Nucleic acid modification profiling method

Abstract

The disclosure relates to methods for determining the modification profiles of nucleic acids. The methods use at least two different binding agents which bind to different types of nucleic acid modification. Provided are methods of analyzing a nucleic acid population comprising nucleic acids with different extents of at least two types of modification.

Description

Atty. Docket No. GH0139WO NUCLEIC ACID MODIFICATION PROFILING METHOD CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application No. 63 / 623,079, filed January 19, 2024, which is incorporated by reference herein in its entirety for all purposes. FIELD OF THE INVENTION

[0002] This disclosure relates to methods for analyzing a nucleic acid population comprising nucleic acids with different extents of at least two types of modification using the simultaneous binding of different types of binding agents for enrichment and separation procedures. INTRODUCTION

[0003] Chemically modified nucleotide bases play an essential role in the epigenetic control of gene expression in both animals and plants. Examples include, but are not limited to, 5- methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5- carboxylcytosine (5caC), N6-methyladenine (6mA), and abasic sites. Modifications can occur de novo or can be inherited. Nucleotide base modifications can be a result of biological processes, such as DNA damage, and abnormal modifications can be indicative of aberrant biological processes. As a consequence, modified nucleosides and modified nucleotide patterns have been highlighted as key biomarkers for diseases, for example cancer.

[0004] The detection of multiple differential epigenetic variants or nucleotide modifications generally requires parallel or completely sequential assays. A general focus on methylation as a key nucleotide modification has resulted in many methods for the detection of DNA methylation, including conversion-based assays. Conversion-based methods for methylation mapping and detecting include variations of bisulfite and oxidative bisulfite and Tet-assisted bisulfide conversion, EM-seq, TAPS and TAPS-β conversion and ACE-seq. See, e.g., Moss et al., Nat Commun.2018; 9: 5068; Booth et al., Science 2012; 336: 934-937; Yu et al., Cell 2012; 149: 1368-80; Liu et al., Nature Biotechnology 2019; 37:424–429; Schutsky, E.K. et al.; and Vaisvila et al. Genome Research 202131(7): 1280-1289. Conversion-based assays are not able to resolve all different types of modification status. For example, bisulfite sequencing and EM-seq are unable to distinguish between 5mC and 5hmC methylation. Detection of multiple differential modification types using conversion-based assays therefore typically requires the input to be split into parallel assays, or requires the use of sequential assays.Atty. Docket No. GH0139WO

[0005] For samples with a limited input amount (e.g. blood volume), parallel assays inherently limit analyte sensitivity as the input amount must be split between the parallel assays. Parallel assays are also costly, as each assay requires a complete set of reagents. Sequential assays take longer to perform than parallel workflows as there are more assay steps involved. Thus, sequential assays are more costly and likely less sensitive than integrated workflow methods.

[0006] There is therefore a need for improved integrated workflows that are able to identify and map the presence or absence of different types of nucleic acid modification. SUMMARY

[0007] The present disclosure aims to meet the need for improved integrated workflows that are able to identify and map the presence or absence of different types of nucleic acid modification, provide other benefits, or at least provide the public with a useful choice. In some embodiments, the present disclosure provides methods that allow for the simultaneous analysis of multiple types of nucleic acid modification in a single integrated workflow. In some embodiments, this is achieved through the simultaneous use of at least two different types of binding agents, wherein each type of binding agent preferentially binds to nucleic acids bearing a specific type of nucleic acid modification, or a derivative thereof. For example, the elution conditions of the nucleic acids (bearing the target modification or a derivative thereof) from the respective binding agents differ between the different types of binding agents. This means that, through the use of different elution conditions, partitions can be obtained which are enriched for nucleic acids bearing the different types of modification or a derivative thereof. Subsequent analysis of these partitions (e.g. through sequencing) enables the identification of the prevalence of the different types of modification in the original sample, and which nucleic acids contain those modifications or a derivative thereof.

[0008] Accordingly, the following exemplary embodiments are provided.

[0009] Embodiment 1 is a method of analyzing a nucleic acid population comprising nucleic acids with different extents of at least two types of modification, including a first type of modification and a second type of modification, wherein the method comprises, in order: i) optionally forming a derivative of the first type of modification and / or the second type of modification; ii) simultaneously contacting the nucleic acid population with:Atty. Docket No. GH0139WO (a) a first binding agent that preferentially binds to nucleic acids bearing the first type of modification, or the derivative thereof; and (b) a second binding agent that preferentially binds to nucleic acids bearing the second type of modification, or the derivative thereof; iii) separating the nucleic acids bound to the first binding agent and the second binding agent from the unbound nucleic acids to produce an unbound partition; iv) subjecting the nucleic acids bound to the first binding agent and the second binding agent to conditions which favour elution of the nucleic acids bound to the first binding agent but not elution of the nucleic acids bound to the second binding agent, thereby providing eluted nucleic acids, wherein the eluted nucleic acids provide a first partition; v) retaining the nucleic acids bound to the second binding agent to provide a second partition; and vi) analyzing one or more of the unbound partition, the first partition, and the second partition.

[0010] Embodiment 2 is the method of embodiment 1, wherein the first type of modification is 5-methylcytosine (5mC).

[0011] Embodiment 3 is the method of embodiment 2, wherein the first binding agent comprises: (i) a methyl-binding domain (MBD) protein; and / or (ii) an anti-5mC antibody, or an antigen-binding fragment thereof.

[0012] Embodiment 4 is the method of any one of embodiments 1 to 3, wherein the second type of modification is 5-hydroxymethyl cytosine (5hmC).

[0013] Embodiment 5 is the method of embodiment 4, wherein the second binding agent comprises an anti-5hmC antibody, or an antigen-binding fragment thereof.

[0014] Embodiment 6 is the method of embodiment 4, wherein the method comprises forming a derivative of 5hmC by glucosylation of 5hmC.

[0015] Embodiment 7 is the method of embodiment 6, wherein the second binding agent comprises J-binding protein-1 (JBP-1).

[0016] Embodiment 8 is the method of embodiment 6, wherein forming a derivative of 5hmC further comprises attaching a capture moiety to the glucosylated 5hmC.

[0017] Embodiment 9 is the method of embodiment 8, wherein the capture moiety is biotin, optionally photocleavable biotin.

[0018] Embodiment 10 is the method of embodiment 9, wherein the second binding agent comprises streptavidin.Atty. Docket No. GH0139WO

[0019] Embodiment 11 is the method of any one of embodiments 1 to 3, wherein the second type of modification is an abasic site.

[0020] Embodiment 12 is the method of embodiment 11, wherein the method comprises forming a derivative of the abasic site, optionally by reacting the nucleic acid with an aldehyde probe, such that the probe covalently binds to the abasic site.

[0021] Embodiment 13 is the method of embodiment 12, wherein the aldehyde probe further comprises a coupling moiety which allows conjugation of the probe to another compound, such as a capture moiety.

[0022] Embodiment 14 is the method of embodiment 13, wherein the capture moiety comprises biotin, optionally wherein the second binding agent comprises streptavidin.

[0023] Embodiment 15 is the method of any one of embodiments 1-14, wherein step (iv) comprises sequentially subjecting the nucleic acids bound to the first binding agent and the second binding agent to a plurality of conditions which increasingly favour elution of the nucleic acids bound to the first binding agent, such that the first partition is separated into a plurality of first sub-partitions, wherein the nucleic acids eluted in the first of the plurality of conditions have a lower extent of the first type of modification compared to the nucleic acids eluted in the last of the plurality of conditions.

[0024] Embodiment 16 is the method of any one of embodiments 1-15, wherein step (v) comprises subjecting the nucleic acids bound to the second binding agent to conditions which favour elution of the nucleic acids bound to the second binding agent, wherein the eluted nucleic acids provide the second partition.

[0025] Embodiment 17 is the method of embodiment 16, wherein step (v) comprises sequentially subjecting the nucleic acids bound to the second binding agent to a plurality of conditions which increasingly favour elution of the nucleic acids bound to the second binding agent, such that the second partition is separated into a plurality of second sub-partitions, wherein the nucleic acids eluted in the first of the plurality of conditions have a lower extent of the second type of modification compared to the nucleic acids eluted in the last of the plurality of conditions.

[0026] Embodiment 18 is the method of any one of the preceding embodiments, further comprising capturing at least an epigenetic target region set of nucleic acids from the nucleic acid population or a subsample thereof, optionally wherein the capturing comprises contactingAtty. Docket No. GH0139WO the nucleic acid population with a plurality of target-specific probes specific for members of the epigenetic target region set.

[0027] Embodiment 19 is the method of the immediately preceding embodiment, comprising determining a methylation level of at least one of the plurality of epigenetic target regions.

[0028] Embodiment 20 is the method of any one of embodiments 18-19, wherein the at least one of the plurality of epigenetic target regions is a differentially methylated region.

[0029] Embodiment 21 is the method of any one of embodiments 18-20, wherein the at least one of the plurality of epigenetic target regions is a fragment.

[0030] Embodiment 22 is the method of any one of embodiments 18-20, wherein the at least one of the plurality of epigenetic target regions is a hypermethylated region, optionally wherein the hypermethylated region is a type-specific hypermethylated region.

[0031] Embodiment 23 is the method of any one of embodiments 18-20, wherein the at least one of the plurality of epigenetic target regions is a hypomethylated region, optionally wherein the hypomethylated region is a type-specific hypomethylated region.

[0032] Embodiment 24 is the method of any one of embodiments 18-20, wherein the at least one of the plurality of epigenetic target regions comprises a CTCF binding site, and / or a transcription start site.

[0033] Embodiment 25 is the method of any one of embodiments 18-24, wherein the at least one of the plurality of epigenetic target regions is at least one type-specific epigenetic target region.

[0034] Embodiment 26 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.

[0035] Embodiment 27 is the method of embodiment 25 or 26, wherein the at least one type- specific epigenetic target region comprises type-specific hypomethylated regions and / or type- specific hypermethylated regions.

[0036] Embodiment 28 is the method of any one of embodiments 25-27, 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.

[0037] Embodiment 29 is the method of any one of embodiments 25-28, wherein the at least one type-specific epigenetic target region comprises type-specific epigenetic target regions that are:Atty. Docket No. GH0139WO 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.

[0038] Embodiment 30 is the method of any one of embodiments 22 or 27-29, 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.

[0039] Embodiment 31 is the method of any one of embodiments 25-29, 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.

[0040] Embodiment 32 is the method of any one of embodiments 25-31, comprising identifying at least one cell type or tissue type from which the at least one type-specific epigenetic target region originated.

[0041] Embodiment 33 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.

[0042] Embodiment 34 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.Atty. Docket No. GH0139WO

[0043] Embodiment 35 is the method of the any one of the preceding embodiments, further comprising capturing at least one sequence-variable target region set of the nucleic acids, optionally wherein the capturing comprises contacting the nucleic acid population with a plurality of target-specific probes specific for the sequence-variable target regions.

[0044] Embodiment 36 is the method of any one of embodiments 18-35, wherein the capturing the at least an epigenetic target region set of nucleic acids and / or the at least one sequence variable target region set of nucleic acids occurs prior to the contacting the nucleic acid population with the first and second binding agents.

[0045] Embodiment 37 is the method of any one of embodiments 18-35, wherein the capturing the at least an epigenetic target region set of nucleic acids and / or the at least one sequence variable target region set of nucleic acids occurs after the contacting the nucleic acid population with the first and second binding agents.

[0046] Embodiment 38 is the method of any one of embodiments 18-35, wherein the at least an epigenetic target region set of nucleic acids and / or the at least one sequence variable target region set of nucleic acids are captured from at least the first partition and / or the second partition.

[0047] Embodiment 39 is the method of any one of embodiments 1-38, wherein the analyzing comprises nucleic acid sequencing.

[0048] Embodiment 40 is the method of any one of embodiments 1-39, wherein the analyzing comprises preparing one or more sequencing libraries from at least a subset of the nucleic acids in one or more of the unbound partition, the first partition, the second partition, one or more of the first sub-partitions, and / or one or more of the second sub-partitions, wherein the one or more sequencing libraries are then subjected to nucleic acid sequencing.

[0049] Embodiment 41 is the method of embodiment 40, wherein the one or more sequencing libraries is obtained using a single-stranded sequencing library preparation method.

[0050] Embodiment 42 is the method of embodiment 40, wherein the one or more sequencing libraries is obtained using a double-stranded sequencing library preparation method.

[0051] Embodiment 43 is the method of any one of embodiments 38-41, wherein the sequencing comprises sequencing the nucleic acids in a manner that distinguishes a nucleobase comprising the first type of modification and a nucleobase comprising the second type of modification.Atty. Docket No. GH0139WO

[0052] Embodiment 44 is the method of any one of embodiments 39-43, wherein two or more of the partitions are pooled prior to the sequencing.

[0053] Embodiment 45 is the method of any one of embodiments 39-44, wherein the sequencing comprises long-read sequencing.

[0054] Embodiment 46 is the method of any one of embodiments 39-45, wherein the sequencing comprises nanopore sequencing.

[0055] Embodiment 47 is the method of any one of embodiments 39-45, wherein the sequencing comprises 5-letter or 6-letter sequencing.

[0056] Embodiment 48 is the method of any one of embodiments 39-44, wherein the sequencing comprises next generation sequencing.

[0057] Embodiment 49 is the method of any one of the preceding embodiments, wherein the method comprises ligating one or more adapters to the nucleic acids of the nucleic acid population, thereby producing adapter-ligated nucleic acids.

[0058] Embodiment 50 is the method of the immediately preceding embodiment, wherein the adapter-ligated nucleic acids are amplified prior to the sequencing.

[0059] Embodiment 51 is the method of embodiment 49 or embodiment 50, wherein the one or more adapters comprises at least one tag.

[0060] Embodiment 52 is the method of the immediately preceding embodiment, wherein the at least one tag comprises a molecular barcode.

[0061] Embodiment 53 is the method of any one of the preceding embodiments, wherein at least nucleic acid molecules of the first partition and nucleic acid molecules of the second partition are differentially tagged.

[0062] Embodiment 54 is the method of any one of the preceding embodiments, wherein the nucleic acid population comprises DNA.

[0063] Embodiment 55 is the method of the immediately preceding embodiment, wherein the DNA comprises cell-free DNA (cfDNA), optionally wherein the cfDNA is from a bodily fluid, such as blood, serum, or plasma.

[0064] Embodiment 56 is the method of any one of the preceding embodiments, wherein the the nucleic acid population is derived from a sample obtained from a subject.

[0065] Embodiment 57 is the method of the immediately preceding embodiment, wherein the sample is from a bodily fluid, such as blood, serum, or plasma.Atty. Docket No. GH0139WO

[0066] Embodiment 58 is the method of embodiment 56 or embodiment 57, wherein the blood sample is a whole blood sample, a plasma sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample.

[0067] Embodiment 59 is the method of any one of embodiments 56-58, wherein the sample is a blood sample.

[0068] Embodiment 60 is the method of embodiment 56, wherein the sample is a tissue sample.

[0069] Embodiment 61 is the method of any one of embodiments 56-60, wherein the subject is an animal.

[0070] Embodiment 62 is the method of embodiment any one of embodiments 56-61, wherein the subject is a human.

[0071] Embodiment 63 is the method of any one of embodiments 56-62, comprising determining a likelihood that the subject has precancer.

[0072] Embodiment 64 is the method of any one of embodiments 56-63, comprising determining a likelihood that the subject has cancer.

[0073] Embodiment 65 is the method of any one of embodiments 39-64, wherein the sequencing comprises generating a plurality of sequencing reads, and wherein the method further comprises mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads, and processing the mapped sequence reads to determine the likelihood that the subject has cancer or precancer.

[0074] Embodiment 66 is the method of any one of embodiments 56-65, wherein the sample is obtained from a subject who was previously diagnosed with a cancer and received one or more previous cancer treatments, optionally wherein the sample is obtained at one or more preselected time points following the one or more previous cancer treatments.

[0075] Embodiment 67 is the method of the immediately preceding embodiment, further comprising determining a cancer recurrence score, optionally 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.

[0076] Embodiment 68 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 cancerAtty. Docket No. GH0139WO 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.

[0077] In some embodiments, the results of the methods disclosed herein are used as an input to generate a report. The report may be in a paper or electronic format. For example, the detection of the detection of methylation profiles of nucleic acids (e.g. cfDNA), as obtained by the methods disclosed herein, or information derived therefrom, can be displayed directly in such a report. Alternatively or additionally, diagnostic information or therapeutic recommendations which are at least in part based on the methods disclosed herein can be included in the report.

[0078] The various steps of the methods disclosed herein may be carried out at the same or different times, in the same or different geographical locations, e.g. countries, and / or by the same or different people.

[0079] Additional advantages will be set forth in part in the description which follows or may be learned by practice. The advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIG 1. illustrates an exemplary methodology and workflow for the methods of the present disclosure.

[0081] FIG 2. illustrates embodiments of the method of the disclosure. This workflow demonstrates the partitioning of a 5mC and 5hmC-containing nucleic acids from cell free DNA (cfDNA).

[0082] FIG 3. illustrates embodiments of the method of the disclosure. The upper panel demonstrates a workflow for the recovering and amplification of double stranded DNA (dsDNA) containing 5hmC after partitioning, for example, as shown in Figure 2. The lower panel demonstrates a workflow for the recovery and amplification of single stranded DNA (ssDNA) containing 5hmC after partitioning, for example, as shown in Figure 2.

[0083] FIG 4. illustrates embodiments of the method of the disclosure. This workflow demonstrates the formation of a 5hmC derivative to attach a capture moiety. This workflow further demonstrates the simultaneous integration of different types of binding agent to facilitate the partitioning of 5mC and 5hmC-containing nucleic acids. This method facilitates theAtty. Docket No. GH0139WO simultaneous identification of 5mC and 5hmC modifications within a nucleic acid population through a single integrated workflow.

[0084] FIG 5. illustrates embodiments of the method of the disclosure. This workflow demonstrates the formation of an abasic site derivative to attach a capture moiety. This workflow further demonstrates the integration of different types of binding agent to facilitate the partitioning of 5mC and abasic site-containing nucleic acids. This method facilitates the simultaneous identification of 5mC and abasic site modifications within a nucleic acid population.

[0085] FIG.6 is a schematic diagram of an example of a system suitable for use with some embodiments of the disclosure. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0086] Reference will now be made in detail to certain embodiments of the disclosure. While the disclosure will be described in conjunction with such embodiments, it will be understood that they are not intended to limit the disclosure to those embodiments. On the contrary, the disclosure is intended to cover all alternatives, modifications, and equivalents, which may be included within the disclosure as defined by the appended claims.

[0087] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of nucleic acids.

[0088] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement.

[0089] Unless specifically noted in the above specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of” or “consisting essentially of” the recited components.

[0090] The section headings used herein are for organizational purposes and are not to be construed as limiting the disclosed subject matter in any way.

[0091] All patents, patent applications, websites, other publications or documents and the like cited herein whether supra or infra, are expressly incorporated by reference in their entirety forAtty. Docket No. GH0139WO all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant, unless otherwise indicated. I. Definitions

[0092] “Solid tissue” or “solid tissue cells” as used herein means tissue or cells, respectively, in or derived from a solid tissue. Solid tissue cells exclude circulating cell types, such as cells normally present in blood or lymph. Examples of solid tissue types include but are not limited to colon, lung, breast, skin, prostate, stomach, pancreas, bladder, kidney, and liver.

[0093] 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-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC), despite them having different modification statuses.

[0094] “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.

[0095] 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-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC), despite them having different modification statuses.Atty. Docket No. GH0139WO

[0096] “Cell-free DNA,” “cfDNA molecules,” or simply “cfDNA” 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) into a fluid found in the organism, and may be obtained from a sample of the fluid without the need to perform an in vitro cell lysis step.

[0097] As used herein, “cellular nucleic acids” means nucleic acids that are located 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.

[0098] A “target region set” or “set of target regions” or “target regions” or “target regions of interest” or “regions of interest” or “genomic 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 complementarity).

