recombined in appropriate proportions to provide material for further processing and analysis such as sequencing.Attorney Ref. No. GH0271WO

[0421] In some embodiments, a captured set of DNA (eg., cfDNA) is provided. With respect to the disclosed methods, the captured set of DNA may be provided, e.g., by performing a capturing step prior to a sequencing step as described herein. The captured set may comprise DNA corresponding to a sequence-variable target region set, an epigenetic target region set, or a combination thereof. In some embodiments, a capture step is performed prior to a conversion step or after a conversion step.

[0422] In some embodiments, a first target region set is captured (e.g., from the first, second, and / or third sample or a subsample thereof), comprising at least epigenetic target regions. The epigenetic target regions captured from the subsample may comprise hypermethylation variable target regions. In some embodiments, the hypermethylation variable target regions are CpG-containing regions that are unmethylated or have low methylation in DNA from healthy subjects (e.g., below-average methylation relative to bulk DNA). In some embodiments, the hypermethylation variable target regions are regions that show lower methylation in healthy DNA than in at least one other tissue type. Without wishing to be bound by any particular theory, cancer cells may shed more DNA into the bloodstream than healthy cells of the same tissue type. As such, the distribution of tissue of origin of cfDNA may change upon carcinogenesis. Thus, an increase in the level of hypermethylation variable target regions in the first subsample can be an indicator of the presence (or recurrence, depending on the history of the subject) of cancer.

[0423] In some embodiments, a second target region set is captured from the (e.g., from the first, second, and / or third sample or a subsample thereof) comprising at least epigenetic target regions. The epigenetic target regions may comprise hypomethylation variable target regions. In some embodiments, the hypomethylation variable target regions are CpG-containing regions that are methylated or have high methylation in DNA from healthy subjects (e.g., above-average methylation relative to bulk DNA). In some embodiments, the hypomethylation variable target regions are regions that show higher methylation in healthy DNA than in at least one other tissue type. Without wishing to be bound by any particular theory, cancer cells may shed more DNA into the bloodstream than healthy cells of the same tissue type. As such, the distribution of tissue of origin of cfDNA may change upon carcinogenesis. Thus, an increase in the level of hypomethylation variable target regions in the second subsample can be an indicator of the presence (or recurrence, depending on the history of the subject) of cancer.Attorney Ref. No. GH0271WO

[0424] In some embodiments the quantity of captured sequence-variable target region DNA is greater than the quantity of the captured epigenetic target region DNA, when normalized for the difference in the size of the targeted regions (footprint size).

[0425] Alternatively, first and second captured sets may be provided, comprising, respectively, DNA corresponding to a sequence-variable target region set and DNA corresponding to an epigenetic target region set. The first and second captured sets may be combined to provide a combined captured set.

[0426] In some embodiments, a probe (e.g., a capture probe) is specific for members of an epigenetic target region set that includes only methylated DNA molecules. In some embodiments, the methylated DNA molecules comprise methylated cytosines. In some embodiments, the methylated cytosines are in CpG dinucleotides. In some embodiments, a probe (e.g., a capture probe) is specific for members of an epigenetic target region set that includes only unmethylated DNA molecules. In some embodiments, the unmethylated DNA molecules comprise uracils as a result of the conversion of unmethylated cytosines. In some embodiments, the unmethylated molecules comprise thymines as a result of the conversion of methylated cytosines. In some embodiments, the uracils are in UpG dinucleotides. In some embodiments, the thymines are in TpG dinucleotides. In some embodiments, a probe (e.g., a capture probe) captures both unmethylated and methylated DNA molecules.

[0427] In some embodiments in which a captured set comprising DNA corresponding to the sequence-variable target region set and the epigenetic target region set includes a combined captured set as discussed above, the DNA corresponding to the sequence-variable target region set may be present at a greater concentration than the DNA corresponding to the epigenetic target region set, e.g., a 1.1 to 1.2-fold greater concentration, a 1.2- to 1.4-fold greater concentration, a 1.4- to 1.6-fold greater concentration, a 1.6- to 1.8-fold greater concentration, a 1.8- to 2.0-fold greater concentration, a 2.0- to 2.2-fold greater concentration, a 2.2- to 2.4-fold greater concentration a 2.4- to 2.6-fold greater concentration, a 2.6- to 2.8-fold greater concentration, a 2.8- to 3.0-fold greater concentration, a 3.0- to 3.5-fold greater concentration, a 3.5- to 4.0, a 4.0-to 4.5-fold greater concentration, a 4.5- to 5.0-fold greater concentration, a 5.0- to 5.5-fold greater concentration, a 5.5- to 6.0-fold greater concentration, a 6.0- to 6.5-fold greater concentration, a 6.5- to 7.0-fold greater, a 7.0- to 7.5-fold greater concentration, a 7.5- to 8.0-fold greater concentration, an 8.0- to 8.5-fold greater concentration, an 8.5- to 9.0-fold greater concentration,Attorney Ref. No. GH0271WOa 9.0- to 9.5-fold greater concentration, 9.5- to 10.0-fold greater concentration, a 10- to 11-fold greater concentration, an 11- to 12-fold greater concentration a 12- to 13-fold greater concentration, a 13- to 14-fold greater concentration, a 14- to 15-fold greater concentration, a 15-to 16-fold greater concentration, a 16- to 17-fold greater concentration, a 17- to 18-fold greater concentration, an 18- to 19-fold greater concentration, a 19- to 20-fold greater concentration, a 20-to 30-fold greater concentration, a 30- to 40-fold greater concentration, a 40- to 50-fold greater concentration, a 50- to 60-fold greater concentration, a 60- to 70-fold greater concentration, a 70-to 80-fold greater concentration, a 80- to 90-fold greater concentrati...

Claims

Attorney Ref. No. GH0271WOCLAIMSWhat is claimed is:

1. A method for monitoring a subject for minimal residual disease, comprising:a) obtaining a first sample from a subject; wherein the first sample comprises a tumor tissue sample;b) profiling nucleic acids obtained from the first sample to identify biologically- linked plasma biomarkers;c) identifying one or more biologically-linked plasma biomarkers specific to the subject;d) determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject; ande) classifying the subject to be positive or negative for minimal residual disease based on the presence or absence of at least a portion of the one or more biologically- linked plasma biomarkers in the second sample.

2. The method of claim 1, further comprising obtaining a third sample from the subject, wherein the third sample is a matched normal tissue sample and / or a huffy coat sample.

3. The method of the immediately preceding claim, wherein the third sample is a buffy coat sample.

4. The method of the immediately preceding claim, further comprising profiling nucleic acids obtained from the third sample.

5. The method of any one of claims 2-4, further comprising, prior to step d), removing variants detected from profiling nucleic acids in the third sample from the one or more biologically-linked plasma biomarkers identified as specific to the subject.

6. The method of any one of the preceding claims, wherein the second sample is a cell-free DNA sample.

7. The method of the immediately preceding claim, wherein the second sample is a plasma sample.

8. The method of any one of the preceding claims, wherein the profiling nucleic acids obtained from the first sample and / or the determining the presence or absence of at least aAttorney Ref. No. GH0271WOportion of the one or more biologically-linked plasma biomarkers in a second sample further comprises a nucleic acid amplification step.

9. The method of any one of the preceding claims, wherein the profiling nucleic acids obtained from the first sample comprises sequencing nucleic acids from the first sample.

10. The method of any one of the preceding claims, wherein the determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject comprises sequencing nucleic acids from the second sample and / or detecting one or more nucleic acids and / or proteins present in the second sample.

11. The method of the immediately preceding claim, wherein the detecting comprises high multiplex PCR, digital PCR, quantitative PCR (qPCR), reverse transcription PCR (RT- PCR), or sequencing.

12. The method of any one of claims 9-11, wherein the sequencing comprises next generation sequencing.

13. The method of any one of claims 9-11, wherein the sequencing comprises long-read sequencing.

14. The method of any one of claims 9-11, wherein the sequencing comprises nanopore sequencing.

15. The method of any one of claims 9-11, wherein the sequencing comprises 5-letter or 6- letter sequencing.

16. The method of any one of claims 9-11, wherein the sequencing comprises sequencing the DNA in a manner that distinguishes the first nucleobase from the second nucleobase.

17. The method of claim 10, wherein the detecting one or more proteins present in the second sample comprises mass spectrometry (MS).

18. The method of any one of the preceding claims, wherein determining the presence or absence of at least a portion of the one or more biologically-linked plasma biomarkers in the second sample comprises enriching biologically-linked plasma biomarkers in the second sample using a custom panel of capture reagents specific to the subject.