[0099] “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). [000100] “Epigenetic target region set” refers to target regions that may show sequence- independent changes in neoplastic cells (e.g., tumor cells or 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 are not limited to, changes in methylation (increases or decreases), nucleosome distribution, CCCTC-binding factor (“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.Atty. Docket No. GH0139WO [000101] As used herein, an “epigenetic feature” refers to any feature of DNA or chromatin other than primary sequence (i.e., the sequence of A, C, G, and T bases). Epigenetic features include covalent modifications of bases, such as methylation, and modifications and positioning of histones and other stably DNA-associated proteins. [000102] 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 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., 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 or from the same cell or tissue type from a healthy subject. In some embodiments, a DMR has a detectably lower degree of methylation (e.g., 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. [000103] As used herein, “type-specific” in the context of an epigenetic variation means an epigenetic variation that is present at a detectably different degree in one cell or tissue type, or in a plurality of related cell or tissue types, relative to other cell or tissue types. Similarly, a “type- specific epigenetic target region” is an epigenetic target region that has a detectably different epigenetic characteristic in one cell or tissue type, or in a plurality of related cell or tissue types, relative to other cell or tissue types. Exemplary epigenetic characteristics are discussed in the definition of epigenetic target regions set forth above. For example, a “type-specific differentially methylated region” is a region of DNA that has a detectably different degree of methylation in one cell or tissue type, or in a plurality of related cell or tissue types, relative to other cell or tissue types. Examples of a type-specific differentially methylated region include tissue-specific differentially methylated regions, including those associated with copy-number gain in early cancer. In some embodiments, capturing, identification, and / or detection of type- specific differentially methylated regions facilitates identification of the cell or tissue type from which the DNA originated. The cell or tissue from which a type-specific differentially methylated region originated may be a wild type cell or tissue or a neoplastic cell or tissue. InAtty. Docket No. GH0139WO another example, a “type-specific fragment” of DNA is a DNA fragment arising from a type- specific fragmentation pattern that is present at a detectably different degree in one cell or tissue type, or in a plurality of related cell or tissue types, relative to other cell or tissue types. In some embodiments, a type-specific fragment is only present in the specific cell or tissue type(s). In some embodiments, a type-specific fragment is present to a detectably greater extent in the specific cell or tissue type(s). [000104] As used herein, a “blood sample” refers to a sample comprising whole blood or a component thereof (e.g., plasma, serum, buffy coat, plasma pellet). [000105] 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. [000106] 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 nucleic acid 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 nineAtty. Docket No. GH0139WO methylated nucleotides, and another partitioned set can have unmethylated nucleic acid molecules (zero methylated nucleotides). [000107] As used herein, the form of the “originally isolated” sample refers to the composition or chemical structure of a sample at the time it was isolated and before undergoing any procedure that changes the chemical structure of the isolated sample. Similarly, a feature that is “originally present” in a molecule refers to a feature present in an “original molecule” or in molecules “originally comprising” the feature before the molecule undergoes any procedure that changes the chemical structure of the molecule. [000108] 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, for example, is irrelevant because uracil nonetheless most preferentially pairs with A among the four standard DNA bases. [000109] “Capturing” one or more target molecules refers to preferentially isolating or separating the one or more target molecules from non-target molecules. [000110] A “captured set” of nucleic acids refers to nucleic acids that have undergone capture. [000111] “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. [000112] As used herein, a “label” is a capture moiety, fluorophore, oligonucleotide, or other moiety that facilitates detection, separation, or isolation of that to which it is attached. [000113] As used herein, a “capture moiety” is a molecule that allows affinity separation of molecules linked to the capture moiety from molecules lacking the capture moiety. Exemplary capture moieties include 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. [000114] As used herein, a “target-specific probe” means a probe that specifically binds to a target region, such as an epigenetic target region or a sequence-variable target region. In some embodiments, target-specific probes comprise a capture moiety to facilitate capture of the target region to which it specifically binds.Atty. Docket No. GH0139WO [000115] As used herein, a “tag” is a molecule, such as a nucleic acid, label, fluorophore, or peptide, containing information that indicates a feature of the molecule to which the tag is associated. For example, molecules can bear a sample tag (which distinguishes molecules in one sample from those in a different sample), a molecular tag / molecular barcode / barcode (which distinguishes different molecules from one another (in both unique and non-unique tagging scenarios), a purification tag, and / or a detectable tag or label. [000116] As used herein, a “target molecule” is a molecule, such as a protein, carbohydrate, nucleic acid, or lipid, that is targeted for capture, identification, and / or detection. In some embodiments, a target molecule is a nucleic acid comprising an epigenetic target region and / or a sequence-variable target region. [000117] “Specifically binds” in the context of a probe or other oligonucleotide and a target sequence means that under appropriate hybridization conditions, the oligonucleotide or probe hybridizes to its target sequence, or 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 non- target 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, 2nded. (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). [000118] 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). [000119] The “capture yield” of a collection of probes for a given target region set refers to the amount (e.g., amount relative to another target region 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 µL) containing stringent hybridization buffer. The capture yield may be expressed in absolute terms or, for a plurality ofAtty. Docket No. GH0139WO 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. [000120] 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 addition of a methyl group to a cytosine at a CpG site (cytosine-phosphate-guanine site (i.e., a cytosine followed by a guanine in 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 5thposition 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-methylcytosine (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 epigeneticAtty. Docket No. GH0139WO regulation may cause diseases, such as cancer. Promoter methylation in DNA may be indicative of cancer. [000121] 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 was separated 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. [000122] 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. [000123] 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%,Atty. Docket No. GH0139WO 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. [000124] 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. [000125] 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. [000126] 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 separate containers 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. [000127] 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 DNA can 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. [000128] As used herein, “type-specific hypermethylation” means an increased level or degree of methylation of nucleic acid molecules in at one cell or tissue type, or in a plurality of related cell or tissue types, relative to other cell or tissue types. In some embodiments, capturing, identification, and / or detection of type-specific hypermethylated regions facilitates identification of the cell or tissue type from which the nucleic acid molecules originated. The cell or tissue from which a type-specific hypermethylated region originated may be a wild type cell or tissue or a neoplastic cell or tissue. [000129] 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.,Atty. Docket No. GH0139WO sample) of nucleic acid molecules. In some embodiments, hypomethylated DNA includes unmethylated DNA molecules. In some embodiments, hypomethylated DNA can 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. [000130] As used herein, “type-specific hypomethylation” means a decreased level or degree of methylation of nucleic acid molecules in at one cell or tissue type, or in a plurality of related cell or tissue types, relative to other cell or tissue types. In some embodiments, capturing, identification, and / or detection of type-specific hypomethylated regions facilitates identification of the cell or tissue type from which the nucleic acid molecules originated. The cell or tissue from which a type-specific hypomethylated region originated may be a wild type cell or tissue or a neoplastic cell or tissue. [000131] 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. [000132] As used herein, “mutation” 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. [000133] As used herein, the terms “neoplasm” and “tumor” are used interchangeably. They refer to abnormal growth of cells in a subject. A neoplasm or tumor can be benign, potentially malignant, or malignant. A malignant tumor is referred to as a cancer or a cancerous tumor. [000134] 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 sequencingAtty. Docket No. GH0139WO includes the use of instruments capable of sequencing single molecules. Examples 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. [000135] 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). This 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 sub-sequence 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 ofAtty. Docket No. GH0139WO 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. Terms such as “library adapters having distinct molecular barcodes” encompass library adapters for uniquely or non-uniquely tagging molecules, in that regardless of whether the adapters are for unique or non-unique tagging, distinct barcodes will be present in the population of adapters. [000136] 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. [000137] The terms “binding agent” and “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 or hmC. A “modified nucleobase,” as discussed elsewhere herein, 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, single-stranded DNA, and both double-stranded and single-stranded DNA. [000138] 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 in 5’ ^ 3’ orderAtty. Docket No. GH0139WO 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. [000139] 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. [000140] 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. A quantitative measure can be a linear combination of quantitative measures. A quantitative measure may be a normalized measure. [000141] 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 or chromosome. Examples of reference sequences include, for example, human genomes, such as, hg19 and hg38. [000142] As used herein, “sample” means anything capable of being analyzed by the methods and / or systems disclosed herein. [000143] 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),Atty. Docket No. GH0139WO 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. [000144] 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. [000145] As used herein “sequence-variable target region set” refers to a set of target regions that may exhibit changes in sequence such as nucleotide substitutions, insertions, deletions, or gene fusions or transpositions in neoplastic cells (e.g., tumor cells and cancer cells). [000146] 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. [000147] 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. Animals include 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.Atty. Docket No. GH0139WO [000148] 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. [000149] 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. [000150] 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. [000151] 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 (double- stranded) 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 insert or 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. [000152] 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, CABABB, 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.Atty. Docket No. GH0139WO [000153] “Buffy coat” refers to the portion of a blood (such as whole blood) or bone marrow sample that contains all or most of the white blood cells and platelets of the sample. The buffy coat fraction of a sample can be prepared from the sample using centrifugation, which separates sample components by density. For example, following centrifugation of a whole blood sample, the buffy coat fraction is situated between the plasma and erythrocyte (red blood cell) layers. The buffy coat can contain both mononuclear (e.g., T cells, B cells, NK cells, dendritic cells, and monocytes) and polymorphonuclear (e.g., granulocytes such as neutrophils and eosinophils) white blood cells. [000154] As used herein, “leukapheresis” refers to a procedure in which white blood cells (leukocytes) are isolated from a sample of blood collected from a subject. Leukapheresis may be performed, e.g., obtain cells for research, diagnostic, prognostic, or monitoring purposes, such as those described herein. Thus, as used herein, a “leukapheresis sample” refers to a sample comprising leukocytes collected from a subject using leukapheresis. [000155] As used herein, “peripheral blood mononuclear cells” or “PBMCs” refers to immune cells having a single, round nucleus that originate in bone marrow and are found in the peripheral circulation. Such cells include, e.g., lymphocytes (T cells, B cells, and NK cells) as well as monocytes, and are isolated from blood samples (such as from a whole blood sample collected from a subject) using density gradient centrifugation. [000156] As used herein, “amplify,” “amplifying,” or “amplification” refers to a process by which extra or multiple copies of a particular polynucleotide are formed. Amplification methods can include any suitable methods known in the art. As used herein, a nucleic acid molecule amplified using “methylation-preserving amplification” substantially maintains its methylation status post-amplification. [000157] The term “type of modification” may refer to a base modification, such as 5mC or 5hmC, or refer to a nucleotide modification, such as an abasic site. [000158] The term “preferentially binds” in the context of the binding agents, such as a binding agent that recognizes 5mC, refers to an agent (e.g. a protein) that binds to nucleic acids comprising a specific type of modification with a higher affinity compared to the equivalent nucleic acid without that modification or with the different type of modification. In some embodiments, the binding agent binds to the nucleic acids bearing the modification with an affinity at least 50X, at least 100X or at least 1000X greater compared to nucleic acids without the modification or with the different type of modification.Atty. Docket No. GH0139WO [000159] The term “simultaneously contacting” refers to the nucleic acid population being contacted by both the first binding agent and the second binding agent at the same time – i.e. in a single reaction mixture. For example, the reaction mixture may contain one set of beads comprising the first binding agent and a second set of beads comprising the second binding agent. Alternatively, the method may employ beads which comprise both the first and the second binding agents. [000160] A “X1nnnX2mutation” in a specified polypeptide as used herein, where X1and X2are amino acids and nnn is a position in an amino acid sequence, refers to a substitution in the polypeptide of amino acid X1present at position nnn of the full-length wild-type polypeptide with amino acid X2. The polypeptide is the human polypeptide unless indicated otherwise. The polypeptide comprising the X1nnnX2mutation 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 “V1900X2mutation” where X2is 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. [000161] “Or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise. II. Exemplary methods A. Overview [000162] The present disclosure provides methods of analyzing a nucleic acid population, such as from a sample, such as a sample from a subject, comprising nucleic acids with different extents of at least two types of modification, including a first type of modification and a second type of modification. Without being bound by a particular theory, multiple DNA modifications of interest can be resolved through a single workflow that enriches analytes (e.g., nucleic acid molecules comprising at least a first and / or a second type of modification) together and subsequently partitions the analytes separately, such as based on differential elution and recoveryAtty. Docket No. GH0139WO mechanisms. Compared to other parallel or sequential enrichment strategies, this integrated enrichment workflow for multiplex analytes can, for example, reduce assay time and / or costs, and can enhance analyte sensitivities for an amount of input material (e.g. blood). For a given input amount (e.g. blood volume), parallel assays generally suffer from limited analyte sensitivity, as the input is split and applied separately to the parallel assays. However, for example, a methylation partitioning (MBD) workflow described herein enables more sensitive detection of the (5mCpG) methylation rate of DNA fragments. Applying this to cfDNA from blood, the methylation pattern determined can be used to screen for early cancer and / or disease recurrence, amongst other oncology applications. Other DNA modifications informative to cancer diagnostics may also be missed by industry standard workflows. Thus, the methods of this disclosure can add additional DNA modification signal detection options and capabilities to assays, while increasing 5mCpG detection sensitivity and reducing assay costs and / or time. [000163] Accordingly, in a first aspect, the disclosure provides a method of analyzing a nucleic acid population comprising nucleic acids with different extents of at least two types of modification, including a first type of modification and a second type of modification, wherein the method comprises, (i) optionally forming a derivative of the first type of modification and / or the second type of modification; (ii) simultaneously contacting the nucleic acid population with (a) a first binding agent that preferentially binds to nucleic acids bearing the first type of modification, or the derivative thereof, and (b) a second binding agent that preferentially binds to nucleic acids bearing the second type of modification, or the derivative thereof; (iii) separating the nucleic acids bound to the first binding agent and the second binding agent from the unbound nucleic acids to produce an unbound partition; (iv) subjecting the nucleic acids bound to the first binding agent and the second binding agent to conditions which favour elution of the nucleic acids bound to the first binding agent but not elution of the nucleic acids bound to the second binding agent, thereby providing eluted nucleic acids, wherein the eluted nucleic acids provide a first partition; (v) retaining the nucleic acids bound to the second binding agent to provide a second partition; and (vi) analyzing one or more of the unbound partition, the first partition, and the second partition. In particular embodiments, a derivative of the first type of modification is formed in step (i). In other particular embodiments, a derivative of the second type of modification is formed in step (i). In other particular embodiments, a derivative of the first type of modification and a derivative of the second type of modification is formed in step (i). InAtty. Docket No. GH0139WO particular embodiments, steps (i) – (vi) of the disclosed method are performed in sequential order. [000164] In some embodiments, the first type of modification is 5-methylcytosine (5mC). In some embodiments, the first binding agent comprises: (i) a methyl-binding domain (MBD) protein; and / or (ii) an anti-5mC antibody, or an antigen-binding fragment thereof. In some embodiments, the first type of modification is 5-methylcytosine (5mC), and the first binding agent comprises: (i) a methyl-binding domain (MBD) protein; and / or (ii) an anti-5mC antibody, or an antigen-binding fragment thereof. [000165] In some embodiments, the second type of modification is 5-hydroxymethyl cytosine (5hmC). In some embodiments, the second binding agent comprises an anti-5hmC antibody, or an antigen-binding fragment thereof. In some embodiments, the second type of modification is 5- hydroxymethyl cytosine (5hmC), and the second binding agent comprises an anti-5hmC antibody, or an antigen-binding fragment thereof. [000166] In some embodiments, the method comprises forming a derivative of 5hmC by glucosylation of 5hmC. In some embodiments, the second binding agent comprises J-binding protein-1 (JBP-1). In some embodiments, forming a derivative of 5hmC further comprises attaching a capture moiety to the glucosylated 5hmC. In certain embodiments, the capture moiety is biotin, such as a photocleavable biotin. In some embodiments, the second binding agent comprises streptavidin. [000167] In some embodiments, the second type of modification is an abasic site. In certain embodiments, the method comprises forming a derivative of the abasic site. In some embodiments, the derivative of the abasic site is formed by reacting the nucleic acid with an aldehyde probe, such that the probe covalently binds to the abasic site. In some embodiments, the aldehyde probe further comprises a coupling moiety which allows conjugation of the probe to another compound, such as a capture moiety, optionally wherein the capture moiety comprises biotin, optionally wherein the second binding agent comprises streptavidin. [000168] In some embodiments, step (iv) comprises sequentially subjecting the nucleic acids bound to the first binding agent and the second binding agent to a plurality of conditions which increasingly favour elution of the nucleic acids bound to the first binding agent, such that the first partition is separated into a plurality of first sub partitions, wherein the nucleic acids eluted in the first of the plurality of conditions have a lower extent of the first type of modification compared to the nucleic acids eluted in the last of the plurality of conditions.Atty. Docket No. GH0139WO [000169] In some embodiments, step (v) comprises subjecting the nucleic acids bound to the second binding agent to conditions which favour elution of the nucleic acids bound to the second binding agent, wherein the eluted nucleic acids provide the second partition. In some embodiments, step (v) comprises sequentially subjecting the nucleic acids bound to the second binding agent to a plurality of conditions which increasingly favour elution of the nucleic acids bound to the second binding agent, such that the second partition is separated into a plurality of second sub partitions, wherein the nucleic acids eluted in the first of the plurality of conditions have a lower extent of the second type of modification compared to the nucleic acids eluted in the last of the plurality of conditions. [000170] In some embodiments, the analyzing comprises preparing one or more sequencing libraries from at least a subset of the nucleic acids in one or more of the unbound partition, the first partition, the second partition, one or more of the first sub-partitions, and / or one or more of the second sub-partitions, wherein the one or more sequencing libraries are then subjected to nucleic acid sequencing. In some embodiments, the one or more sequencing libraries is obtained using a single-stranded sequencing library preparation method. In some embodiments, the one or more sequencing libraries is obtained using a double-stranded sequencing library preparation method. [000171] Some embodiments of the disclosed methods further comprise capturing at least an epigenetic target region set of nucleic acids from the nucleic acid population or a subsample thereof, comprising contacting the nucleic acid population with a plurality of target-specific probes specific for members of the epigenetic target region set. In some embodiments, a methylation level of the at least one of the plurality of epigenetic target regions is determined. In some embodiments, at least one of the plurality of epigenetic target regions is a differentially methylated region. In some embodiments, at least one of the plurality of epigenetic target regions is a fragment or a fragmentation-variable target region. In some embodiments, at least one of the plurality of epigenetic target regions is a hypermethylated region. In some embodiments, the hypermethylated region is a type-specific hypermethylated region. In some embodiments, the at least one of the plurality of epigenetic target regions is a hypomethylated region. In some embodiments, the hypomethylated region is a type-specific hypomethylated region. In some embodiments, the at least one of the plurality of epigenetic target regions comprises a CTCF binding site, and / or a transcription start site.Atty. Docket No. GH0139WO [000172] In some embodiments, the at least one of the plurality of 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 type specific 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. [000173] 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. [000174] In some embodiments, 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. [000175] 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. [000176] Some embodiments of the disclosed methods comprise identifying at least one cell type or tissue type from which the at least one type-specific epigenetic target region originated. InAtty. Docket No. GH0139WO 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 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. [000177] Some embodiments of the disclosed methods further comprise capturing at least one sequence-variable target region set of the nucleic acids, comprising contacting the nucleic acid population with a plurality of target-specific probes specific for the sequence-variable target regions. In some embodiments, the capturing the at least an epigenetic target region set of nucleic acids and / or the at least one sequence variable target region set of nucleic acids occurs prior to the contacting the nucleic acid population with the first and second binding agents. In some embodiments, the capturing the at least an epigenetic target region set of nucleic acids and / or the at least one sequence variable target region set of nucleic acids occurs after the contacting the nucleic acid population with the first and second binding agents. In some embodiments, the at least an epigenetic target region set of nucleic acids and / or the at least one sequence variable target region set of nucleic acids are captured from at least the first partition and / or the second partition. In some embodiments, the at least an epigenetic target region set of nucleic acids and / or the at least one sequence variable target region set of nucleic acids are captured from at least the first partition. In some embodiments, the at least an epigenetic target region set of nucleic acids and / or the at least one sequence variable target region set of nucleic acids are captured from at least the second partition. [000178] In some embodiments of the disclosed methods, the analyzing comprises nucleic acid sequencing. In particular embodiments, the analyzing comprises preparing one or more sequencing libraries from at least a subset of the nucleic acids in one or more of the unbound partition, the first partition, the second partition, one or more of the first sub-partitions, and / or one or more of the second sub-partitions, wherein the one or more sequencing libraries are then subjected to nucleic acid sequencing. In some embodiments, the one or more sequencing libraries is obtained using a single-stranded sequencing library preparation method. In some embodiments, the one or more sequencing libraries is obtained using a double-stranded sequencing library preparation method. In some embodiments, the sequencing comprises sequencing the nucleic acids in a manner that distinguishes a nucleobase comprising the first type of modification and a nucleobase comprising the second type of modification. In some embodiments, two or more of the partitions are pooled prior to the sequencing.Atty. Docket No. GH0139WO [000179] 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 next generation sequencing. [000180] In some embodiments, the method comprises ligating one or more adapters to the nucleic acids of the nucleic acid population, thereby producing adapter-ligated nucleic acids. In some embodiments, the adapter-ligated nucleic acids are amplified prior to the sequencing. In some embodiments, the one or more adapters comprises at least one tag. In some embodiments, the at least one tag comprises a molecular barcode. In some embodiments, at least nucleic acid molecules of the first partition and nucleic acid molecules of the second partition are differentially tagged. [000181] In some embodiments, the nucleic acid population comprises DNA. In some embodiments, the DNA comprises cell-free DNA (cfDNA), optionally wherein the cfDNA is from a bodily fluid, such as blood, serum, or plasma. In some embodiments, the the nucleic acid population is derived from a sample obtained from a subject. In some embodiments, the sample is from a bodily fluid, such as blood, serum, or plasma. In some embodiments, the blood sample is a whole blood sample, a plasma sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a tissue sample. [000182] In some embodiments, the nucleic acid population comprises cell-free DNA (cfDNA), optionally wherein the cfDNA is from a bodily fluid, such as blood, serum, or plasma. In some embodiments, the analyzing step comprises determining the presence or absence of genetic variants in the one or more partitions. The genetic variants may include, for example, single nucleotide variants (SNVs), indels, fusions, and / or copy number variation. In some embodiments, the genetic variants are SNVs. [000183] In some embodiments, the subject is an animal. In some embodiments, the subject is a human. [000184] Some embodiments of the disclosed methods comprise determining a likelihood that the subject has precancer. Some embodiments comprise determining a likelihood that the subject has cancer. [000185] In some embodiments, the sequencing comprises generating a plurality of sequencing reads, and wherein the method further comprises mapping the plurality of sequence reads to oneAtty. Docket No. GH0139WO or more reference sequences to generate mapped sequence reads, and processing the mapped sequence reads to determine the likelihood that the subject has cancer or precancer. In some embodiments, the sample is obtained from a subject who was previously diagnosed with a cancer and received one or more previous cancer treatments In some embodiments, the sample is obtained at one or more preselected time points following the one or more previous cancer treatments. [000186] Some embodiments of the disclosed methods comprise determining a cancer recurrence score, optionally 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. In some embodiments, the cancer recurrence score of the subject is compared 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. [000187] 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-methylcytosine.Atty. Docket No. GH0139WO B. Samples and Subjects

[0001] The disclosure relates to methods of analyzing the modification profiles of nucleic acids in a sample, e.g. DNA in a sample. In particular, the methods of the disclosure can be used to analyze the modification status of cell free DNA (cfDNA). In some cases, the nucleic acid is obtained or has been obtained from a subject. In some embodiments, the nucleic acid sample may comprise or consist of nucleic acids, e.g. DNA, from a biological sample obtained from a subject. 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. 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 biologics. 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 sample is a polynucleotide 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, rectal (or colorectal), kidney, breast, prostate, or liver, or other type of cancer as described herein. In some embodiments, the 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). In any of the foregoing embodiments, the precancer, cancer, tumor, or neoplasia or suspected precancer, 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 precancer, cancer, tumor, or neoplasia or suspected precancer, cancer, tumor, or neoplasia is of the lung. In some embodiments, the precancer, cancer, tumor, or neoplasia or suspected precancer, cancer, tumor, or neoplasia is of the colon or rectum. In some embodiments, the precancer, cancer, tumor, or neoplasia or suspected precancer, cancer, tumor, or neoplasia is of the breast. In some embodiments, the precancer, cancer, tumor, or neoplasia or suspected precancer, 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 sample is obtained from a subject having a stage I cancer, stage II cancer, stage III cancer or stage IV cancer.Atty. Docket No. GH0139WO