19. The method of the immediately preceding claim, wherein the custom panel of capture reagents are configured to capture nucleic acids and / or proteins comprising the biologically-linked plasma biomarkers.Attorney Ref. No. GH0271WO20. The method of the immediately preceding claim, wherein the custom panel of capture reagents comprises at least 5, 7, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 capture reagents.

21. The method of any one of claims 19-20, wherein the custom panel of capture reagents comprise a plurality of capture reagents, wherein the plurality of capture reagents comprise one or more DNA capture probes, one or more RNA capture probes, and / or one or more protein capture reagents.

22. The method of the immediately preceding claim, wherein the one or more protein capture reagents is selected from antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp.

23. The method of the immediately preceding claim, wherein the one or more protein capture reagents comprises an antibody.

24. The method of any one of claims 21-23, wherein the one or more protein capture reagents comprise an oligonucleotide-capture reagent conjugate.

25. The method of the immediately preceding claim, wherein the oligonucleotide-capture reagent conjugate comprises a molecular tag.

26. The method of the immediately preceding claim, wherein the molecular tag is a molecular barcode.

27. The method of any one of the preceding claims, wherein markers comprising somatic variants, DNA methylation and / or RNA expression markers are identified by profiling the nucleic acids obtained from the first sample.

28. The method of any one of the preceding claims, wherein the biologically-linked plasma biomarkers comprise differentially methylated DNA, cell-free RNA (cfRNA), extracellular vesicle (EV)-surface protein, and / or circulating protein.

29. The method of any one of the preceding claims, wherein markers comprising somatic variants and / or epigenetic variants are identified by profiling the nucleic acids obtained from the first sample.

30. The method of the immediately preceding claim, wherein the biologically-linked plasma biomarkers comprise cell-free RNA (cfRNA), extracellular vesicle (EV)-surface protein, and / or circulating protein.Attorney Ref. No. GH0271WO31. The method of any one of the preceding claims, wherein markers comprising RNA expression markers are identified by profiling the nucleic acids obtained from the first sample.

32. The method of the immediately preceding claim, wherein the biologically-linked plasma biomarkers comprise differentially methylated DNA, extracellular vesicle (EV)-surface protein, and / or circulating protein.

33. The method of any one of claims 27-32, wherein the biologically-linked plasma biomarkers comprise one or more biomarkers resulting from the markers identified by profiling the nucleic acids obtained from the first sample.

34. The method of any one of claims 27-33, wherein nucleic acid data is obtained from the first sample comprising hypomethylated DNA and the biologically-linked plasma biomarkers comprise cfRNA and / or circulating protein corresponding to one or more hypomethylated DNA regions.

35. The method of any one of claims 27-34, wherein nucleic acid data is obtained from the first sample comprising an increase in RNA expression of one or more target genes and the biologically-linked plasma biomarkers comprise one or more circulating proteins corresponding to the one or more target genes.

36. The method of the immediately preceding claim, wherein the target genes correspond to cell-surface proteins, optionally wherein the cell-surface proteins are extracellular vesicle (EV)-proteins.

37. The method of any one of claims 27-36, wherein nucleic acid data is obtained from the first sample comprising an increase in DNA copy number of one or more target regions and the biologically-linked plasma biomarkers comprise one or more circulating proteins corresponding to the one or more target regions.

38. The method of any one of claims 27-37, wherein nucleic acid data is obtained from the first sample comprising one or more alternatively spliced mRNA molecules and the biologically-linked plasma markers comprise one or more circulating protein isoforms corresponding to the alternatively spliced mRNA.

39. The method of any one of claims 27-38, wherein nucleic acid data is obtained from the first sample comprising one or more somatic DNA mutations and the biologically-linkedAttorney Ref. No. GH0271WOplasma markers comprise one or more mutated circulating proteins corresponding to the one or more somatic DNA mutations.

40. The method of any one of claims 27-39, wherein nucleic acid data is obtained from the first sample comprising one or more structural variants and the biologically-linked plasma markers comprise one or more mutated circulating proteins and / or one or more cell free RNA (cfRNA) molecules corresponding to the one or more structural variants.

41. The method of any one of claims 27-40, wherein one or more, or each, of the biologically-linked plasma biomarkers do not comprise DNA.