[0002] 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 biologics. 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. [000188] The sample can be any biological sample isolated from a subject. The sample can be a bodily sample. Biological samples can include body tissues, such as known or suspected solid tumors (such as carcinomas, adenocarcinomas, or sarcomas), whole blood, platelets, serum, plasma, stool, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies, cerebrospinal fluid synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid, the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, pleural effusions, cerebrospinal fluid, saliva, mucous, sputum, semen, sweat, and urine. In some embodiments, biological samples are body fluids, particularly blood and fractions thereof (e.g., plasma and / or serum) or urine.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 as cells, or enrich for one component relative to another. 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, or -80°C. A sample can be isolated or obtained from a subject at the site of the sample analysis. [000189] In some embodiments, a population of nucleic acids is obtained from a serum, plasma or blood sample from a subject 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, epigenetic variation, and / or post- replication or transcriptional modifications. Post-replication modifications include modifications of cytosine, particularly at the 5-position of the nucleobase, e.g., 5-methylcytosine, 5- hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. [000190] 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.Atty. Docket No. GH0139WO [000191] In a particular embodiment, the DNA sample comprises cell-free DNA. In another particular embodiment the DNA sample is a DNA sample from a formalin fixed paraffin embedded (FFPE) sample. [000192] The subject can be a human, a mammal, an animal, a companion animal, a service animal, or a pet. The subject may have a cancer, precancer, infection, 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 of being susceptible to cancer or any cancer-associated genetic mutations / disorders. [000193] In some embodiments, the sample comprises 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 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 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. [000194] 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. [000195] In some embodiments, the 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 0.3-0.5 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 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. [000196] In some embodiments, the 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 sampledAtty. Docket No. GH0139WO 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. [000197] In some embodiments, the 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. [000198] A sample can comprise various amounts of nucleic acid 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 cfDNA, about 200 billion (2x1011) 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. [000199] A sample can comprise nucleic acids from different sources, e.g., nucleic acids from cells and cell-free nucleic acids of the same subject, and nucleic acids from cells and cell-free nucleic acids of different subjects. In some embodiments, the nucleic acid may be DNA. A sample can comprise nucleic acids (e.g., DNA) carrying mutations. For example, a sample can comprise DNA carrying germline mutations and / or somatic mutations. Germline mutations refer to mutations existing in germline DNA of a subject. Somatic mutations refer to mutations originating in somatic cells of a subject, e.g., cancer cells. A sample can comprise DNA carrying cancer-associated mutations (e.g., cancer-associated somatic mutations). A sample can comprise an epigenetic variant (i.e., a chemical or protein modification), 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. [000200] The sample may comprise cell free nucleic acids, such as 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,Atty. Docket No. GH0139WO 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 cfDNA not contained within or otherwise bound to a cell. Cell-free nucleic acids include DNA, RNA, and hybrids thereof, including cfDNA derived from 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-free nucleic 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. [000201] Exemplary amounts of cell-free nucleic acids in a sample before amplification range from about 1 fg to about 1 μg, e.g., 1 pg to 200 ng, 1 ng to 100 ng, 10 ng to 1000 ng. For example, the amount can be up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acid molecules. The amount can be at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of cell-free nucleic acid molecules. The amount can be up to 1 femtogram (fg), 10 fg, 100 fg, 1 picogram (pg), 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng, or 200 ng of cell-free nucleic acid molecules. The method can comprise obtaining 1 femtogram (fg) to 200 ng cell-free nucleic acid molecules from samples. [000202] Cell-free nucleic acids have an exemplary size distribution of about 100-500 nucleotides, with molecules of 110 to about 230 nucleotides representing about 90% of molecules, with a mode of about 168 nucleotides and a second minor peak in a range between 240 to 440 nucleotides. [000203] Cell-free nucleic acids can be isolated from bodily fluids through a fractionation step in which cell-free nucleic acids, as found in solution, are separated from intact cells and other non-Atty. Docket No. GH0139WO soluble components of the bodily fluid. Fractionation may include techniques such as centrifugation or filtration. Alternatively, cells in bodily fluids can be lysed and cell-free and cellular nucleic acids processed together. Generally, after addition of buffers and wash steps, nucleic acids can be precipitated with an alcohol. Further clean up steps may be used such as silica-based columns to remove contaminants or salts. Non-specific bulk carrier nucleic acids, such as C1 DNA, DNA or protein for bisulfite sequencing, hybridization, and / or ligation, may be added throughout the reaction to optimize certain aspects of the procedure such as yield. [000204] 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. [000205] 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. [000206] 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 can lead 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. [000207] 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. C. Derivatives of Modifications [000208] The methods of the disclosure employ procedures for optionally forming a derivative of the first type of modification and / or the second type of modification.Atty. Docket No. GH0139WO [000209] In some embodiments, derivatives of the first type of modification and / or the second type of modification are formed such that the derivative can be preferentially bound by a binding agent. In some embodiments, the derivative may comprise a capture moiety, wherein the corresponding binding agent preferentially binds to the capture moiety. Capture moieties include, without limitation, biotin, avidin, streptavidin, a hapten recognized by an antibody, and magnetically attractable particles. The capture moiety can be a member of a binding pair, such as biotin / streptavidin or hapten / antibody. In some embodiments, a capture moiety is captured by its binding pair which is attached to an isolatable moiety, such as a magnetically attractable particle. The capture moiety can be any type of molecule that allows affinity separation of nucleic acids bearing the capture moiety from nucleic acids lacking the capture moiety. Exemplary capture moieties are biotin which allows affinity separation by binding to streptavidin linked or linkable to a solid phase. In some embodiments, the capture moiety comprises biotin and the corresponding binding agent comprises streptavidin. In some embodiments, biotin is photocleavable biotin. [000210] In some embodiments, 5hmC is one type of nucleic acid modification analyzed. Derivatization procedures can be used to form a derivative of 5hmC. In some embodiments, derivatization procedures form a derivative of 5hmC through enzymatic glucosylation of 5hmC. In some embodiments, enzymatic glucosylation of 5hmC may produce the derivative 5- glucosylhydroxymethylcytosine (β-glu-5hmC). In some embodiments, β-glu-5hmC may be derivatized from 5hmC through incubation of a 5hmC-containing nucleic acid with a β- glucosyltransferase (BGT) and UDP glucose, wherein the BGT transfers the glucose moiety of UDP glucose to the 5hmC. In some embodiments, the BGT may be T4 Phage β- glucosyltransferase (T4-BGT). [000211] In some embodiments, formation of a derivative of 5hmC further comprises attaching a capture moiety to glucosylated 5hmC. In some embodiments, the capture moiety is biotin, optionally photocleavable biotin. In some embodiments, the capture moiety is derivatized from β-glu-5hmC through copper-free ‘click chemistry’. ‘Click chemistry’ facilitates the conjugation of two molecules with a high yield. Copper-free ‘click chemistry’ is a biorthogonal reaction that proceeds at a lower activation barrier to typical click chemistry and is free of cytotoxic transition metals. Copper-free ‘click chemistry’ can be used for the labelling of biomolecules. In some embodiments, copper-free ‘click chemistry’ is used to form the capture moiety. Copper-free ‘click chemistry’ is used to conjugate a biotin probe to the derivatized nucleic-acid modification.Atty. Docket No. GH0139WO In some embodiments, this biotin probe is DBCO-Biotin. Copper-free ‘click chemistry’ can be used to conjugate DBCO-Biotin with glucosylated 5hmC to create biotin labelled nucleic acids. This can be performed, for example, as follows. βGT is first applied to DNA with a UDP-6-N3- Glu substrate. The UDP-6-N3-Glu substrate reacts selectively with 5hmC bases, resulting in a glucose moiety and N3 being transferred. Standard copper-free click chemistry with DBCO- Biotin then is performed, in which the DBCO and N3 react, transferring the biotin to the 5hmC- originating base. [000212] In some embodiments of the method, abasic sites are one type of nucleic acid modification analyzed. Abasic sites are indicative of DNA damage. Abasic sites are formed through spontaneous depurination due to DNA damage and block the activity of polymerases to maintain genome integrity. In some embodiments, derivatization procedures form a derivative from abasic sites on nucleic acids, optionally wherein the derivative comprises a capture moiety. In some embodiments, this derivative from abasic sites may be chemical labelling of these abasic sites. In some embodiments, an aldehyde probe is used to chemically label abasic sites on nucleic acids. The aldehyde probe may be a nitrogen nucleophile, which forms a nitrogen adduct with the abasic site. The aldehyde probe forms the nitrogen adduct with an aldehyde of the abasic site, as described in more detail in WO2020021099A1. In some embodiments, the aldehyde probe is of the formula: Ar-L-A-NR1R2 where -Ar is optionally substituted aryl, such as heteroaryl, such as indolyl, -L- is alkylene, such as methylene, -A- is -CR3R4-, -N(R5)- or -O-, such as -N(R5)-, where each of -R3 and -R4 is independently hydrogen or alkyl, and -R5 is hydrogen or alkyl, such as alkyl, -R1 is hydrogen, -R2 is hydrogen or alkyl, such as hydrogen, and salts, solvates and protected forms thereof. In some embodiments, the probe has the below formula: [000213] In somefurther comprises coupling a capture moiety to the probe. In some embodiments, the capture moiety is coupled to the probe before reaction to the abasic site. [000214] In some embodiments, formation of a derivative of abasic sites on nucleic acids further comprises attaching a capture moiety to the abasic site, e.g. through an aldehyde probe as described above. In some embodiments, the method further comprises coupling a capture moietyAtty. Docket No. GH0139WO to the probe. In some embodiments, the capture moiety is coupled to the probe before reaction to the abasic site. The aldehyde probe may further comprise a coupling moiety which allows conjugation of the probe to another compound, such as a capture moiety, e.g. biotin. In some embodiments, forming a derivative of abasic sites on nucleic acids further comprises attaching a capture moiety to the abasic site, wherein the capture moiety is biotin. The aldehyde probe can be used to chemically label the abasic site modified nucleic acids with biotin, thereby forming a derivative of the abasic site. [000215] In some embodiments, 5mC is one type of nucleic acid modification analyzed. Derivatization procedures can be used to form a derivative of 5mC, optionally wherein the derivative comprises a capture moiety such as biotin. The capture moiety comprised in the derivative of the first type of modification must, however, be different from the capture moiety present in any derivative of the second type of modification. D. Binding Agents [000216] The methods of the disclosure employ the use of the simultaneous contact of the nucleic acid population with at least two binding agents for use in the partitioning of the nucleic acid population. The first binding agent will preferentially bind to nucleic acids bearing the first type of modification, or derivatives thereof. The second binding agent will preferentially bind to nucleic acids bearing the second type of modification, or derivatives thereof. In some embodiments, elution conditions from the respective binding agents differ such that, through the use of different elution conditions, partitions can be obtained which are enriched for nucleic acids containing a particular type of modification. [000217] The first binding agents used in the disclosed methods preferentially bind nucleic acids comprising the first type of modification, or derivative thereof, over nucleic acids comprising the second type of modification, or derivative thereof, for instance with an affinity at least 50X, at least 100X or at least 1000X greater compared to nucleic acids comprising the second type of modification, or derivative thereof. [000218] The second binding agents used in the disclosed methods preferentially bind nucleic acids comprising the second type of modification over nucleic acids comprising the first type of modification, for instance with an affinity at least 50X, at least 100X or at least 1000X greater compared to nucleic acids comprising the first type of modification. In some embodiments, one type of modification may be 5mC.5mC can be preferentially bound by the first binding agent. Wherein the modification is 5mC, the corresponding binding agent may comprise an MBDAtty. Docket No. GH0139WO domain, such as methyl-CpG binding domain protein 1 (MBD1) or MBD2. In alternative embodiments, the corresponding binding agent may be an anti-5mC antibody or an antigen- binding fragment thereof. For an exemplary description of mCpG binding domain proteins, see, e.g., Du et al., Methyl-CpG-binding domain proteins: readers of the epigenome. Epigenomics. 2015;7(6):1051-73. [000219] In some embodiments, one type of modification may be 5hmC. In some embodiments, the first type of modification may be 5mC and the second type of modification may be 5hmC. In some embodiments, wherein the modification is 5hmC, the corresponding binding agent may comprise an anti-5hmC antibody and / or an antigen binding fragment of an anti-5hmC antibody. [000220] In some embodiments, one type of modification may be 5hmC. In some embodiments, the first type of modification may be 5mC and the second type of modification may be 5hmC. Wherein the modification is 5hmC, the modification may be derivatized to form β-glu-5hmC by enzymatic glucosylation of 5hmC. In some embodiments, β-glu-5hmC may be derivatized from 5hmC through incubation of 5hmC with a BGT and UDP glucose, wherein the BGT transfers the glucose moiety of UDP glucose to the 5hmC. In some embodiments, the BGT may be T4 Phage β-glucosyltransferase (T4-BGT). In some embodiments, β-glu-5hmC may be derivatized from 5hmC through incubation of 5hmC with a BGT and UDP-N3-glucose, wherein the BGT transfers the N3-glucose moiety of UDP-N3-glucose to the 5hmC. A capture moiety can then be added to the derivative through the N3 group. For example, standard copper-free click chemistry with DBCO-Biotin can be performed, in which the DBCO and N3 react, transferring the biotin to the 5hmC-originating base, thereby forming a derivative comprising a capture moiety. In embodiments wherein the derivative of 5hmC comprises biotin, the corresponding binding agent can comprise streptavidin. In some embodiments, the corresponding binding agent may comprise streptavidin agarose beads, streptavidin magnetic beads, or streptavidin coated wells. [000221] In some embodiments, wherein one type of modification is 5hmC that has been derivatized to form β-glu-5hmC, the corresponding binding agent may comprise JBP-1. This is described in further detail in Robertson et al., Nat Protoc 2012; 7: 340-350. [000222] In some embodiments, one type of modification may comprise an abasic site. In some embodiments, the first type of modification may comprise 5mC and the second type of modification may comprise an abasic site. Wherein the type of modification is an abasic site, the modification may be derivatized to attach a capture moiety. In some embodiments, the method may comprise forming a derivative of the abasic site, optionally by reacting the nucleic acid withAtty. Docket No. GH0139WO an aldehyde probe, as described elsewhere herein. The aldehyde probe can be used to chemically label the abasic site with a capture moiety such as biotin. In some embodiments wherein the type of modification is an abasic site which has been derivatized to comprise a biotin label, the corresponding binding agent may comprise streptavidin. In some embodiments, the corresponding binding agent may comprise streptavidin agarose beads, streptavidin magnetic beads, or streptavidin coated wells. [000223] In some embodiments, the binding agent may be conjugated to an isolatable particle, such as a magnetically attractable particle or a large particle that can be sedimented through centrifugation. In some embodiments, the binding agent may be linked or linkable to a solid phase or other surface for forming a functionalized surface. E. Partitioning [000224] The methods of the disclosure employ multiple partitioning steps. For example, nucleic acids bound to the first binding agent and the second binding agent can be separated from the unbound nucleic acids to produce an unbound partition. The nucleic acids bound to the first binding agent and the second binding agent are subsequently subjected to conditions which favour elution of the nucleic acids bound to the first binding agent but not the elution of the nucleic acids bound to the second binding agents, wherein the eluted nucleic acids provide a first partition. The nucleic acids that remain bound to the second binding agent provide a second partition. [000225] In some embodiments, the nucleic acids bound to the first binding agent and the second binding agent are sequentially subjected to a plurality of conditions which increasingly favour elution of the nucleic acids bound to the first binding agent such that the first partition is separated into a plurality of first sub partitions, wherein the nucleic acids eluted in the first of the plurality of conditions have a lower extent of the first type of modification compared to the nucleic acids eluted in the last of the plurality of conditions. Optionally, the plurality of conditions may be a plurality of NaCl concentrations. For example, successive rounds of elution with increasing NaCl concentration would result in the generation of multiple sub partitions, wherein each subsequent partition comprises nucleic acids with a higher extent of the first type of modification compared to the previous sub partition. [000226] In some embodiments, the remaining nucleic acids bound to the second binding agents are subjected to conditions which favour elution of the nucleic acids bound to the second binding agent, wherein the eluted nucleic acids provide the second partition. In some embodiments, theAtty. Docket No. GH0139WO nucleic acids bound to the second binding agents may be subjected to a plurality of conditions which increasingly favour elution of the nucleic acids bound to the second binding agent, such that the second partition is separated into a plurality of second sub partitions, wherein the nucleic acids eluted in the first of the plurality of conditions have a lower extent of the second type of modification compared to the nucleic acids eluted in the last of the plurality of conditions. In some embodiments, the nucleic acids which remain bound to the second binding agent can be further processed while remaining attached to the second binding agent. [000227] When multiple partitions and / or sub-partitions are carried forward, adapters comprising partition tags can be applied to each of the partitions and / or sub-partitions such that the partitions and / or sub-partitions can be sequenced in the same sequencing reaction while still allowing the sequencing data from each subsample to be distinguished. Tagged partitions and / or sub- partitions can therefore be pooled together for collective sample preparation and / or sequencing. After partitioning, nucleic acids from any or all of the partitions and / or sub-partitions can be recovered, amplified and sequenced. [000228] 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. [000229] 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). In some embodiments, the DNA of interest is a copy number variant. In some embodiments, theAtty. Docket No. GH0139WO detecting or quantifying the DNA of interest comprises quantifying and / or detecting a level of a copy number variant of the DNA. In some embodiments, quantifying and / or detecting the level of a copy number variant of the DNA comprises quantitative PCR (qPCR). [000230] In some embodiments, methylation levels can be determined using partitioning, modification-sensitive conversion such as bisulfite conversion, direct detection during sequencing, methylation-sensitive restriction enzyme digestion, methylation-dependent restriction enzyme digestion, or any other suitable 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 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. [000231] 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. [000232] 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 setAtty. Docket No. GH0139WO 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). [000233] 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). [000234] 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. [000235] For further details regarding portioning nucleic acid samples based on characteristics such as methylation, see WO2018 / 119452, which is incorporated herein by reference. [000236] 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. [000237] 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 forAtty. Docket No. GH0139WO 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). [000238] 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. [000239] 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. [000240] 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 of molecules 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 DNA is 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 DNA is eluted using a high salt concentration, e.g., at least about 2000 mM.Atty. Docket No. GH0139WO [000241] 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. [000242] In some embodiments, the partitions of DNA are desalted and concentrated in preparation for enzymatic steps of library preparation. [000243] Sequences that comprise aberrantly high copy numbers may tend to be hypermethylated. Accordingly, in some embodiments, the DNA contacted with target-specific 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. [000244] 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 the methods 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. [000245] 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 and whether certain hypermethylated regions overlap with regions with copy number variants. 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 aliquotAtty. Docket No. GH0139WO 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. [000246] 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. F. Library preparation [000247] The method of the disclosure can employ an analyzing step of one or more of the unbound partition, the first partition, and the second partition. In some embodiments, the method comprises preparing one or more sequencing libraries from the nucleic acids in one or more of the unbound partition, the first partition, the second partition, one or more of the first sub- partitions, and / or one or more of the second sub-partitions, wherein the one or more sequencing libraries are then subjected to nucleic acid sequencing. [000248] Double-stranded nucleic acids e.g. DNA molecules in a sample, and single stranded nucleic acid molecule converted to double stranded molecules, can be linked to adapters at either one end or both ends. In the methods of the disclosure, adapters can be ligated to sample nucleic acids prior to the derivatization, and / or partitioning steps. In some embodiments, adapters may be ligated to the sample nucleic acids after the derivatization, and partitioning steps, but before the step of amplifying the nucleic acids which have been subjected to derivatization, and partitioning steps. [000249] DNA ligase and adapters can be added to ligate DNA molecules (e.g. cfDNA) 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 nucleic acid molecule. In some instances, two adapters canAtty. Docket No. GH0139WO be ligated to a single sample nucleic acid molecule, with one adapter ligated to each end of the sample nucleic acid molecule. [000250] Adapters can include nucleic acid primer binding sites to permit amplification of a sample 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 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 nucleic acid molecule. Adapters of the same or different sequence can be linked to the respective ends of a sample nucleic acid molecule. In some cases, adapters of the same or different sequence are linked to the respective ends of the nucleic acid molecule except that the sample index and / or molecular barcode differs in its sequence. [000251] In some embodiments, DNA molecules can be subjected to blunt-end ligation with blunt-ended adapters. In some embodiments, DNA molecules can be subjected to sticky-end ligation with sticky-ended adapters. DNA molecules can be ligated to adapters at either one end or both ends. DNA molecules can be ligated with at least partially double stranded adapter (e.g., a Y shaped or bell-shaped adapter). [000252] 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. [000253] 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 generationAtty. Docket No. GH0139WO 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 an asymmetric adapter, such as 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 hairpin shaped adapters and bubble 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 digestion-resistant adapters comprise one or more methylated nucleotides (e.g., 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof), 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, theAtty. Docket No. GH0139WO 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 digestion-resistant 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. [000254] 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. [000255] In some embodiments, adapters may be added to the DNA 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. 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 first sample, than the DNA used as a template to generate capture probes. In some embodiments, the adapters ligated to DNA captured by the capture probes. [000256] 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. [000257] In some embodiments, the disclosed methods comprise analyzing DNA in a sample. 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 isAtty. Docket No. GH0139WO 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 double-stranded 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. [000258] In some embodiments, the one or more sequencing libraries are obtained using a single-stranded sequencing library preparation method. In some embodiments, only one strand of dsDNA is bound by the binding agent. In some embodiments, the strand of DNA that is not bound by the binding agent is eluted from the binding agent within a partition through denaturation of the dsDNA. The bound strand ssDNA may remain bound to the binding agent of the partition whereas the eluted ssDNA undergoes single-stranded sequencing library preparation. In some embodiments, both strands remain bound to the binding agent. In some embodiments, both strands may be eluted from the binding agent. Optionally, the binding agent may be streptavidin and the bound nucleic acid strand may possess a biotin label, optionally photocleavable biotin. In some embodiments, ssDNA library preparation may use a method similar to library preparation for ancient or damaged DNA, see, e.g., Gansauge et al, Nat Protoc 2013; 8: 737-748. In some embodiments, ssDNA library preparation may use a splint-adapter ligation method. Within splint-adapter ligation methods for ssDNA library preparation, splint oligonucleotides are used to create short stretches of dsDNA fragments that facilitate ligation ofAtty. Docket No. GH0139WO the sequencing adapters using standard dsDNA ligation opposed to a less efficient ssDNA ligation. Split-adapter ligation methods for ssDNA library preparation include SPLAT and SRSLY, see, e.g., Raine et al, Nucleic Acids Research 2017; 45: 36 and Troll et al, BMC Genomics 2019; 20: 1023. In some embodiments, ssDNA library preparation may use a ssDNA tailing method. Within ssDNA tailing methods for ssDNA library preparation, a tail is attached to the 3’ end of the ssDNA to be used as a priming site to convert the ssDNA to dsDNA for adaptor ligation, as described in more detail in WO2015117040. [000259] 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 / s12864-019-6355-0. This method, called Single 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 single-stranded 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. [000260] 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 ssDNA and dsDNA molecules and has no activity on the phosphorylation state of proteins. Simultaneously, the randomAtty. Docket No. GH0139WO 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. [000261] In some embodiments, analysis may comprise sequencing one or more sequencing libraries obtained using a double-stranded sequencing library preparation method. In some embodiments, end repair and dA-tailing of nucleic acids within a partition may occur while the nucleic acids are still bound to the binding agent. Optionally, the binding agent may be streptavidin and the bound nucleic acid strand may possess a biotin label, optionally photocleavable biotin. In some embodiments, adapters are ligated to one or both ends of the dsDNA nucleic acids of the partition. In some embodiments, the adapter is a Y-shaped adapter in which one end of the adapter is blunt ended or tailed as described herein, for conjugating 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 some embodiments, the adapted non-bound strands are eluted from the binding agent and undergo a clean-up reaction. Optionally, clean-up methods could use Solid Phase Reversible Immobilisation (SPRI) beads, see, e.g., DeAngelis et al, Nucleic Acids Res 1995; 23: 4742-4743. Eluted and adapted ssDNA can be amplified within a solution. In some embodiments, the adapted bound strand can be retained bound to the binding agent. ssDNA clean-up can occur while bound to the binding agent to remove the non-bound strand. Bound adapted ssDNA can be amplified while bound to binding agent. In some embodiments, clean up reactions may retain the adapted dsDNA. The adapted dsDNA may then be amplified. [000262] 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, whether bearing the same or different tags, can include the same or different primer binding sites, but preferably adapters include the same primer binding site. [000263] 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.Atty. Docket No. GH0139WO [000264] 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. [000265] In some embodiments, the nucleic acids are linked at both ends to Y-shaped adapters including primer binding sites and tags. The molecules are amplified. G. Molecular Tagging [000266] In some embodiments, the nucleic acid molecules of the sample may be tagged with sample indexes, partition and / or sub-partition tags and / or molecular barcodes (referred to generally as “tags”). Tags can form part of an adapter. In some embodiments, the DNA molecules of the sample comprise barcodes. 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, molecules can bear a sample tag or sample index (which distinguishes molecules in one sample from those in a different sample), a partition or sub-partition tag (which distinguishes molecules in one partition or sub-partition from those in a different partition or sub-partition) and / or a molecular tag / molecular barcode (which distinguishes different molecules from one another (in both unique and non-unique tagging scenarios). [000267] 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, startAtty. Docket No. GH0139WO 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. [000268] In certain embodiments, a tag can comprise one or a combination of barcodes, such as partition-specific 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, different sets of molecular barcodes can be used such that the barcodes serve as a molecular tag through their individual sequences and also serve to identify the partition to which they correspond based the set of which they are a member. [000269] In certain embodiments of the disclosure, partitioning results in the generation of multiple partitions and / or sub-partitions based on the presence or absence of modifications in the sample nucleic acids. For example, barcodes can be used to allow the origin of the DNA (e.g., the subject, biological sample (e.g., samples collected at various time points), 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 nucleic acids in each partition and / or sub-partition so as to correlate the tag (or tags) with a specific partition and / or sub-partition. For example, if multiple partitions and / or sub-partitions are carried forward after the partitioning step, tags can be used to label each of the subsamples such that they corresponding sequence data deriving from each partitions and / or sub-partitions can be identified. In some embodiments, a single tag can be used to label a specific partition and / or sub- partition. In some embodiments, multiple different tags can be used to label a specific partition and / or sub-partition. In embodiments employing multiple different tags to label a specificAtty. Docket No. GH0139WO partition and / or sub-partition, the set of tags used to label one partition and / or sub-partition can be readily differentiated for the set of tags used to label other partitions and / or sub-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 ONE,11: e0146638 (2016)) or used as non-unique molecular 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). [000270] 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, partition and / or sub-partition tags 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 the partitioning procedure. In some embodiments, molecular barcodes and / or sample indexes are introduced prior to and / or after the partitioning step. 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 sequence capturing steps, if present. In some embodiments, the sample indexes are introduced after sequence capturing steps are performed, if present. In some embodiments, sample indexes are incorporated through overlap extension polymerase chain reaction (PCR).Atty. Docket No. GH0139WO [000271] In some embodiments, the tags may be located at one end or at both ends of the sample nucleic acids. In some embodiments, tags are predetermined or random or semi-random sequences. In some embodiments, the tag(s) may together 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. [000272] In some embodiments, each sample is distinctly tagged with a sample index or a combination of sample indexes. In some examples, when multiple partitions and / or sub- partitions are subsequently processed after the partitioning step, each partition can be distinctly tagged with a partition and / or sub-partition tag or a combination of partition tags. 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) to individual nucleic acid molecules such that the combination of the molecular barcode and the sequence of the sample nucleic acid that it is attached to creates a unique sequence that may be individually tracked. Detection of non-unique molecular barcodes in combination with endogenous sequence information typically allows for the assignment of a unique identity to a particular molecule. Endogenous sequence information includes 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. In some embodiments, the beginning region comprises the first 1, the 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, the 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 toAtty. Docket No. GH0139WO assign a unique identity to a given 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, and / or a complementary strand. [000273] 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). [000274] 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, 19*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 of receiving (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 suchAtty. Docket No. GH0139WO molecules. To uniquely tag all 3000 molecules mapping across a nucleotide coordinate, about 1 million to about 20 million different tags would be required. [000275] 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.2001 / 0053519, 2003 / 0152490, and 2011 / 0160078, and U.S. Patent Nos. 6,582,908, 7,537,898, 9,598,731, and 9,902,992, each of which is hereby incorporated by reference in its entirety. 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, sub-sequences of one or both ends of a sequence, and / or lengths). The addition of tags (e.g. sample indexes, partition and / or sub-partition tags and / or molecular barcodes) to nucleic acids can be done through amplification, wherein the tags are comprised in primers used for amplification. Tags can be linked to sample nucleic acids randomly or non-randomly. [000276] In some embodiments, the nucleic acids are ligated to adapters comprising molecular barcodes. These molecular barcodes (optionally in combination with endogenous sequence information) can then be used when analyzing the sequencing data to group sequence reads deriving from the same parent nucleic acids (i.e. those nucleic acids prior to any amplification). The grouped sequence reads can then be analyzed, for example, to determine a consensus sequence for parent nucleic acids. Some methods are particularly suited to methods which identify the presence or absence of genetic variants in addition to the modification status of the nucleic acids. This is because the determination of a consensus sequence can distinguish true genetic variants from sequencing and / or amplification errors. [000277] 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, 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 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,Atty. Docket No. GH0139WO 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. [000278] 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. [000279] 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. [000280] 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 consist of 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)Atty. Docket No. GH0139WO 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. H. Amplification [000281] In some embodiments, DNA is amplified. In some embodiments, the DNA can be subjected to a plurality of distinct amplification reactions. For example, adapted DNA can be amplified (e.g. by PCR) prior to, or as part of, sequencing. For example, in sequencing procedures which comprise a conversion step, the adapted DNA may be amplified after the conversion step. In sequencing procedures which involve single molecule sequencing (such a nanopore-based sequencing or SMRT sequencing), there may be no amplification step. In some embodiments, the DNA is amplified prior to a step of subjecting adapter-ligated DNA to sequencing. In some embodiments, DNA is amplified after ligating adapters to the DNA and / or before sequencing the DNA. Amplification can be performed after the partitioning steps. Amplification may be performed before or after any sequence capture step. In some embodiments, the ligating occurs before or simultaneously with amplification. In some embodiments, amplification is primed by primer binding to primer binding site(s) in the adapter(s). [000282] Sample nucleic acids flanked by adapters can be amplified by PCR and / or other amplification methods. Amplification is typically primed by primers binding to primer binding sites in adapters flanking a DNA molecule to be amplified. Amplification methods can involve cycles of denaturation, annealing and extension, resulting from thermocycling or can be isothermal as in transcription-mediated amplification. For example, sample nucleic acids flanked by adapters can be amplified by PCR and other amplification methods. Amplification methods of use herein can include any suitable methods, such as known to those of ordinary skill in the art. In some embodiments, amplification is primed by primers binding to primer binding sites in adapters flanking a DNA molecule to be amplified. Amplification methods can involve cycles ofAtty. Docket No. GH0139WO denaturation, annealing and extension, resulting from thermocycling, such as polymerase chain reaction (PCR), 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 amplification methods include the ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustained sequence-based replication. [000283] In some embodiments, the amplification of the DNA (e.g., adapter ligated DNA) comprises using a DNA polymerase. In some embodiments, the DNA polymerase may be Q5® High-Fidelity DNA Polymerase, Q5U® Hot Start High-Fidelity DNA Polymerase, Phusion® High-Fidelity DNA Polymerase, OneTaq®DNA Polymerase, Taq DNA Polymerase, LongAmp®Taq DNA Polymerase, Hemo KlenTaq, 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, Sulfolobus DNA Polymerase IV, Therminator™ DNA Polymerase, T7 DNA Polymerase, DNA Polymerase I (E. coli), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”), Klenow Fragment (3′→5′ exo-), T4 DNA Polymerase, Vent® DNA Polymerase, Vent® (exo-) DNA Polymerase, Deep Vent® DNA Polymerase, Deep Vent® (exo-) DNA Polymerase, or any combination thereof. [000284] 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 an mCpG-binding protein. For an exemplary description of mCpG binding domain proteins, see, e.g., Du et al., Methyl-CpG-binding domain proteins: readers of the epigenome. Epigenomics.2015;7(6):1051-73. [000285] Amplification, including methylation-preserving amplification, is typically primed by primers binding to primer binding sites in adapters flanking a DNA molecule to be amplified. Amplification methods can involve cycles of denaturation, annealing and extension, resulting from thermocycling or can be isothermal as in transcription-mediated amplification. For example, DNA flanked by adapters added to the DNA as described herein can be amplified by PCR or other amplification methods. Amplification methods of use herein, includingAtty. Docket No. GH0139WO methylation-preserving amplification, can include any suitable methods, such as known to those of ordinary skill in the art. In some embodiments, amplification is primed by primers binding to primer binding sites in adapters flanking a DNA molecule to be amplified. Amplification methods can involve cycles of denaturation, annealing and extension, resulting from thermocycling, such as polymerase chain reaction (PCR), 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 amplification methods 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. [000286] In some embodiments, methylation-preserving amplification comprises amplification performed in the presence of a methyltransferase. Methylating agents of use in methylation- preserving 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). [000287] 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 aAtty. Docket No. GH0139WO 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. [000288] In some embodiments, enriching methylated DNA in a sample comprises amplification, such as embodiments comprising quantitative PCR (qPCR) or digital PCR. 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. [000289] 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%.Capturing using capture probes [000290] Nucleic acids may be subject to a sequence capture step, in which molecules having target sequences are captured for subsequent analysis. This allows nucleic acids derived from target regions of the genome to be isolated and analyzed, thus avoiding the need for whole genome analysis. Capture can be performed before or after the partitioning step. In some embodiments, capture is performed after partitioning and after an amplification step. [000291] Capture may be performed using any suitable approach known in the art. Target capture can involve use of a bait set comprising oligonucleotide baits labeled with a capture moiety, such as the 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 can be captured by bead-Atty. Docket No. GH0139WO 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. [000292] 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. 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 capture moieties are biotin that 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. [000293] In some embodiments, the probes specific for the target regions (i.e., target-specific probes) comprise a capture moiety that facilitates the enrichment or capture of the DNA hybridized to the probes. In some embodiments, the capture moiety is biotin. In some such embodiments, streptavidin attached to a solid support, such as magnetic beads, is used to bind to the biotin. Nonspecifically bound DNA that does not comprise a target region is washed away from the captured DNA. In some embodiments, DNA is then dissociated from the probes and eluted from the solid support using salt washes or buffers comprising another DNA denaturing agent. In some embodiments, the probes are also eluted from the solid support by, e.g., disrupting the biotin-streptavidin interaction. In some embodiments, captured DNA is amplified following elution from the solid support. In some such embodiments, DNA comprising adapters is amplified using PCR primers that anneal to the adapters. In some embodiments, captured DNA is amplified while attached to the solid support. In some such embodiments, the amplificationAtty. Docket No. GH0139WO comprises use of a PCR primer that anneals to a sequence within an adapter and a PCR primer that anneals to a sequence within a probe annealed to the target region of the DNA. [000294] In some embodiments, the methods herein comprise capturing nucleic acids comprising epigenetic and / or sequence-variable target regions. In some embodiments, the methods herein comprise capturing nucleic acids comprising epigenetic target regions, such as differentially methylated regions. Such regions may be captured from a sample (e.g., a subsample) that has undergone attachment of adapters, derivatization, partitioning, and / or amplification. Enriching for or capturing DNA comprising epigenetic and / or sequence-variable target regions may comprise contacting the DNA 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 partitions and / or sub-partitions prepared during methods disclosed herein. When capturing is performed on multiple partitions and / or sub-partitions, the capture probes used for each partition and / or sub-partition may be different. In some embodiments, DNA is captured from the first partition and / or the second partition and / or the unbound partition. In some embodiments, the partitions and / or sub-partitions are differentially tagged (e.g., as described herein) and then pooled before undergoing capture. [000295] 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. [000296] In some embodiments, methods described herein comprise capturing nucleic acids, e.g., obtained from a subject, for a plurality of sets of target regions. The target regions may comprise intronic regions or VDJ regions that may comprise rearrangements. The target regions may comprise epigenetic target regions, which may show differences in methylation levels depending on whether they originated from a tumor or from healthy cells. The target regions may comprise sequence-variable regions, which may show differences in sequence, other than rearrangements, depending on whether they originated from a tumor or from healthy cells. The target regions may comprise both epigenetic target regions and sequence-variable regions. The capturing step produces a captured set of DNA molecules. In some embodiments, the DNA molecules corresponding to the sequence-variable target region set are captured at a greaterAtty. Docket No. GH0139WO capture yield in the captured set of DNA molecules than DNA molecules corresponding to the epigenetic target region set. In some embodiments, a method described herein comprises contacting DNA with a set of target-specific probes, wherein the set of target-specific probes is configured to capture cfDNA corresponding to the sequence-variable target region set at a greater capture yield than DNA corresponding to the epigenetic target region set. For additional discussion of capturing steps, capture yields, and related aspects, see WO2020 / 160414, which is incorporated herein by reference for all purposes. [000297] In some embodiments, a method described herein comprises contacting nucleic acids obtained from a subject with a set of target-specific probes, wherein the set of target-specific probes is configured to capture nucleic acids corresponding to the sequence-variable target region set at a greater capture yield than nucleic acids corresponding to the epigenetic target region set.