42. The method of any one of claims 27-40, wherein nucleic acid data is obtained from the first sample comprising one or more cancer associated somatic mutations associated with a gain-of-function phenotype and the biologically-linked plasma markers comprise one or more biomarkers resulting from the gain-of-function mutations.

43. The method of the immediately preceding claim, wherein the somatic mutations associated with a gain-of-function phenotype are one or more mutations in the BRAF gene.

44. The method of the immediately preceding claim, wherein the biologically-linked plasma markers comprise one or more hypermethylated DNA regions.

45. The method of the immediately preceding claim, where the one or more hypermethylated DNA regions are associated with CpG island methylator phenotype (CIMP).

46. The method of any one of the preceding claims, wherein the biologically-linked plasma biomarkers comprise biomarkers of a different type than the nucleic acids profiled to identify the biologically-linked plasma biomarkers.

47. The method of any one of the preceding claims, wherein determining the presence or absence of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject comprises determining the amounts of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject.

48. The method of any one of the preceding claims, further comprising determining the presence or absence of minimum residual disease based on the amounts of at least a portion of the one or more biologically-linked plasma biomarkers in a second sample obtained from the subject.Attorney Ref. No. GH0271WO49. The method of the immediately preceding claim, wherein the method further comprises obtaining a third sample from the subject, wherein the third sample is a matched normal tissue sample and / or a buffy coat sample, and determining the presence or absence of minimum residual disease is not based on variants detectable in the third sample.

50. The method of the immediately preceding claim, wherein the variants detectable in the third sample comprise clonal hematopoiesis of indeterminate potential (CHIP) variants.

51. The method of any one of claims 49-50, wherein the one or more biologically-linked plasma biomarkers are not present in the third sample.

52. The method of any one of the preceding claims, wherein the subject was previously diagnosed with a cancer and received one or more previous cancer treatments.

53. The method of the immediately preceding claim, wherein the first sample is obtained at one or more preselected time points prior to the one or more previous cancer treatments.

54. The method of any one of claims 52-53, wherein a third sample is obtained at one or more preselected time points prior to the one or more previous cancer treatments, wherein the third sample is a matched normal tissue sample and / or a buffy coat sample.

55. The method of any one of claims 52-54, wherein the second sample is obtained at one or more preselected time points following the one or more previous cancer treatments.

56. The method of any one of claims 52-55, further comprising determining a cancer recurrence score.

57. The method of any one of claims 52-56, wherein the cancer recurrence status of the subject is determined to be at risk for cancer recurrence when a cancer recurrence score is determined to be at or above a predetermined threshold or the cancer recurrence status of the subject is determined to be at lower risk for cancer recurrence when the cancer recurrence score is below the predetermined threshold.

58. The method of the immediately preceding claim, further comprising comparing the cancer recurrence score of the subject with a predetermined cancer recurrence threshold, wherein the subject is classified as a candidate for a subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold or not a candidate for a subsequent cancer treatment when the cancer recurrence score is below the cancer recurrence threshold.Attorney Ref. No. GH0271WO59. The method of any one of the preceding claims, further comprising partitioning at least a portion of the DNA from the first sample into at least first and second subsamples, wherein the first subsample is a hypermethylated subsample comprising methylated DNA to a greater extent than the second subsample, and the second subsample is a hypomethylated subsample.

60. The method of any one of the preceding claims, further comprising partitioning at least a portion of the DNA from the second sample into at least first and second subsamples, wherein the first subsample is a hypermethylated subsample comprising methylated DNA to a greater extent than the second subsample, and the second subsample is a hypomethylated subsample.

61. The method of any one of claims 59-60, wherein the partitioning comprises contacting the DNA with an agent that recognizes methyl cytosine in the DNA.

62. The method of the immediately preceding claim, wherein the agent that recognizes methyl cytosine is a methyl binding reagent.

63. The method of the immediately preceding claim, wherein the methyl binding reagent is a methyl binding domain (MBD) protein or an antibody.

64. The method of any one of claims 59-63, wherein the methyl binding reagent specifically recognizes 5-methylcytosine.

65. The method of any one of claims 59-64, wherein the methyl binding reagent is immobilized on a solid support.

66. The method of any one of claims 59-65, wherein the partitioning comprises immunoprecipitation of methylated DNA.

67. The method of any one of claims 59-66, further comprising contacting the DNA from the hypermethylated subsample with at least one nuclease, optionally wherein the at least one nuclease comprises at least one restriction enzyme.