[0003] It can be beneficial 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 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 methylation status is generally less than the volume of data needed to determine the presence or absence of genetic variants, such as 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). Although copy number variations such as focal amplifications are somatic mutations, they can be detected by sequencing based on read frequency in a manner analogous to approaches for detecting certain epigenetic changes such as changes in methylation. Thus, they can be considered epigenetic target regions for functional reasons. Additionally, regions showing copy number variation that are also hypermethylation-variable or fragmentation-variable target regions are considered epigenetic target regions because they may show epigenetic variation.

[0004] 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.Atty. Docket No. GH0139WO [000298] In some embodiments, amplification is performed before the capturing step. In some embodiments, amplification is performed after the capturing step. In some embodiments, an amplification step is performed before and after the capturing step. In some embodiments, the methods further comprise sequencing the captured DNA to different degrees of sequencing depth for the epigenetic and sequence-variable target region sets and for rearrangements, consistent with the discussion herein. [000299] 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. [000300] 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 embodiments, a capturing step is performed with probes for a sequence-variable target region set and probes for an 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 that the concentration of the probes for the epigenetic target region set. [000301] Alternatively, a capturing step is performed with a sequence-variable target region probe set in a first vessel and with an epigenetic target region probe set in a second vessel, or a contacting step is performed with a sequence-variable target region probe set at a first time and a first vessel and an 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 a sequence-variable target region set and captured DNA corresponding to an epigenetic target region set. The compositions can be processed separately as desired (e.g., partitioning based on modification as described herein). These can then be pooled in appropriate proportions to provide material for further processing and analysis such as sequencing. [000302] In some embodiments, a captured set of DNA (e.g., cfDNA) is provided. With respect to the disclosed methods, the captured set of DNA may be provided, e.g., by performing aAtty. Docket No. GH0139WO 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.

[0005] In some embodiments, a first target region set is captured (e.g., from a sample or a first subsample), comprising at least epigenetic target regions. The epigenetic target regions captured from the first 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 cfDNA from healthy subjects (e.g., below-average methylation relative to bulk cfDNA). In some embodiments, the hypermethylation variable target regions are regions that are regions that show lower methylation in healthy cfDNA 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 that 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.

[0006] In some embodiments, a second target region set is captured from the second subsample 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 cfDNA from healthy subjects (e.g., above-average methylation relative to bulk cfDNA). In some embodiments, the hypomethylation variable target regions are regions that show higher methylation in healthy cfDNA 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.

[0007] 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).

[0008] Alternatively, first and second captured sets may be provided, comprising, respectively, DNA corresponding to a sequence-variable target region set and DNA corresponding to anAtty. Docket No. GH0139WO epigenetic target region set. The captured sets may be combined to provide a combined captured set.

[0009] 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, a 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 concentration, or a 90- to 100-fold greater concentration. The degree of difference in concentrations accounts for normalization for the footprint sizes of the target regions, as discussed in the definition section.

[0010] In some embodiments, the DNA that is captured comprises intronic regions. In some embodiments, the intronic regions comprise one or more introns likely to differentiate DNA from neoplastic (e.g., tumor or cancer) cells and from healthy cells, e.g., non-neoplastic circulating cells. For example, an intron comprising a rearrangement known to be present in some neoplastic cells and absent from healthy cells can be used to differentiate DNA from neoplastic (e.g., tumorAtty. Docket No. GH0139WO or cancer) cells and from healthy cells. In some embodiments, the rearrangement is a translocation.

[0011] In some embodiments, captured intronic regions have a footprint of at least 30 bp, e.g., at least 100 bp, at least 200 bp, at least 500 bp, at least 1 kb, at least 2 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 50 kb, at least 200 kb, at least 300 kb, or at least 400 kb. In some embodiments, the intronic target region set has a footprint in the range of 30 bp-1000 kb, e.g., 30 bp-100 bp, 100 bp-200 bp, 200 bp-500 bp, 500 bp-1kb, 1 kb-2 kb, 2 kb-5 kb, 5 kb-10 kb, 10 kb- 20 kb, 20 kb-50 kb, 50 kb-100 kb, 100-200 kb, 200-300 kb, 300-400 kb, 400-500 kb, 500-600 kb, 600-700 kb, 700-800 kb, 800-900 kb, and 900-1,000 kb.

[0012] Exemplary rearrangements, such as intronic translocations that can be detected using the methods described herein include but are not limited to translocations wherein at least one of the two genes involved in the translocation is a receptor tyrosine kinase. Exemplary translocation products are the BCR-ABL fusion. and fusions comprising any of ALK, FGFR2, FGFR3, NTRK1, RET, or ROS1.

[0013] In some embodiments, the DNA that is captured comprises target regions having a type- specific epigenetic variation and / or a copy number variation. In some embodiments, an epigenetic target region set consists of target regions having a type-specific epigenetic variation and / or a copy number variation. In some embodiments, the type-specific epigenetic variations, e.g., differential methylation or a type-specific fragmentation pattern, are likely to differentiate DNA from one or more related cell or tissue types cells from DNA from other cell or tissue types present in a sample or in a subject.

[0014] In some embodiments, nucleic acids captured or enriched using a method described herein comprise captured DNA, such as one or more captured sets of DNA. In some embodiments, the captured DNA comprise target regions that are differentially methylated in different immune cell types. In some embodiments, the immune cell types comprise rare or closely related immune cell types, such as activated and naïve lymphocytes or myeloid cells at different stages of differentiation.

[0015] In some embodiments, a captured epigenetic target region set captured from a sample or first subsample comprises hypermethylation variable target regions. In some embodiments, the hypermethylation variable target regions are differentially or exclusively hypermethylated in one or more related cell or tissue types. In some embodiments, the hypermethylation variable target regions are differentially or exclusively hypermethylated in one cell type or in one immune cellAtty. Docket No. GH0139WO type, or in one immune cell type within a cluster. In some embodiments, the hypermethylation variable target regions are hypermethylated to an extent that is distinguishably higher or exclusively present in one cell type or one immune cell type or one immune cell type within a cluster. Such hypermethylation variable target regions may be hypermethylated in other cell or tissue types but not to the extent observed in the one or more related cell or tissue types. In some embodiments, the hypermethylation variable target regions show lower methylation in healthy cfDNA than in at least one other tissue type. In some embodiments, the hypermethylation variable target regions show even higher methylation in cfDNA from a diseased cell of the one or more related cell or tissue types. In some embodiments, target regions comprise hypermethylated regions with aberrantly high copy number. In some such embodiments, the target regions are hypermethylated in healthy and diseased colon tissue and have aberrantly high copy number in precancerous or cancerous colon tissue. Examples of such target regions are shown in Tables 1 and 2 below. A gene is considered to comprise a DMR when the DMR is located within an untranslated region (UTR), intron, or exon of the gene, or within 5000 nucleotides of either the 5’ end of the sense strand of the 5’ UTR or the 3’ end of the sense strand of the 3’ UTR. Table 1: Hypermethylated target regions with aberrantly high copy number in colon cancer or precancer Chromosomal region Genes comprising DMRs within the chromosomal region , B,Atty. Docket No. GH0139WO 0q12-13.33 MAFB, LPIN3, EMILIN3, PTPRT, TOX2, GDAP1L1, HNF4A, MMP9, CDH22, PREX1, ZNFX1, PARD6B, ZFP64, DOK5, 4,Gene Name Chromosome MARCH11 h 5Atty. Docket No. GH0139WO

[0017] In some embodiments, a captured epigenetic target region set captured from a sample or subsample comprises hypomethylation variable target regions. In some embodiments, the hypomethylation variable target regions are exclusively hypomethylated in one or more related cell or tissue types. In some embodiments, the hypomethylation variable target regions are exclusively hypomethylated in one cell type or in one immune cell type or in one immune cell type within a cluster. In some embodiments, the hypomethylation variable target regions are hypomethylated to an extent that is exclusively present in one cell type or one immune cell type or in one immune cell type within a cluster. Such hypomethylation variable target regions may be hypomethylated in other cell or tissue types but not to the extent observed in the one or more cell or tissue types. In some embodiments, the hypomethylation variable target regions show higher methylation in healthy cfDNA than in at least one other tissue type. Without wishing to be bound by any particular theory, in an individual with cancer, proliferating or activated immune cells and / or dying cancer cells may shed more DNA into the bloodstream than cells (e.g., immune cells) in a healthy individual and / or healthy cells of the same tissue type, respectively. As such, the distribution of cell type and / or tissue of origin of cfDNA may change upon carcinogenesis. Variations in hypermethylation and / or hypomethylation can be an indicator of disease. Thus, the presence and / or levels of cfDNA originating from certain cell or tissue types can be an indicator of disease. For example, an increase in the level of hypermethylation variable target regions and / or hypomethylation variable target regions in a subsample following a partitioning step can be an indicator of the presence (or recurrence, depending on the history of the subject) of cancer.

[0018] Exemplary hypermethylation variable target regions and hypomethylation variable target regions useful for distinguishing between various cell types, including but not limited to immune cell types, have been identified by analyzing DNA obtained from various cell types via whole genome bisulfite sequencing, as described, e.g., in Scott, C.A., Duryea, J.D., MacKay, H. et al., “Identification of cell type-specific methylation signals in bulk whole genome bisulfite sequencing data,” Genome Biol 21, 156 (2020) (doi.org / 10.1186 / s13059-020-02065-5). Whole- genome bisulfite sequencing data is available from the Blueprint consortium, available on the internet at dcc.blueprint-epigenome.eu.

[0019] In some embodiments, first and second captured target region sets comprise, respectively, DNA corresponding to a sequence-variable target region set and DNA corresponding to an epigenetic target region set, for example, as described in WO 2020 / 160414. The first and secondAtty. Docket No. GH0139WO captured sets may be combined to provide a combined captured set. In some embodiments, the sequence-variable target region set and epigenetic target region set may have any of the features described for such sets in WO 2020 / 160414, which is incorporated by reference herein in its entirety. In some embodiments, the epigenetic target region set comprises a hypermethylation variable target region set. In some embodiments, the epigenetic target region set comprises a hypomethylation variable target region set. In some embodiments, the epigenetic target region set comprises CTCF binding regions. In some embodiments, the epigenetic target region set comprises fragmentation variable target regions. In some embodiments, the epigenetic target region set comprises transcriptional start sites. In some embodiments, the epigenetic target region set comprises regions that may show focal amplifications in cancer, e.g., one or more of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAF1. For example, in some embodiments, the epigenetic target region set comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the foregoing targets.

[0020] In some embodiments, the sequence-variable target region set comprises a plurality of regions known to undergo somatic mutations in cancer. In some aspects, the sequence-variable target region set targets a plurality of different genes or genomic regions (“panel”) selected such that a determined proportion of subjects having a cancer exhibits a genetic variant or tumor marker in one or more different genes or genomic regions in the panel. The panel may be selected to limit a region for sequencing to a fixed number of base pairs. The panel may be selected to sequence a desired amount of DNA, e.g., by adjusting the affinity and / or amount of the probes as described elsewhere herein. The panel may be further selected to achieve a desired sequence read depth. The panel may be selected to achieve a desired sequence read depth or sequence read coverage for an amount of sequenced base pairs. The panel may be selected to achieve a theoretical sensitivity, a theoretical specificity, and / or a theoretical accuracy for detecting one or more genetic variants in a sample.

[0021] Probes for detecting the panel of regions can include those for detecting genomic regions of interest (hotspot regions). Information about chromatin structure can be taken into account in designing probes, and / or probes can be designed to maximize the likelihood that particular sites (e.g., KRAS codons 12 and 13) can be captured, and may be designed to optimize capture based on analysis of cfDNA coverage and fragment size variation impacted by nucleosome bindingAtty. Docket No. GH0139WO patterns and GC sequence composition. Regions used herein can also include non-hotspot regions optimized based on nucleosome positions and GC models.

[0022] Probes for detecting the panel of regions can include those for detecting genomic regions of interest (hotspot regions). Information about chromatin structure can be taken into account in designing probes, and / or probes can be designed to maximize the likelihood that particular sites (e.g., KRAS codons 12 and 13) can be captured, and may be designed to optimize capture based on analysis of cfDNA coverage and fragment size variation impacted by nucleosome binding patterns and GC sequence composition. Regions used herein can also include non-hotspot regions optimized based on nucleosome positions and GC models.

[0023] Examples of listings of genomic locations of interest may be found in Table 3 and Table 4 of WO 2020 / 160414. In some embodiments, a sequence-variable target region set used in the methods of the present disclosure comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 of the genes of Table 3 of WO 2020 / 160414. In some embodiments, a sequence-variable target region set used in the methods of the present disclosure comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the genes of Table 4 of WO 2020 / 160414. Additionally or alternatively, suitable target region sets are available from the literature. For example, Gale et al., PLoS One 13: e0194630 (2018), which is incorporated herein by reference, describes a panel of 35 cancer-related gene targets that can be used as part or all of a sequence-variable target region set. These 35 targets are AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MED12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, and U2AF1.

[0024] In some embodiments, the sequence-variable target region set comprises target regions from at least 10, 20, 30, or 35 cancer-related genes, such as the cancer-related genes listed above and in WO 2020 / 160414.

[0025] In some embodiments, a collection of capture probes is used in methods described herein, e.g., comprising capture probes prepared by any method disclosed herein for doing so. In some embodiments, the collection of capture probes further comprises target-binding probes specific for a sequence-variable target region set and / or target-binding probes specific for a sequence-variable target region set and / or target-binding probes specific an epigenetic target region set. In someAtty. Docket No. GH0139WO embodiments, the capture yield of the capture probes specific for the sequence-variable target region set is higher (e.g., at least 2-fold higher) than the capture yield of the target-binding probes specific for the epigenetic target region set. In some embodiments, the collection of capture probes is configured to have a capture yield specific for the sequence-variable target region set higher (e.g., at least 2-fold higher) than its capture yield specific for the epigenetic target region set.

[0026] In some embodiments, the capture yield of the target-binding probes specific for the sequence-variable target region set is at least 1.25-, 1.5-, 1.75-, 2-, 2.25-, 2.5-, 2.75-, 3-, 3.5-, 4-, 4.5-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, or 15-fold higher than the capture yield of the target- binding probes specific for the epigenetic target region set. In some embodiments, the capture yield of the target-binding probes specific for the sequence-variable target region set is 1.25- to 1.5-, 1.5- to 1.75-, 1.75- to 2-, 2- to 2.25-, 2.25- to 2.5-, 2.5- to 2.75-, 2.75- to 3-, 3- to 3.5-, 3.5- to 4-, 4- to 4.5-, 4.5- to 5-, 5- to 5.5-, 5.5- to 6-, 6- to 7-, 7- to 8-, 8- to 9-, 9- to 10-, 10- to 11-, 11- to 12-, 13- to 14-, or 14- to 15-fold higher than the capture yield of the target-binding probes specific for the epigenetic target region set.

[0027] In some embodiments, the collection of capture probes is configured to have a capture yield specific for the sequence-variable target region set at least 1.25-, 1.5-, 1.75-, 2-, 2.25-, 2.5-, 2.75-, 3-, 3.5-, 4-, 4.5-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, or 15-fold higher than its capture yield for the epigenetic target region set. In some embodiments, the collection of capture probes is configured to have a capture yield specific for the sequence-variable target region set is 1.25- to 1.5-, 1.5- to 1.75-, 1.75- to 2-, 2- to 2.25-, 2.25- to 2.5-, 2.5- to 2.75-, 2.75- to 3-, 3- to 3.5-, 3.5- to 4-, 4- to 4.5-, 4.5- to 5-, 5- to 5.5-, 5.5- to 6-, 6- to 7-, 7- to 8-, 8- to 9-, 9- to 10-, 10- to 11-, 11- to 12-, 13- to 14-, or 14- to 15-fold higher than its capture yield specific for the epigenetic target region set.

[0028] The collection of probes can be configured to provide higher capture yields for the sequence-variable target region set in various ways, including concentration, different lengths and / or chemistries (e.g., that affect affinity), and combinations thereof. Affinity can be modulated by adjusting probe length and / or including nucleotide modifications as discussed below.

[0029] In some embodiments, the capture probes specific for the sequence-variable target region set are present at a higher concentration than the capture probes specific for the epigenetic target region set. In some embodiments, concentration of the target-binding probes specific for the sequence-variable target region set is at least 1.25-, 1.5-, 1.75-, 2-, 2.25-, 2.5-, 2.75-, 3-, 3.5-, 4-, 4.5-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, or 15-fold higher than the concentration of the target-Atty. Docket No. GH0139WO binding probes specific for the epigenetic target region set. In some embodiments, the concentration of the target-binding probes specific for the sequence-variable target region set is 1.25- to 1.5-, 1.5- to 1.75-, 1.75- to 2-, 2- to 2.25-, 2.25- to 2.5-, 2.5- to 2.75-, 2.75- to 3-, 3- to 3.5-, 3.5- to 4-, 4- to 4.5-, 4.5- to 5-, 5- to 5.5-, 5.5- to 6-, 6- to 7-, 7- to 8-, 8- to 9-, 9- to 10-, 10- to 11-, 11- to 12-, 13- to 14-, or 14- to 15-fold higher than the concentration of the target-binding probes specific for the epigenetic target region set. In such embodiments, concentration may refer to the average mass per volume concentration of individual probes in each set.

[0030] In some embodiments, the capture probes specific for the sequence-variable target region set have a higher affinity for their targets than the capture probes specific for the epigenetic target region set. Affinity can be modulated in any way known to those skilled in the art, including by using different probe chemistries. For example, certain nucleotide modifications, such as cytosine 5-methylation (in certain sequence contexts), modifications that provide a heteroatom at the 2’ sugar position, and LNA nucleotides, can increase stability of double-stranded nucleic acids, indicating that oligonucleotides with such modifications have relatively higher affinity for their complementary sequences. See, e.g., Severin et al., Nucleic Acids Res. 39: 8740–8751 (2011); Freier et al., Nucleic Acids Res. 25: 4429–4443 (1997); US Patent No. 9,738,894. Also, longer sequence lengths will generally provide increased affinity. Other nucleotide modifications, such as the substitution of the nucleobase hypoxanthine for guanine, reduce affinity by reducing the amount of hydrogen bonding between the oligonucleotide and its complementary sequence. In some embodiments, the capture probes specific for the sequence-variable target region set have modifications that increase their affinity for their targets. In some embodiments, alternatively or additionally, the capture probes specific for the epigenetic target region set have modifications that decrease their affinity for their targets. In some embodiments, the capture probes specific for the sequence-variable target region set have longer average lengths and / or higher average melting temperatures than the capture probes specific for the epigenetic target region set. These embodiments may be combined with each other and / or with differences in concentration as discussed above to achieve a desired fold difference in capture yield, such as any fold difference or range thereof described above.

[0031] In some embodiments, the capture probes comprise a capture moiety. The capture moiety may be any of the capture moieties described herein, e.g., biotin. In some embodiments, the target- specific probes are linked to a solid support, e.g., covalently or non-covalently such as through theAtty. Docket No. GH0139WO interaction of a binding pair of capture moieties. In some embodiments, the solid support is a bead, such as a magnetic bead.

[0032] In some embodiments, the capture probes specific for the sequence-variable target region set and / or the capture probes specific for the epigenetic target region set are a capture probe set as discussed above, e.g., probes comprising capture moieties and sequences selected to tile across a panel of regions, such as genes.

[0033] In some embodiments, the capture probes are provided in a single composition. The single composition may be a solution (liquid or frozen). Alternatively, it may be a lyophilizate.

[0034] Alternatively, the capture probes may be provided as a plurality of compositions, e.g., comprising a first composition comprising probes specific for the epigenetic target region set and a second composition comprising probes specific for the sequence-variable target region set. These probes may be mixed in appropriate proportions to provide a combined probe composition with any of the foregoing fold differences in concentration and / or capture yield. Alternatively, they may be used in separate capture procedures (e.g., with aliquots of a sample or sequentially with the same sample) to provide first and second compositions comprising captured epigenetic target regions and sequence-variable target regions, respectively. 1. Probes specific for epigenetic target regions

[0035] The probes for the epigenetic target region set may comprise probes specific for one or more types of target regions likely to differentiate DNA from neoplastic (e.g., tumor or cancer) cells from healthy cells, e.g., non-neoplastic circulating cells. Exemplary types of such regions are discussed in detail herein, e.g., in the sections above concerning captured sets. The probes for the epigenetic target region set may also comprise probes for one or more control regions, e.g., as described herein.

[0036] In some embodiments, the probes for the epigenetic target region set have a footprint of at least 100 kbp, e.g., at least 200 kbp, at least 300 kbp, or at least 400 kbp. In some embodiments, the epigenetic target region set has a footprint in the range of 100-20 Mbp, e.g., 100-200 kbp, 200- 300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900- 1,000 kbp, 1-1.5 Mbp, 1.5-2 Mbp, 2-3 Mbp, 3-4 Mbp, 4-5 Mbp, 5-6 Mbp, 6-7 Mbp, 7-8 Mbp, 8-9 Mbp, 9-10 Mbp, or 10-20 Mbp. In some embodiments, the epigenetic target region set has a footprint of at least 20 Mbp.Atty. Docket No. GH0139WO a. Hypermethylation variable target regions

[0037] In some embodiments, the probes for the epigenetic target region set comprise probes specific for one or more hypermethylation variable target regions. Hypermethylation variable target regions may also be referred to herein as hypermethylated DMRs (differentially methylated regions). The hypermethylation variable target regions may be any of those set forth above. For example, in some embodiments, the probes specific for hypermethylation variable target regions comprise probes specific for a plurality of loci listed in Table 1, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 1. In some embodiments, the probes specific for hypermethylation variable target regions comprise probes specific for a plurality of loci listed in Table 2, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 2. In some embodiments, the probes specific for hypermethylation variable target regions comprise probes specific for a plurality of loci listed in Table 1 or Table 2, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 1 or Table 2. In some embodiments, for each locus included as a target region, there may be one or more probes with a hybridization site that binds between the transcription start site and the stop codon (the last stop codon for genes that are alternatively spliced) of the gene. In some embodiments, the one or more probes bind within 300 bp of the listed position, e.g., within 200 or 100 bp. In some embodiments, a probe has a hybridization site overlapping the position listed above. In some embodiments, the probes specific for the hypermethylation target regions include probes specific for one, two, three, four, or five subsets of hypermethylation target regions that collectively show hypermethylation in one, two, three, four, or five of breast, colon, kidney, liver, and lung cancers. b. Hypomethylation variable target regions

[0038] In some embodiments, the probes for the epigenetic target region set comprise probes specific for one or more hypomethylation variable target regions. Hypomethylation variable target regions may also be referred to herein as hypomethylated DMRs (differentially methylated regions). The hypomethylation variable target regions may be any of those set forth above. For example, the probes specific for one or more hypomethylation variable target regions may include probes for regions such as repeated elements, e.g., LINE1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and satellite DNA, and intergenic regions that are ordinarily methylated in healthy cells may show reduced methylation in tumor cells.Atty. Docket No. GH0139WO

[0039] In some embodiments, probes specific for hypomethylation variable target regions include probes specific for repeated elements and / or intergenic regions. In some embodiments, probes specific for repeated elements include probes specific for one, two, three, four, or five of LINE1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and / or satellite DNA.

[0040] Exemplary probes specific for genomic regions that show cancer-associated hypomethylation include probes specific for nucleotides 8403565-8953708 and / or 151104701- 151106035 of human chromosome 1. In some embodiments, the probes specific for hypomethylation variable target regions include probes specific for regions overlapping or comprising nucleotides 8403565-8953708 and / or 151104701-151106035 of human chromosome 1. c. CTCF binding regions

[0041] In some embodiments, the probes for the epigenetic target region set include probes specific for CTCF binding regions. In some embodiments, the probes specific for CTCF binding regions comprise probes specific for at least 10, 20, 50, 100, 200, or 500 CTCF binding regions, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 CTCF binding regions, e.g., such as CTCF binding regions described above or in one or more of CTCFBSDB or the Cuddapah et al., Martin et al., or Rhee et al. articles cited above. In some embodiments, the probes for the epigenetic target region set comprise at least 100 bp, at least 200 bp at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, or at least 1000 bp upstream and downstream regions of the CTCF binding sites. d. Transcription start sites

[0042] In some embodiments, the probes for the epigenetic target region set include probes specific for transcriptional start sites. In some embodiments, the probes specific for transcriptional start sites comprise probes specific for at least 10, 20, 50, 100, 200, or 500 transcriptional start sites, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 transcriptional start sites, e.g., such as transcriptional start sites listed in DBTSS. In some embodiments, the probes for the epigenetic target region set comprise probes for sequences at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, or at least 1000 bp upstream and downstream of the transcriptional start sites.Atty. Docket No. GH0139WO e. Focal amplifications

[0043] As noted above, although focal amplifications are somatic mutations, they can be detected by sequencing based on read frequency in a manner analogous to approaches for detecting certain epigenetic changes such as changes in methylation. As such, regions that may show focal amplifications in cancer can be included in the epigenetic target region set, as discussed above. In some embodiments, the probes specific for the epigenetic target region set include probes specific for focal amplifications. In some embodiments, the probes specific for focal amplifications include probes specific for one or more of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAF1. For example, in some embodiments, the probes specific for focal amplifications include probes specific for one or more of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the foregoing targets. f. Control regions

[0044] It can be useful to include control regions to facilitate data validation. In some embodiments, the probes specific for the epigenetic target region set include probes specific for control methylated regions that are expected to be methylated in essentially all samples. In some embodiments, the probes specific for the epigenetic target region set include probes specific for control hypomethylated regions that are expected to be hypomethylated in essentially all samples. 2. Probes specific for sequence-variable target regions

[0045] The probes for the sequence-variable target region set may comprise probes specific for a plurality of regions known to undergo somatic mutations in cancer. The probes may be specific for any sequence-variable target region set described herein. Exemplary sequence-variable target region sets are discussed in detail herein, e.g., in the sections above concerning captured sets.

[0046] In some embodiments, the sequence-variable target region probe set has a footprint of at least 0.5 kb, e.g., at least 1 kb, at least 2 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, or at least 40 kb. In some embodiments, the epigenetic target region probe set has a footprint in the range of 0.5-100 kb, e.g., 0.5-2 kb, 2-10 kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50 kb, 50-60 kb, 60-70 kb, 70-80 kb, 80-90 kb, and 90-100 kb. In some embodiments, the sequence-variable target region probe set has a footprint of at least 50 kbp, e.g., at least 100 kbp, at least 200 kbp, at least 300 kbp, or at least 400 kbp. In some embodiments, the sequence-variable target region probe set has a footprint in the range of 100-2000 kbp, e.g., 100-200 kbp, 200-300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900-1,000 kbp, 1-1.5 MbpAtty. Docket No. GH0139WO or 1.5-2 Mbp. In some embodiments, the sequence-variable target region set has a footprint of at least 2 Mbp.

[0047] In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at 70 of the genes of Table 3. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for the at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 of the SNVs of Table 3. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least 1, at least 2, at least 3, at least 4, at least 5, or 6 of the fusions of Table 3. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 1, at least 2, or 3 of the indels of Table 3. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the genes of Table 4. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the SNVs of Table 4. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least 1, at least 2, at least 3, at least 4, at least 5, or 6 of the fusions of Table 4. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or 18 of the indels of Table 4. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 of the genes of Table 5.

[0048] In some embodiments, the probes specific for the sequence-variable target region set comprise probes specific for target regions from at least 10, 20, 30, or 35 cancer-related genes, such as AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS,Atty. Docket No. GH0139WO MED12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, and U2AF1.

[0049] Table 3 Point Mutations (SNVs) Fusions AKT1 ALK APC AR ARAF ARID1A ALK ATM BRAF BRCA1 BRCA2 CCND1 CCND2 FGFR2 3 1s AKT1 ALK APC AR ARAF ARID1A ALK 2 3 1

[0051] Table 5 Start Stop Length ExonsAtty. Docket No. GH0139WO BRAF chr7 140453064 140453203 139 15 BRAF V600 CTNNB1 chr3 41266007 41266254 247 3 S37 s , , F,Atty. Docket No. GH0139WO KRAS chr12 25378537 25378717 180 4 A146 KRAS chr12 25380157 25380356 199 3 Q61I. Sequencing [000303] In general, sample nucleic acids flanked by adapters can be subject to sequencing after amplification. Thus, in some embodiments, the method comprises sequencing at least a portion of the DNA in the sample, e.g., from any one or more, or each, of the first partition, the second partition, and the unbound partition. In some embodiments, sequencing comprises sequencing the DNA in a manner that distinguishes the first nucleobase from the second nucleobase. In some embodiments, subsamples are pooled prior to the sequencing. In some embodiments, subsamples are produced using a partitioning step. In general, sample nucleic acids, including nucleic acidsAtty. Docket No. GH0139WO flanked by adapters, with or without prior amplification can be subject to sequencing. Sequencing methods include, for example, Sanger sequencing, high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, long-read sequencing (also known as single-molecule sequencing or third generation sequencing), nanopore sequencing (a type of long-read sequencing), 5-letter sequencing or 6-letter sequencing, semiconductor sequencing, sequencing- by-ligation, sequencing-by-hybridization, Digital Gene Expression (Helicos), Next generation sequencing (NGS), Single Molecule Sequencing by Synthesis (SMSS) (Helicos), massively- parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Ion Torrent, Oxford Nanopore, Roche Genia, Maxim-Gilbert sequencing, primer walking, and sequencing using PacBio, SOLiD, Ion Torrent, or Nanopore platforms. Sequencing reactions can be performed in a variety of sample processing units, which may include multiple lanes, multiple channels, multiple wells, or other mean of processing multiple sample sets substantially simultaneously. Sample processing unit can also include multiple sample chambers to enable processing of multiple runs simultaneously. [000304] In some embodiments, sequencing comprises detecting and / or distinguishing unmodified and modified nucleobases. For example, long-read sequencing (also referred to herein as third generation sequencing) methods include those that can generate longer sequencing reads, such as reads in excess of 10 kilobases, as compared to short-read sequencing methods, which generally produce reads of up to about 600 bases in length. Compared to short reads, long reads can improve de novo assembly, transcript isoform identification, and detection and / or mapping of structural variants. Furthermore, long-read sequencing of native DNA or RNA molecules reduces amplification bias and preserves base modifications, such as methylation status. Long-read sequencing technologies useful herein can include any suitable long-read sequencing methods, including, but not limited to, Pacific Biosciences (PacBio) single- molecule real-time (SMRT) sequencing, Oxford Nanopore Technologies (ONT) nanopore sequencing, and synthetic long-read sequencing approaches, such as linked reads, proximity ligation strategies, and optical mapping. Synthetic long-read approaches comprise assembly of short reads from the same DNA molecule to generate synthetic long reads, and may be used in conjunction with “true” long-read sequencing technologies, such as SMRT and nanopore sequencing methods. [000305] Single-molecule real-time (SMRT) sequencing can facilitate direct detection of, e.g., 5- methylcytosine and 5-hydroxymethylcytosine as well as unmodified cytosine (Weirather JL, etAtty. Docket No. GH0139WO al., “Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis,” F1000Research, 6:100, 2017). Whereas next- generation sequencing methods detect augmented signals from a clonal population of amplified DNA fragments, SMRT sequencing captures a single DNA molecule, maintaining base modification during sequencing. The error rate of raw PacBio SMRT sequencing-generated data is about 13–15%, as the signal-to-noise ratio from single DNA molecules not high. To increase accuracy, this platform uses a circular DNA template by ligating hairpin adapters to both ends of target double-stranded DNA. As the polymerase repeatedly traverses and replicates the circular molecule, the DNA template is sequenced multiple times to generate a continuous long read (CLR). The CLR can be split into multiple reads (“subreads”) by removing adapter sequences, and multiple subreads generate circular consensus sequence (“CCS”) reads with higher accuracy. The average length of a CLR is >10 kb and up to 60 kb, with length depending on the polymerase lifetime. Thus, the length and accuracy of CCS reads depends on the fragment sizes. PacBio sequencing has been utilized for genome (e.g., de novo assembly, detection of structural variants and haplotyping) and transcriptome (e.g., gene isoform reconstruction and novel gene / isoform discovery) studies. [000306] SMRT sequencing relies on sequencing-by-synthesis, where the sequence of a circular DNA template is determined from the succession of fluorescence pulses, each resulting from the addition of one labelled nucleotide by a polymerase fixed to the bottom of a well. Base modifications do not affect the base-called sequence, but they affect the kinetics of the polymerase. By considering the inter-pulse duration (IPD), base modifications can be inferred from the comparison of a modified template to an in silico model or an unmodified template. Such methods can therefore use the pulse width of a signal from sequencing bases, the interpulse duration (IPD) of bases, and the identity of the bases in order to detect a modification in a base or in a neighboring base. (See e.g., Weirather et al., F1000Research, 6:100, 2017.) SMRT sequencing can thus be used to detect base modifications such as 5-caC, 4mC, 5mC, 5hmC, 6mA, and 8oxoG (Gouil & Keniry Essays in Biochemistry (2019) 63639–648). Accordingly, in some embodiments, the sequencing comprises SMRT sequencing. [000307] Some sequencing reactions involve use of an enzyme to control passage of a nucleic acid through a nanopore, and in such cases reaction data can include both kinetics and other behavior of the enzyme and fluctuations in current through the nanopore. For example, ratchet proteins, helicases, or motor proteins can be used to push or pull a nucleic acid molecule throughAtty. Docket No. GH0139WO a hole in a biological or synthetic membrane. The kinetics of these proteins can vary depending on the sequence context of a nucleic acid on which they are acting. For example, they may slow down or pause at a modified base, and this behavior, captured as a part of the reaction data, is indicative of the presence of the modified base even where the modified base is not within the sensing portion of the nanopore. [000308] One example of a nanopore-based single molecule sequencing system is that commercialized by Oxford Nanopore Technologies (ONT). (Weirather JL, et al., F1000Research, 6:100, 2017). ONT directly sequences a native single-stranded DNA (ssDNA) molecule by measuring characteristic current changes as the bases are threaded through the nanopore by a molecular motor protein. ONT uses a hairpin library structure similar to the PacBio circular DNA template: the DNA template and its complement are bound by a hairpin adapter. Therefore, the DNA template passes through the nanopore, followed by a hairpin and finally the complement. The raw read can be split into two “1D” reads (“template” and “complement”) by removing the adapter. The consensus sequence of two “1D” reads is a “2D” read with a higher accuracy. [000309] Nanopore sequencing can be used to detect base modifications including 5-caC, 5mC, 5hmC, 6mA, BrdU, FldU, IdU, and EdU (see e.g., Gouil & Keniry Essays in Biochemistry (2019) 63639–648; Kutyavin, Biochemistry (2008), 47, 51, 13666–1367; Müller et al., Nature Methods (2019), volume 16, pages 429–436; Hennion et al., Genome Biology (2020), volume 21, Article number: 125). Accordingly, in some embodiments, the sequencing comprises nanopore sequencing. [000310] 5-letter and 6-letter sequencing methods include whole genome sequencing methods capable of sequencing A, C, T, and G in addition to 5mC and 5hmC to provide a 5-letter (A, C, T, G, and either 5mC or 5hmC) or 6-letter (A, C, T, G, 5mC, and 5hmC) digital readout in a single workflow. The processing of the DNA sample is entirely enzymatic and avoids the DNA degradation and genome coverage biases of bisulfite treatment. In an exemplary 5-letter sequencing method developed by Cambridge Epigenetix, the sample DNA is first fragmented via sonication and then ligated to short, synthetic DNA hairpin adapters at both ends (Füllgrabe, et al.2022, bioRxiv doi: https: / / doi.org / 10.1101 / 2022.07.08.499285). The construct is then split to separate the sense and antisense sample strands. For each original sample strand a complementary copy strand is synthesized by DNA polymerase extension of the 3’-end to generate a hairpin construct with the original sample DNA strand connected to itsAtty. Docket No. GH0139WO complementary strand, lacking epigenetic modifications, via a synthetic loop. Sequencing adapters are then ligated to the end. Modified cytosines are enzymatically protected. The unprotected Cs are then deaminated to uracil, which is subsequently read as thymine. In any such embodiments, amplification methods may comprise uracil- and / or dihydrouracil-tolerant amplification methods, such as PCR using a uracil- and / or dihydrouracil-tolerant DNA polymerase (i.e., a DNA polymerase that can read and amplify templates comprising uracil and / or dihydrouracil bases). The deaminated constructs are no longer fully complementary and have substantially reduced duplex stability, thus the hairpins can be readily opened and amplified by PCR. The constructs can be sequenced in paired-end format whereby read 1 (P1 primed) is the original stand and read 2 (P2 primed) is the copy stand. The read data is pairwise aligned so read 1 is aligned to its complementary read 2. Cognate residues from both reads are computationally resolved to produce a single genetic or epigenetic letter. Pairings of cognate bases that differ from the permissible five are the result of incomplete fidelity at some stage(s) comprising sample preparation, amplification, or erroneous base calling during sequencing. As these errors occur independently to cognate bases on each strand, substitutions result in a non- permissible pair. Non-permissible pairs are masked (marked as N) within the resolved read and the read itself is retained, leading to minimal information loss and high accuracy at read-level. The resolved read is aligned to the reference genome. Genetic variants and methylation counts are produced by read-counting at base-level. [000311] 5hmC has been shown to have value as a marker of biological states and disease which includes early cancer detection from cell-free DNA. In adapting 5-letter to 6-letter sequencing, 5mC is disambiguated from 5hmC without compromising genetic base calling within the same sample fragment. The first three steps of the workflow are identical to 5-letter sequencing described above, to generate the adapter ligated sample fragment with the synthetic copy strand. Methylation at 5mC is enzymatically copied across the CpG unit to the C on the copy strand, whilst 5hmC is enzymatically protected from such a copy. Thus, unmodified C, 5mC and 5hmC in each of the original CpG units are distinguished by unique 2-base combinations. The unmodified cytosines are then deaminated to uracil, which is subsequently read as thymine. The DNA is subjected to PCR amplification and sequencing as described earlier. The reads are pairwise aligned and resolved using a 2-base code. Each of unmodified C, 5mC, and 5hmC can be resolved as the three CpG units are distinct sequencing environments of the 2-base code.Atty. Docket No. GH0139WO [000312] In some embodiments, sequencing comprises an enzymatic conversion process, e.g., as in EM-Seq. See, e.g., Vaisvila R, et al. (2019) EM-seq: Detection of DNA methylation at single base resolution from picograms of DNA. bioRxiv; DOI: 10.1101 / 2019.12.20.884692, available at www.biorxiv.org / content / 10.1101 / 2019.12.20.884692v1. For example, TET2 and T4-βGT or 5-hydroxymethylcytosine carbamoyltransferase (described in Yang et al., Bio- protocol, 2023; 12(17): e4496) 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 (e.g., APOBEC3A), and then a deaminase (e.g., APOBEC3A) 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, V1900I, or V1900P TET mutant. In some embodiments, the one or more TET enzymes comprise a V1900 TET2 mutant, such as a V1900A, V1900C, V1900G, V1900I, or V1900P TET2 mutant. [000313] 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 APOBEC enzymes such as APOBEC3A. 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., inAtty. Docket No. GH0139WO 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. [000314] In some embodiments, the one or more TET enzymes comprise 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. [000315] In some embodiments, sequencing comprises an enzymatic conversion process, e.g., as in SEM-seq. See, e.g., Vaisvila et al. (2023) 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. bioRxiv; DOI: 10.1101 / 2023.06.29.547047, available at biorxiv.org / content / 10.1101 / 2023.06.29.547047v1. SEM-Seq employs a non-specific, modification-sensitive double-stranded DNA deaminase (MsddA) or a modification-sensitive DNA deaminase A (MsddA)-like deaminase in a nondestructive single-enzyme 5-methylctyosine sequencing (SEM-seq) method that deaminates unmodified cytosines. Accordingly, SEM-seq does not require the TET2 / T4-βGT protection and denaturing steps that are of use, e.g., in APOEC3A-based protocols. Additionally, MsddA does not deaminate 5-formylated cytosines (5fC) or 5-carboxylated cytosines (5caC). 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.Atty. Docket No. GH0139WO [000316] 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 dcm- 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 1X NEBNext Sample Purification Beads according to the manufacturer’s instructions and the purified library can be analyzed and quantified by an Agilent Bioanalyzer 2100 DNA Highsensitivity chip. The libraries can be sequenced using the Illumina NextSeq and NovaSeq 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. [000317] In some embodiments, sequence coverage of the genome may be, for example, less than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or 100%. In some embodiments, the sequence reactions may provide for sequence coverage of at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the genome. SequenceAtty. Docket No. GH0139WO coverage can be performed on at least 5, 10, 20, 70, 100, 200 or 500 different genes, or up to, for example, 5000, 2500, 1000, 500 or 100 different genes. [000318] Simultaneous sequencing reactions may be performed using multiplex sequencing. In some cases, cell-free nucleic acids may be sequenced with at least, for example, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions. In other cases, cell-free nucleic acids may be sequenced with less than, for example, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions. Sequencing reactions may be performed sequentially or simultaneously. Subsequent data analysis may be performed on all or part of the sequencing reactions. In some cases, data analysis may be performed on at least, for example, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions. In other cases, data analysis may be performed on less than, for example, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions. An exemplary read depth is 1,000-50,000 or 1,000-10,000 or 1,000-20,000 reads per locus (base). [000319] In general, sequencing of nucleic acid modifications and epigenetic target regions, e.g. to analyze a methylation profile of DNA, requires a lesser depth of sequencing than sequencing of a sequence-variable target region, e.g. for analysis of mutations. Hence, lesser sequencing depths, as described herein, may in some cases be adequate for the methods described herein. 1. Differential depth of sequencing