68. The method of the immediately preceding claim, wherein the at least one restriction enzyme comprises at least one methylation-sensitive restriction enzyme (MSRE).

69. The method of the immediately preceding claim, wherein the MSRE cleaves an unmethylated CpG sequence.

70. The method of any one of claims 68-69, wherein the MSRE comprises one or more of Aatll, AccII, Acil, Aorl3HI, Aor51HI, BspT104I, BssHII, BstUI, CfrlOI, Clal, Cpol,Attorney Ref. No. GH0271WOEco52T, HaelT, HapII, Hhal, Hin6T, HpalT, HpyCH4TV, Mlul, Nael, NotT, Nrul, Nsbl, PmaCI, Psp 14061, Pvul, Sadi, Sall, Smal, and SnaBI.

71. The method of any one of claims 59-70, further comprising contacting the DNA from the hypomethylated subsample with at least one nuclease, optionally wherein the at least one nuclease comprises at least one restriction enzyme.

72. The method of the immediately preceding claim, wherein the at least one restriction enzyme comprises at least one methylation-dependent restriction enzyme (MORE).

73. The method of the immediately preceding claim, wherein the MDRE cleaves a methylated CpG sequence.

74. The method of the immediately preceding claim, wherein the MDRE comprises one or more of MspJI, LpnPI, FspEI, or McrBC.

75. The method of any one of claims 1-58, further comprising partitioning at least a portion of the DNA from the first sample into at least first and second subsamples on the basis of binding to a protein, optionally wherein the protein is a methylated protein, an acetylated protein, an unmethylated protein, or an unacetylated protein; and / or optionally wherein the protein is a histone.

76. The method of the immediately preceding claim, wherein the partitioning comprises contacting the nucleic acids of the sample with a binding reagent which is specific for the protein and is immobilized on a solid support.

77. The method of any one of the preceding claims, further comprising capturing a sequencevariable target region set from the first sample.

78. The method of the immediately preceding claim, wherein the capturing is performed before the profiling step and at least a portion of the sequence-variable target region set is profiled to identify the biologically-linked plasma biomarkers.

79. The method of any one of claims 77-78, wherein the capturing is performed after a partitioning step, if present.

80. The method of the immediately preceding claim, wherein the sequence-variable target region set comprises at least 10, at least 20, at least 50, at least 75, at least 100, at least 200, at least 500, at least 750, at least 1,000, at least 2,000, at least 5,000, at least 7,500 or at least 10,000 target regions.Attorney Ref. No. GH0271WO81. The method of any one of claims 77-80, wherein the sequence-variable target region set has a footprint of at least 100 megabases.

82. The method of any one of the preceding claims, further comprising capturing an epigenetic target region set from the first sample.

83. The method of the immediately preceding claim, wherein the capturing is performed before the profiling step and at least a portion of the epigenetic target region set is profiled to identify the biologically-linked plasma biomarkers.

84. The method of the immediately preceding claim, wherein the capturing is performed prior to a partitioning step, if present.

85. The method of any one of claims 82-84, wherein the epigenetic target region set comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 target regions.

86. The method of any one of claims 82-85, wherein the epigenetic target region set has a footprint of at least 2000 kilobases.

87. The method of any one of claims 82-86, comprising determining a methylation level of the captured epigenetic target regions.

88. The method of any one of claims 82-87, wherein at least one of the captured epigenetic target regions is a differentially methylated region.

89. The method of any one of claims 82-88, wherein at least one of the captured epigenetic target regions is a fragment.

90. The method of any one of claims 82-89, wherein at least one of the captured epigenetic target regions is a hypermethylated region, optionally wherein the hypermethylated region is a type-specific hypermethylated region.

91. The method of any one of claims 82-90, wherein at least one of the captured epigenetic target regions is a hypomethylated region, optionally wherein the hypomethylated region is a type-specific hypomethylated region.

92. The method of any one of claims 82-91, wherein at least one of the captured epigenetic target regions comprises a CTCF binding site, and / or a transcription start site.

93. The method of any one of claims 82-92, wherein at least one of the captured epigenetic target regions is at least one type-specific epigenetic target region.Attorney Ref. No. GH0271WO94. The method of the immediately preceding claim, wherein the at least one type-specific epigenetic target region comprises type-specific differentially methylated regions and / or type specific fragments.