[0052] In some embodiments, nucleic acids corresponding to the sequence-variable target region set are sequenced to a greater depth of sequencing than nucleic acids corresponding to the epigenetic target region set. In some embodiments, nucleic acids corresponding to the hydroxymethylation-variable target region set are sequenced to a greater depth of sequencing than nucleic acids corresponding to at least one other target region set. For example, the depth of sequencing for nucleic acids corresponding to the sequence-variable and / or hydroxymethylation- variable target region sets may be at least 1.25-, 1.5-, 1.75-, 2-, 2.25-, 2.5-, 2.75-, 3-, 3.5-, 4-, 4.5-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, or 15-fold greater, or 1.25- to 1.5-, 1.5- to 1.75-, 1.75- to 2-, 2- to 2.25-, 2.25- to 2.5-, 2.5- to 2.75-, 2.75- to 3-, 3- to 3.5-, 3.5- to 4-, 4- to 4.5-, 4.5- to 5-, 5- to 5.5-, 5.5- to 6-, 6- to 7-, 7- to 8-, 8- to 9-, 9- to 10-, 10- to 11-, 11- to 12-, 13- to 14-, 14- to 15-fold, or 15- to 100-fold greater, than the depth of sequencing for nucleic acids corresponding to the epigenetic target region set or to at least one other target region set. In someAtty. Docket No. GH0139WO embodiments, said depth of sequencing is at least 2-fold greater. In some embodiments, said depth of sequencing is at least 5-fold greater. In some embodiments, said depth of sequencing is at least 10-fold greater. In some embodiments, said depth of sequencing is 4- to 10-fold greater. In some embodiments, said depth of sequencing is 4- to 100-fold greater. Each of these embodiments refer to the extent to which nucleic acids corresponding to the sequence-variable target region set are sequenced to a greater depth of sequencing than nucleic acids corresponding to the epigenetic target region set.

[0053] In some embodiments, the captured cfDNA corresponding to the sequence-variable target region set and the captured cfDNA corresponding to the epigenetic target region set are sequenced concurrently, e.g., in the same sequencing cell (such as the flow cell of an Illumina sequencer) and / or in the same composition, which may be a pooled composition resulting from recombining separately captured sets or a composition obtained by capturing the cfDNA corresponding to the sequence-variable target region set and the captured cfDNA corresponding to the epigenetic target region set in the same vessel.

[0054] In some embodiments, the captured cfDNA corresponding to the hydroxymethylation variable target region set and the captured cfDNA corresponding to the at least one other target region set are sequenced concurrently, e.g., in the same sequencing cell (such as the flow cell of an Illumina sequencer) and / or in the same composition, which may be a pooled composition resulting from recombining separately captured sets or a composition obtained by capturing the cfDNA corresponding to the hydroxymethylation variable target region set and the captured cfDNA corresponding to the at least one other target region set in the same vessel. J. Analysis [000320] The partitions obtained by the methods of the present disclosure can be analyzed to determine whether nucleic acids comprise the at least two types of modification. The nucleic acids in a population will partition based on the modifications present on that nucleic acid. For example, if the second type of modification is 5hmC, nucleic acids comprising 5hmC will partition to the second partition. Subsequent analysis of the second partition (e.g. by sequencing) will identify that that nucleic acid comprises 5hmC. [000321] The partitioning procedures used in the methods of the present disclosure allow for multiple types of modification to be identified in a single integrated workflow. For example, sequencing nucleic acids derived from one or more partition and aligning the sequence reads to a reference sequence (e.g. a reference genome) can identify the presence or absence of differentAtty. Docket No. GH0139WO types of modification in the original nucleic acid population, depending on which partition the sequences are identified in. [000322] Nucleic acids which do not contain either type of modification will primarily partition to the unbound partition. Analysis of the unbound partition can thus be used to identify nucleic acids in the nucleic acid population which do not include either type of modification. [000323] Nucleic acids which comprise only the first type of modification will primarily partition to the first partition, or the plurality of first sub-partitions. Analysis of the first partition or the plurality of first sub-partitions can thus be used to identify nucleic acids in the nucleic acid population which comprise the first type of modification. In embodiments which generate a plurality of first sub-partitions, the first sub-partitions can be analyzed such that nucleic acids with different extents of the first type of modification can be identified based on which of the first sub-partitions they are present in. [000324] Nucleic acids which comprise the second type of modification will primarily partition to the second partition, or the plurality of second sub-partitions. Analysis of the second partition or the plurality of first sub-partitions can thus be used to identify nucleic acids in the nucleic acid population which comprise the second type of modification. In embodiments which generate a plurality of second sub-partitions, the second sub-partitions can be analyzed such that nucleic acids with different extents of the second type of modification can be identified based on which of the second sub-partitions they are present in. Nucleic acids bearing both the first type of modification and the second type of modification will primarily partition to the second partition because the presence of the second type of modification would mean that the nucleic acid remains bound to the second binding agent when subjecting the binding agents to conditions which favour elution of the nucleic acids bound to the first binding agent but not the elution of the nucleic acids bound to the second binding agent. As such, nucleic acids present in the second partition may comprise both the first type of modification and the second type of modification. [000325] The methods disclosed herein can be used to detect the presence or absence of 5hmC. Typically, nucleic acids will contain at most one 5hmC nucleic acid base due to their scarcity in nature. The position of the 5hmC nucleic acid base in the nucleic acid can be identified using base coverage analysis. Base coverage analysis can involve aligning sequence reads (e.g. individual sequence reads, or consensus sequence reads for parent nucleic acids, as described elsewhere) from the corresponding partition to a reference sequence. Analysis of the frequency (e.g. the proportion) of sequence reads which align to a specific C position in a referenceAtty. Docket No. GH0139WO sequence can identify the C position which comprised a 5hmC nucleic acid base in the nucleic acid population. Specifically, the C position that comprised a 5hmC nucleic acid base in the sample nucleic acids would be expected to have a higher base coverage in the partition enriched for nucleic acids comprising 5hmC nucleic acid bases compared to those C positions that did not comprise a 5hmC nucleic acid base in the nucleic acid population. [000326] The identification of the modification status of a nucleic acid population has a variety of utilities. For example, methylation status can be used to characterize disease states, including for example, identifying the presence or absence of cancer, identification of cancer type, and / or identifying the tissue of origin of cfDNA molecules. [000327] Analyzing the sequence data may also include the analysis of non-modification features, such as fragmentation patterns (e.g. in the case of cfDNA analysis) or genetic variants (such as SNVs, indels and / or CNVs). Fragmentation patterns of DNA molecules in cfDNA samples carry information about the chromatin organization of the cells or tissues from which the cfDNA fragments originate. In particular, DNA fragments released to the bloodstream is often fragmented or cleaved around nucleosomes and / or other DNA bound proteins in the cells or tissues of origin. Further, nucleosome positioning and the location of DNA binding proteins is highly tissue specific and thus is used herein to amplify signal coming from the cells or tissues from which the cfDNA fragments originate (e.g., tumor cells as well as cells in the tumor microenvironment and cells involved in the immune response). Accordingly, in some embodiments, analyzing the sequencing data may comprise analyzing the modification profile and the fragmentation pattern of cfDNA. Such analysis can be used to identify the tissue of origin of the cfDNA and / or diagnose or prognose cancer. In some embodiments, analyzing the sequencing data may comprise analyzing the modification profile and the presence or absence of genetic variants in cfDNA. Such analysis can be used to identify the tissue of origin of the cfDNA and / or diagnose or prognose cancer. [000328] In some embodiments, analyzing the sequencing data may comprise analyzing: (i) the modification profile; (ii) the fragmentation pattern; and (iii) the presence or absence of genetic variants in cfDNA. Such analysis can be used to identify the tissue of origin of the cfDNA and / or diagnose or prognose cancer. [000329] In some embodiments, a method described herein comprises identifying the presence of DNA produced by a tumor (or neoplastic cells, or cancer cells) or by precancer cells. In some embodiments, a method described herein comprises identifying the presence of DNA producedAtty. Docket No. GH0139WO by cells (such as immune cells) that are not tumor cells, cancer cells, or precancer cells. In some embodiments, a method described herein comprises determining the levels of particular cell types (such as at least one cancer cell type and / or at least one immune cell type) from which DNA originated. Exemplary immune cell types may comprise naïve lymphocytes, activated lymphocytes, myeloid cells at different points of differentiation, and / or other types. In some such embodiments, determination of immune cell distribution facilitates detection or diagnosis or cancer or precancer, or determination of cancer prognosis or cancer treatment options (such as prediction of a clinical outcome for a therapy, such as a chemotherapy or an immunotherapy, in a subject). In some embodiments, the determination of levels of cell types (such as at least one cancer cell type and / or at least one immune cell type) facilitates determination of the likelihood that the subject from which the DNA was obtained has a disease or disorder, such as a cancer or precancer, and / or a disease or disorder related to the immune system, such as an infection, or transplant rejection. [000330] For example, determining the ratios of different cell types may facilitate such detection or determination. In some embodiments, wherein the cell types are immune cell types, the ratio numerator is the number or relative number of neutrophils, monocytes, or both, and the ratio denominator is the number or relative number of T cells, B cells, NK cells, or all lymphocytes. In some embodiments, the ratio numerator is the number or relative number of neutrophils, and the ratio denominator is the number or relative number of T cells, B cells, NK cells, or all lymphocytes. In some embodiments, the ratio numerator is the number or relative number of monocytes, and the ratio denominator is the number or relative number of T cells, B cells, NK cells, or all lymphocytes. In some embodiments, the ratio numerator is the number or relative number of neutrophils and monocytes, and the ratio denominator is the number or relative number of T cells, B cells, NK cells, or all lymphocytes. In some embodiments, the ratio is a neutrophil to lymphocyte ratio. In some embodiments, the ratio is a NK cell to total lymphocytes ratio. In some embodiments, the ratio numerator is the number or relative number of NK cells and the ratio denominator is the number or relative number of total lymphocytes. In some embodiments, the ratio is an M1 macrophage to M2 macrophage ratio. In some embodiments, the ratio numerator is the number or relative number of M1 macrophages and the ratio denominator is the number or relative number of M2 macrophages. In some embodiments, the ratio is a monocyte to T cell ratio. In some embodiments, elevations in such ratios are associated with cancer. In other embodiments, reductions in such ratios are associated with cancer.Atty. Docket No. GH0139WO [000331] The present methods can be used to diagnose presence of conditions, particularly cancer or precancer, in a subject, to characterize conditions (e.g., staging cancer or determining heterogeneity of a cancer), monitor response to treatment of a condition, effect prognosis risk of developing a condition or subsequent course of a condition. The present disclosure can also be useful in determining the efficacy of a particular treatment option. Successful treatment options may increase the amount of copy number variation or rare mutations detected in subject's blood if the treatment is successful as more cancers may die and shed DNA. In other examples, this may not occur. In another example, perhaps certain treatment options may be correlated with genetic profiles of cancers over time. This correlation may be useful in selecting a therapy. [000332] Additionally, if a cancer is observed to be in remission after treatment, the present methods can be used to monitor residual disease or recurrence of disease. [000333] The types and number of cancers that may be detected may include blood cancers, brain cancers, lung cancers, skin cancers, nose cancers, throat cancers, liver cancers, bone cancers, lymphomas, pancreatic cancers, skin cancers, bowel cancers, rectal cancers, thyroid cancers, bladder cancers, kidney cancers, mouth cancers, stomach cancers, solid state tumors, heterogeneous tumors, homogenous tumors and the like. Type and / or stage of cancer can be detected from genetic variations including mutations, rare mutations, indels, copy number variations, transversions, translocations, recombination, 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 abnormal changes in nucleic acid 5- methylcytosine. [000334] Genetic data can also be used for characterizing a specific form of cancer. Cancers are often heterogeneous in both composition and staging. Genetic profile data may allow characterization of specific sub-types of cancer that may be important in the diagnosis or treatment of that specific sub-type. This information may also provide a subject or practitioner clues regarding the prognosis of a specific type of cancer and allow either a subject or practitioner to adapt treatment options in accord with the progress of the disease. Some cancers can progress to become more aggressive and genetically unstable. Other cancers may remain benign, inactive or dormant. The system and methods of this disclosure may be useful in determining disease progression.Atty. Docket No. GH0139WO [000335] Further, the methods of the disclosure may be used to characterize the heterogeneity of an abnormal condition in a subject. Such methods can include, e.g., generating a genetic profile of extracellular polynucleotides derived from the subject, wherein the genetic profile comprises a plurality of data resulting from copy number variation and rare mutation analyses. In some embodiments, an abnormal condition is cancer or precancer. In some embodiments, the abnormal condition may be one resulting in a heterogeneous genomic population. In the example of cancer, some tumors are known to comprise tumor cells in different stages of the cancer. In other examples, heterogeneity may comprise multiple foci of disease. Again, in the example of cancer, there may be multiple tumor foci, perhaps where one or more foci are the result of metastases that have spread from a primary site. [000336] The present methods can be used to generate a profile, fingerprint, or set of data that is a summation of genetic information derived from different cells in a heterogeneous disease. Such a set of data may comprise copy number variation, epigenetic variation, or other mutation analyses alone or in combination. [000337] The present methods can be used to diagnose, prognose, monitor or observe cancers, or other diseases. In some embodiments, the methods herein do not involve the diagnosing, prognosing or monitoring a fetus and as such are not directed to non-invasive prenatal testing. In other embodiments, these methodologies may be employed in a pregnant subject to diagnose, prognose, monitor or observe cancers or other diseases in an unborn subject whose DNA and other polynucleotides may co-circulate with maternal molecules. [000338] In general, after sequencing, analysis of reads can be performed on a partition-by- partition level, as well as a whole DNA population level. Tags can be 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). [000339] An exemplary method for analyzing a nucleic acid population comprising nucleic acids with different extents of at least two types of modification comprises the following steps: 1. Optionally forming a derivative of the first type of modification and / or the second type of modification.Atty. Docket No. GH0139WO 2. Simultaneously contacting the nucleic acid population with (a) a first binding agent that preferentially binds to nucleic acids bearing the first type of modification, or the derivative thereof, and (b) a second binding agent that preferentially binds to nucleic acids bearing the second type of modification, or the derivative thereof. 3. Separating the nucleic acids bound to the first binding agent and the second binding agent from the unbound nucleic acids to produce an unbound partition. 4. Subjecting the nucleic acids bound to the first binding agent and the second binding agent to conditions which favour elution of the nucleic acids bound to the first binding agent but not the elution of the nucleic acids bound to the second binding agent, wherein the eluted nucleic acids provide a first partition. 5. Retaining the nucleic acids bound to the second binding agent to provide a second partition. 6. Analyzing one or more of the unbound partition, the first partition, and the second partition, optionally wherein the analyzing comprises sequencing the nucleic acids from the one or more partitions. [000340] Another exemplary method for analyzing a nucleic acid population comprising nucleic acids with different extents of at least two types of modification comprises the following steps: 1. Forming a derivative of the second type of modification, wherein the derivative comprises a capture moiety, such as biotin. 2. Simultaneously contacting the nucleic acid population with (a) a first binding agent that preferentially binds to nucleic acids bearing the first type of modification, for example wherein the first binding agent comprises an MBD protein, and (b) a second binding agent that preferentially binds to nucleic acids bearing the derivative thereof of the second type of modification, for example wherein the second binding agent comprises streptavidin. 3. Separating the nucleic acids bound to the first binding agent and the second binding agent from the unbound nucleic acids to produce an unbound partition. 4. Subjecting the nucleic acids bound to the first binding agent and the second binding agent to conditions which favour elution of the nucleic acids bound to the first binding agent but not the elution of the nucleic acids bound to the second binding agent, wherein the eluted nucleic acids provide a first partition. 5. Retaining the nucleic acids bound to the second binding agent to provide a second partition. 6. Analyzing one or more of the unbound partition, the first partition and the second partition,Atty. Docket No. GH0139WO optionally wherein the analyzing comprises sequencing the nucleic acids from the one or more partitions. [000341] An exemplary method for analyzing a nucleic acid population comprising nucleic acids with different extents of at least two types of modification, wherein the first type of modification is 5mC and the second type of modification is 5hmC, comprises the following steps: 1. Forming a derivative of 5hmC (e.g. through glucosylation), wherein the derivative comprises a capture moiety, such as biotin. 2. Simultaneously contacting the nucleic acid population with (a) a first binding agent that preferentially binds to nucleic acids bearing 5mC, for example wherein the first binding agent comprises an MBD protein, and (b) a second binding agent that preferentially binds to nucleic acids bearing the derivative of 5hmC, for example wherein the second binding agent comprises streptavidin. 3. Separating the nucleic acids bound to the first binding agent and the second binding agent from the unbound nucleic acids to produce an unbound partition. 4. Subjecting the nucleic acids bound to the first binding agent and the second binding agent to conditions that favor elution of the nucleic acids bound to the first binding agent but not the elution of the nucleic acids bound to the second binding agent, wherein the eluted nucleic acids provide a first partition. 5. Retaining the nucleic acids bound to the second binding agent to provide a second partition. 6. Analyzing one or more of the unbound partition, the first partition and the second partition, wherein the analyzing comprises sequencing the nucleic acids from the one or more partitions. K. Computer Systems [000342] Methods of the present disclosure can be implemented using, or with the aid of, computer systems. FIG.6 shows a computer system 601 that is programmed or otherwise configured to implement the methods of the present disclosure. The computer system 601 can regulate various aspects sample preparation, sequencing, and / or analysis. In some examples, the computer system 601 is configured to perform sample preparation and sample analysis, including (where applicable) nucleic acid sequencing, e.g., according to any of the methods disclosed herein. [000343] The computer system 601 includes a central processing unit (CPU, also "processor" and "computer processor" herein) 605, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 601 also includes memoryAtty. Docket No. GH0139WO or memory location 610 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 615 (e.g., hard disk), communication interface 620 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 625, such as cache, other memory, data storage, and / or electronic display adapters. The memory 610, storage unit 615, interface 620, and peripheral devices 625 are in communication with the CPU 105 through a communication network or bus (solid lines), such as a motherboard. The storage unit 615 can be a data storage unit (or data repository) for storing data. The computer system 601 can be operatively coupled to a computer network 630 with the aid of the communication interface 620. The computer network 630 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet. The computer network 630 in some cases is a telecommunication and / or data network. The computer network 630 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The computer network 630, in some cases with the aid of the computer system 601, can implement a peer-to-peer network, which may enable devices coupled to the computer system 601 to behave as a client or a server. [000344] The CPU 605 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 610. Examples of operations performed by the CPU 605 can include fetch, decode, execute, and writeback. [000345] The storage unit 615 can store files, such as drivers, libraries, and saved programs. The storage unit 615 can store programs generated by users and recorded sessions, as well as output(s) associated with the programs. The storage unit 615 can store user data, e.g., user preferences and user programs. The computer system 601 in some cases can include one or more additional data storage units that are external to the computer system 601, such as located on a remote server that is in communication with the computer system 601 through an intranet or the Internet. Data may be transferred from one location to another using, for example, a communication network or physical data transfer (e.g., using a hard drive, thumb drive, or other data storage mechanism). [000346] The computer system 601 can communicate with one or more remote computer systems through the network 630. For embodiment, the computer system 601 can communicate with a remote computer system of a user (e.g., operator). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad,Atty. Docket No. GH0139WO Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 601 via the network 630. [000347] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 601, such as, for example, on the memory 610 or electronic storage unit 615. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 605. In some cases, the code can be retrieved from the storage unit 615 and stored on the memory 610 for ready access by the processor 605. In some situations, the electronic storage unit 615 can be precluded, and machine-executable instructions are stored on memory 610. [000348] In an aspect, the present disclosure provides a non-transitory computer-readable medium comprising computer-executable instructions which, when executed by at least one electronic processor, perform at least a portion of a method described herein. For example, the method may comprise: i) optionally forming a derivative of the first type of modification and / or the second type of modification; ii) simultaneously contacting the nucleic acid population with: (a) a first binding agent that preferentially binds to nucleic acids bearing the first type of modification, or the derivative thereof, and (b) a second binding agent that preferentially binds to nucleic acids bearing the second type of modification, or the derivative thereof; iii) separating the nucleic acids bound to the first binding agent and the second binding agent from the unbound nucleic acids to produce an unbound partition; iv) subjecting the nucleic acids bound to the first binding agent and the second binding agent to conditions which favour elution of the nucleic acids bound to the first binding agent but not elution of the nucleic acids bound to the second binding agent, thereby providing eluted nucleic acids, wherein the eluted nucleic acids provide a first partition; v) retaining the nucleic acids bound to the second binding agent to provide a second partition; andvi) analyzing one or more of the unbound partition, the first partition, and the second partition. [000349] The code can be pre-compiled and configured for use with a machine have a processer adapted to execute the code or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as- compiled fashion.Atty. Docket No. GH0139WO [000350] Aspects of the systems and methods provided herein, such as the computer system 601, can be embodied in programming. Various aspects of the technology may be thought of as "products" or "articles of manufacture" typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. "Storage" type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. [000351] All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical landline networks, and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links, or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution. [000352] Hence, a machine-readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, anyAtty. Docket No. GH0139WO other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards, paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution. [000353] The computer system 601 can include or be in communication with an electronic display that comprises a user interface (UI) for providing, for example, one or more results of sample analysis. Examples of UIs include, without limitation, a graphical user interface (GUI) and web-based user interface. [000354] Additional details relating to computer systems and networks, databases, and computer program products are also provided in, for example, Peterson, Computer Networks: A Systems Approach, Morgan Kaufmann, 5th Ed. (2011), Kurose, Computer Networking: A Top-Down Approach, Pearson, 7thEd. (2016), Elmasri, Fundamentals of Database Systems, Addison Wesley, 6th Ed. (2010), Coronel, Database Systems: Design, Implementation, & Management, Cengage Learning, 11thEd. (2014), Tucker, Programming Languages, McGraw-Hill Science / Engineering / Math, 2nd Ed. (2006), and Rhoton, Cloud Computing Architected: Solution Design Handbook, Recursive Press (2011), each of which is hereby incorporated by reference in its entirety. L. Applications [000355] The methods presented herein may be used as part of any method that benefits from obtaining a modification profile (e.g. profile of 5mC, 5hmC and / or abasic sites) of DNA in any sample. [000356] One exemplary application of the methods of the present disclosure is using a modification profile in diagnosing and / or prognosing cancer. Hence, in some embodiments, a method described herein comprises identifying and / or predicting the presence or absence of DNA produced by a tumor (or neoplastic cells, or cancer cells), determining the probability that a test subject has a tumor or cancer, and / or characterizing a tumor, neoplastic cells, or cancer as described herein.Atty. Docket No. GH0139WO 1. Cancer and other diseases; cell type quantification [000357] The present methods can be used to diagnose the presence of a condition, e.g., cancer or precancer, in a subject, to characterize a condition (such as to determine a cancer stage or determining heterogeneity of a cancer), to monitor a subject’s response to receiving a treatment for a condition (such as a response to a chemotherapeutic or immunotherapeutic), assess prognosis of a subject (such as to predict a survival outcome in a subject having a cancer), to determine a subject’s risk of developing a condition, to predict a subsequent course of a condition in a subject, to determine metastasis or recurrence of a cancer in a subject (or a risk of cancer metastasis or recurrence), and / or to monitor a subject’s health as part of a preventative health monitoring program (such as to determine whether and / or when a subject is in need of further diagnostic screening). The present disclosure can also be useful in determining the efficacy of a particular treatment option. Successful treatment options may increase the amount of copy number variation, rare mutations, and / or cancer-related epigenetic signatures (such as hypermethylated regions or hypomethylated regions) detected in a subject's blood (such as in DNA isolated from a buffy coat sample or any other sample comprising cells, such as a blood sample (e.g., a whole blood sample, a leukapheresis sample, or a PBMC sample) from the subject) if the treatment is successful as more cancer cells may die and shed DNA, or, e.g., if a successful treatment results in an increase or decrease in the quantity of a specific immune cell type in the blood and an unsuccessful treatment results in no change. In other examples, this may not occur. In another example, certain treatment options may be correlated with genetic profiles of cancers over time. This correlation may be useful in selecting a therapy for a subject. [000358] In some embodiments, the present methods are used for screening for a cancer, such as a metastasis, or in a method for screening cancer, such as in a method of detecting the presence or absence of a metastasis. For example, the sample can be a sample from a subject who has not been previously diagnosed with cancer. In some embodiments, one or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more samples are collected from a subject as described herein, such as before and / or after the subject is diagnosed with a cancer. In some embodiments, the subject may or may not have cancer. In some embodiments, the subject may or may not have an early-stage cancer. In some embodiments, the subject has one or more risk factors for cancer, such as tobacco use (e.g., smoking), being overweight or obese, having a high body mass index (BMI), being of advanced age, poor nutrition, high alcohol consumption, or a family history of cancer.Atty. Docket No. GH0139WO [000359] In some embodiments, the subject has used tobacco, e.g., for at least 1, 5, 10, or 15 years. In some embodiments, the subject has a high BMI, e.g., a BMI of 25 or greater, 26 or greater, 27 or greater, 28 or greater, 29 or greater, or 30 or greater. In some embodiments, the subject is at least 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old. In some embodiments, the subject has poor nutrition, e.g., high consumption of one or more of red meat and / or processed meat, trans fat, saturated fat, and refined sugars, and / or low consumption of fruits and vegetables, complex carbohydrates, and / or unsaturated fats. High and low consumption can be defined, e.g., as exceeding or falling below, respectively, recommendations in Dietary Guidelines for Americans 2020-2025, available at dietaryguidelines.gov / sites / default / files / 2021- 03 / Dietary_Guidelines_for_Americans-2020-2025.pdf. In some embodiments, the subject has high alcohol consumption, e.g., at least three, four, or five drinks per day on average (where a drink is about one ounce or 30 mL of 80-proof hard liquor or the equivalent). In some embodiments, the subject has a family history of cancer, e.g., at least one, two, or three blood relatives were previously diagnosed with cancer. In some embodiments, the relatives are at least third-degree relatives (e.g., great-grandparent, great aunt or uncle, first cousin), at least second- degree relatives (e.g., grandparent, aunt or uncle, or half-sibling), or first-degree relatives (e.g., parent or full sibling). [000360] Typically, the disease under consideration is a type of cancer. Non-limiting examples of such cancers include biliary ...

Claims

Atty. Docket No. GH0139WO What is claimed is:

1. A method of analyzing a nucleic acid population comprising nucleic acids with different extents of at least two types of modification, including a first type of modification and a second type of modification, wherein the method comprises, in order: i) optionally forming a derivative of the first type of modification and / or the second type of modification; ii) simultaneously contacting the nucleic acid population with: (a) a first binding agent that preferentially binds to nucleic acids bearing the first type of modification, or the derivative thereof; and (b) a second binding agent that preferentially binds to nucleic acids bearing the second type of modification, or the derivative thereof; iii) separating the nucleic acids bound to the first binding agent and the second binding agent from the unbound nucleic acids to produce an unbound partition; iv) subjecting the nucleic acids bound to the first binding agent and the second binding agent to conditions which favour elution of the nucleic acids bound to the first binding agent but not elution of the nucleic acids bound to the second binding agent, thereby providing eluted nucleic acids, wherein the eluted nucleic acids provide a first partition; v) retaining the nucleic acids bound to the second binding agent to provide a second partition; and vi) analyzing one or more of the unbound partition, the first partition, and the second partition.

2. The method of claim 1, wherein the first type of modification is 5-methylcytosine (5mC).

3. The method of claim 2, wherein the first binding agent comprises: (i) a methyl-binding domain (MBD) protein; and / or (ii) an anti-5mC antibody, or an antigen-binding fragment thereof.

4. The method of any one of claims 1 to 3, wherein the second type of modification is 5- hydroxymethyl cytosine (5hmC).

5. The method of claim 4, wherein the second binding agent comprises an anti-5hmC antibody, or an antigen-binding fragment thereof.Atty. Docket No. GH0139WO 6. The method of claim 4, wherein the method comprises forming a derivative of 5hmC by glucosylation of 5hmC.

7. The method of claim 6, wherein the second binding agent comprises J-binding protein-1 (JBP-1).

8. The method of claim 6, wherein forming a derivative of 5hmC further comprises attaching a capture moiety to the glucosylated 5hmC.

9. The method of claim 8, wherein the capture moiety is biotin, optionally photocleavable biotin.

10. The method of claim 9, wherein the second binding agent comprises streptavidin.

11. The method of any one of claims 1 to 3, wherein the second type of modification is an abasic site.

12. The method of claim 11, wherein the method comprises forming a derivative of the abasic site, optionally by reacting the nucleic acid with an aldehyde probe, such that the probe covalently binds to the abasic site.

13. The method of claim 12, wherein the aldehyde probe further comprises a coupling moiety which allows conjugation of the probe to another compound, such as a capture moiety.

14. The method of claim 13, wherein the capture moiety comprises biotin, optionally wherein the second binding agent comprises streptavidin.

15. The method of any one of claims 1-14, wherein step (iv) comprises sequentially subjecting the nucleic acids bound to the first binding agent and the second binding agent to a plurality of conditions which increasingly favour elution of the nucleic acids bound to the first binding agent, such that the first partition is separated into a plurality of first sub-partitions,Atty. Docket No. GH0139WO wherein the nucleic acids eluted in the first of the plurality of conditions have a lower extent of the first type of modification compared to the nucleic acids eluted in the last of the plurality of conditions.

16. The method of any one of claims 1-15, wherein step (v) comprises subjecting the nucleic acids bound to the second binding agent to conditions which favour elution of the nucleic acids bound to the second binding agent, wherein the eluted nucleic acids provide the second partition.

17. The method of claim 16, wherein step (v) comprises sequentially subjecting the nucleic acids bound to the second binding agent to a plurality of conditions which increasingly favour elution of the nucleic acids bound to the second binding agent, such that the second partition is separated into a plurality of second sub-partitions, wherein the nucleic acids eluted in the first of the plurality of conditions have a lower extent of the second type of modification compared to the nucleic acids eluted in the last of the plurality of conditions.

18. The method of any one of the preceding claims, further comprising capturing at least an epigenetic target region set of nucleic acids from the nucleic acid population or a subsample thereof, optionally wherein the capturing comprises contacting the nucleic acid population with a plurality of target-specific probes specific for members of the epigenetic target region set.

19. The method of the immediately preceding claim, comprising determining a methylation level of at least one of the plurality of epigenetic target regions.

20. The method of any one of claims 18-19, wherein the at least one of the plurality of epigenetic target regions is a differentially methylated region.

21. The method of any one of claims 18-20, wherein the at least one of the plurality of epigenetic target regions is a fragment.

22. The method of any one of claims 18-20, wherein the at least one of the plurality of epigenetic target regions is a hypermethylated region, optionally wherein the hypermethylated region is a type-specific hypermethylated region.Atty. Docket No. GH0139WO 23. The method of any one of claims 18-20, wherein the at least one of the plurality of epigenetic target regions is a hypomethylated region, optionally wherein the hypomethylated region is a type-specific hypomethylated region.

24. The method of any one of claims 18-20, wherein the at least one of the plurality of epigenetic target regions comprises a CTCF binding site, and / or a transcription start site.

25. The method of any one of claims 18-24, wherein the at least one of the plurality of epigenetic target regions is at least one type-specific epigenetic target region.

26. 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.

27. The method of claim 25 or 26, wherein the at least one type-specific epigenetic target region comprises type-specific hypomethylated regions and / or type-specific hypermethylated regions.

28. The method of any one of claims 25-27, 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.

29. The method of any one of claims 25-28, 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;Atty. Docket No. GH0139WO 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.

30. The method of any one of claims 22 or 27-29, 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.

31. The method of any one of claims 25-29, 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.

32. The method of any one of claims 25-31, comprising identifying at least one cell type or tissue type from which the at least one type-specific epigenetic target region originated.

33. 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.

34. 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.

35. The method of the any one of the preceding claims, further comprising capturing at least one sequence-variable target region set of the nucleic acids, optionally wherein the capturing comprises contacting the nucleic acid population with a plurality of target-specific probes specific for the sequence-variable target regions.Atty. Docket No. GH0139WO 36. The method of any one of claims 18-35, wherein the capturing the at least an epigenetic target region set of nucleic acids and / or the at least one sequence variable target region set of nucleic acids occurs prior to the contacting the nucleic acid population with the first and second binding agents.

37. The method of any one of claims 18-35, wherein the capturing the at least an epigenetic target region set of nucleic acids and / or the at least one sequence variable target region set of nucleic acids occurs after the contacting the nucleic acid population with the first and second binding agents.

38. The method of any one of claims 18-35, wherein the at least an epigenetic target region set of nucleic acids and / or the at least one sequence variable target region set of nucleic acids are captured from at least the first partition and / or the second partition.

39. The method of any one of claims 1-38, wherein the analyzing comprises nucleic acid sequencing.

40. The method of any one of claims 1-39, wherein the analyzing comprises preparing one or more sequencing libraries from at least a subset of the nucleic acids in one or more of the unbound partition, the first partition, the second partition, one or more of the first sub-partitions, and / or one or more of the second sub-partitions, wherein the one or more sequencing libraries are then subjected to nucleic acid sequencing.

41. The method of claim 40, wherein the one or more sequencing libraries is obtained using a single-stranded sequencing library preparation method.

42. The method of claim 40, wherein the one or more sequencing libraries is obtained using a double-stranded sequencing library preparation method.

43. The method of any one of claims 38-41, wherein the sequencing comprises sequencing the nucleic acids in a manner that distinguishes a nucleobase comprising the first type of modification and a nucleobase comprising the second type of modification.Atty. Docket No. GH0139WO 44. The method of any one of claims 39-43, wherein two or more of the partitions are pooled prior to the sequencing.

45. The method of any one of claims 39-44, wherein the sequencing comprises long-read sequencing.

46. The method of any one of claims 39-45, wherein the sequencing comprises nanopore sequencing.

47. The method of any one of claims 39-45, wherein the sequencing comprises 5-letter or 6- letter sequencing.

48. The method of any one of claims 39-44, wherein the sequencing comprises next generation sequencing.

49. The method of any one of the preceding claims, wherein the method comprises ligating one or more adapters to the nucleic acids of the nucleic acid population, thereby producing adapter-ligated nucleic acids.

50. The method of the immediately preceding claim, wherein the adapter-ligated nucleic acids are amplified prior to the sequencing.

51. The method of claim 49 or claim 50, wherein the one or more adapters comprises at least one tag.

52. The method of the immediately preceding claim, wherein the at least one tag comprises a molecular barcode.

53. The method of any one of the preceding claims, wherein at least nucleic acid molecules of the first partition and nucleic acid molecules of the second partition are differentially tagged.Atty. Docket No. GH0139WO 54. The method of any one of the preceding claims, wherein the nucleic acid population comprises DNA.

55. The method of the immediately preceding claim, wherein the DNA comprises cell-free DNA (cfDNA), optionally wherein the cfDNA is from a bodily fluid, such as blood, serum, or plasma.

56. The method of any one of the preceding claims, wherein the the nucleic acid population is derived from a sample obtained from a subject.

57. The method of the immediately preceding claim, wherein the sample is from a bodily fluid, such as blood, serum, or plasma.

58. The method of claim 56 or claim 57, wherein the blood sample is a whole blood sample, a plasma sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample.

59. The method of any one of claims 56-58, wherein the sample is a blood sample.

60. The method of claim 56, wherein the sample is a tissue sample.

61. The method of any one of claims 56-60, wherein the subject is an animal.

62. The method of claim any one of claims 56-61, wherein the subject is a human.

63. The method of any one of claims 56-62, comprising determining a likelihood that the subject has precancer.

64. The method of any one of claims 56-63, comprising determining a likelihood that the subject has cancer.

65. The method of any one of claims 39-64, wherein the sequencing comprises generating a plurality of sequencing reads, and wherein the method further comprises mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads, andAtty. Docket No. GH0139WO processing the mapped sequence reads to determine the likelihood that the subject has cancer or precancer.

66. The method of any one of claims 56-65, wherein the sample is obtained from a subject who was previously diagnosed with a cancer and received one or more previous cancer treatments, optionally wherein the sample is obtained at one or more preselected time points following the one or more previous cancer treatments.

67. The method of the immediately preceding claim, further comprising determining a cancer recurrence score, optionally 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.

68. 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.

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