95. The method of the immediately preceding claim, wherein the at least one type-specific epigenetic target region comprises type-specific hypomethylated regions and / or typespecific hypermethylated regions.

96. The method of any one of claims 94-95, wherein the at least one type-specific epigenetic target region comprises cell-type specific, cell cluster-type specific, tissue-type specific, and / or cancer-type specific epigenetic target regions.

97. The method of any one of claims 94-96, wherein the at least one type-specific epigenetic target region comprises type-specific epigenetic target regions that are: hypermethylated in immune cells relative to non-immune cell types present in a blood sample;differentially methylated in colon relative to other tissue types;differentially methylated in lung relative to other tissue types;differentially methylated in breast relative to other tissue types;differentially methylated in liver relative to other tissue types;differentially methylated in kidney relative to other tissue types;differentially methylated in pancreas relative to other tissue types;differentially methylated in prostate relative to other tissue types;differentially methylated in skin relative to other tissue types; ordifferentially methylated in bladder relative to other tissue types.

98. The method of any one of claims 94-97, wherein the type-specific hypermethylated region or the hypermethylated regions are methylated to an extent that is at least 10%, 20%, 30%, or at least 40% greater than the average methylation of the target regions in the sample.

99. The method of any one of claims 94-98, wherein the at least one type-specific epigenetic target region comprises target regions that are:hypomethylated in non-immune blood cells relative to the methylation level of the target regions in a different cell or tissue type in the sample;fragments specific to immune cells relative to non-immune cell types present in theAttorney Ref. No. GH0271WOsample; orfragments specific to colon, lung, breast, liver, kidney, pancreas, prostate, skin, or bladder relative to other tissue types.

100. The method of any one of claims 94-99, comprising identifying at least one cell type or tissue type from which the at least one type-specific epigenetic target region originated.

101. The method of the immediately preceding claim, wherein the level of the at least one type-specific epigenetic target region that originated from a cell or tissue type is determined.

102. The method of the immediately preceding claim, wherein the level of the at least one type-specific epigenetic target regions that originated from immune cells, non-immune blood cells, colon, lung, breast, liver, kidney, prostate, skin, bladder, or pancreas are determined.

103. The method of any one of the preceding claims, further comprising subjecting the DNA of the first sample or one or more subsamples thereof to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase, 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.

104. The method of the immediately preceding claim, wherein the procedure that affects a first nucleobase in the DNA differently from a second nucleobase comprises a conversion procedure that changes the base pairing specificity of the base or does not change the base pairing specificity of the base, depending on the modification status of the base.

105. The method of any one of claims 103-104, wherein the first nucleobase is an unmodified cytosine and the second nucleobase is a modified cytosine, optionally wherein the modified cytosine is 5-methylcytosine or 5-hydroxymethylcytosine.

106. The method of any one of claims 104-105, wherein the procedure that affects a first nucleobase of the DNA differently from a second nucleobase of the DNA is methylationsensitive conversion.

107. The method of the immediately preceding claim, wherein the methylation-sensitive conversion is bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, APOBEC-coupled epigenetic (ACE) conversion, enzymaticAttorney Ref. No. GH0271WOmethyl-seq (EM-seq) conversion, single-enzyme 5-methylcytosine sequencing (SEM- seq) conversion, or direct methylation sequencing (DM-seq).

108. The method of the immediately preceding claim, wherein the Tet-assisted conversion further comprises a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.

109. The method of any one of claims 104-108, wherein the conversion procedure comprises contacting the DNA with a CpG-specific DNA methyltransferase (MTase) or a CpG- specific carboxymethyltransferase (CxMTase), a methyl donor or a carboxymethyl donor, and a cytosine deaminase.

110. The method of the immediately preceding claim, wherein the cytosine deaminase is an APOBEC enzyme, optionally wherein the APOBEC enzyme is APOBEC3A.

111. The method of any one of claims 104-110, wherein the procedure that affects a first nucleobase of the DNA differently from a second nucleobase of the DNA is performed before the profiling step and at least a portion of the DNA subjected to the procedure is profiled to identify the biologically-linked plasma biomarkers.

112. The method of any one of the preceding claims, wherein the subject is a human.

113. The method of any one of the preceding claims, further comprising determining a likelihood that the subject has precancer.

114. The method of any one of claims 1-113, further comprising determining a likelihood that the subject has cancer.

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