Methods involving multi-modal tumor variant identification and tracking of tumor molecules

Abstract

Provided herein are methods related to analyzing DNA in a sample from a subject, comprising measuring in the sample or a subsample thereof a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation that the one or more corresponding DMRs do not comprise.

Description

Atty. Docket No. GH0213WO / 01228-0067-00PCTMETHODS INVOLVING MULTI-MODAL TUMOR VARIANT IDENTIFICATION AND TRACKING OF TUMOR MOLECULESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application No. 63 / 703,112, filed October 3, 2024, and US Provisional Patent Application No. 63 / 752,333, filed January 31, 2025, each which is incorporated by reference herein in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present disclosure provides methods related to analyzing DNA in a sample from a subject, comprising measuring in the sample or a subsample thereof a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation that the one or more corresponding DMRs do not comprise. In some embodiments, the method comprises subjecting the DNA or a subsample thereof to a conversion procedure that converts a first nucleobase in the DNA and does not convert a second nucleobase, wherein the first nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine different from the first nucleobase, thereby providing a converted sample. In some embodiments, the subject has or is suspected of having a disease or disorder, such as cancer.INTRODUCTION AND SUMMARY

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

[0004] Improperly controlled cell growth is a hallmark of cancer that generally results from an accumulation of genetic and epigenetic changes, such as copy number variations (CNVs), single nucleotide variations (SNVs), gene fusions, insertions and / or deletions (indels), epigenetic variations including modification of cytosine (e.g., 5-methylcytosine, 5- hydroxymethylcytosine, and other more oxidized forms) and association of DNA with chromatin proteins and transcription factors. Thus, cancer can be indicated by sequence modifications and / or non-sequence modifications, such as methylation. Examples of methylation changes in cancer include local gains of DNA methylation in the CpG islands at the transcription start site of genes involved in normal growth control, DNA repair, cell cycleAtty. Docket No. GH0213WO / 01228-0067-00PCT regulation, and / or cell differentiation. Hypermethylation can be associated with an aberrant loss of transcriptional capacity of involved genes and occurs at least as frequently as point mutations and deletions as a cause of altered gene expression. Furthermore, without wishing to be bound by any particular theory, cells in or around a cancer or neoplasm may shed more DNA than cells of the same tissue type in a healthy subject. The DNA from such cells may differ epigenetically from shed DNA in a healthy subject. As such, the distribution of epigenetically modified (e.g., methylated) DNA in certain DNA samples, such as cell-free DNA (cfDNA), may change upon carcinogenesis. Thus, sufficiently sensitive epigenetic (e.g., DNA methylation) profiling can be used to detect aberrant methylation in DNA of a sample.

[0005] Tumor-informed somatic assays are subject to significant technical and biological noise, which can be mostly mitigated through increased assay costs (deep tumor, normal, plasma sequencing). Tumor-naive methylation-only assays can be cost effective but subject to biological noise that theoretically limits performance, which cannot be overcome with tumor methylation information alone.

[0006] Accordingly, there is a continued need for tracking of tumor molecules to improve performance and / or cost of cancer tests, including minimum residual disease (MRD) and monitoring tests. Identifying and leveraging co-occurring methylation and somatic variants could reduce biological and technical noise in each signal type alone.

[0007] The present disclosure aims to meet the need for tracking of tumor molecules to improve performance of MRD and monitoring tests, and / or provide other benefits. In some embodiments, the disclosure provides methods related to analyzing DNA in a sample from a subject, comprising measuring in the sample or a subsample thereof a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation that the one or more corresponding DMRs do not comprise. In some embodiments, the method comprises subjecting the DNA or a subsample thereof to a conversion procedure that converts a first nucleobase in the DNA and does not convert a second nucleobase, wherein the first nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine different from the first nucleobase, thereby providing a converted sample. In some embodiments, the subject has or is suspected of having a disease or disorder, such as cancer.

[0008] The following exemplary embodiments are provided.Atty. Docket No. GH0213WO / 01228-0067-00PCTEmbodiment 1. A method of analyzing DNA in a sample from a subject, comprising: measuring in the sample or a subsample thereof a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation that the one or more corresponding DMRs do not comprise.Embodiment 2. The method of embodiment 1, wherein the one or more variant DMRs and the one or more corresponding DMRs are sequenced.Embodiment 3. The method of embodiment 1 or 2, wherein the one or more variant DMRs and the one or more corresponding DMRs are sequenced via whole-genome sequencing (WGS). Embodiment 4. A method of analyzing DNA in a sample from a subject, comprising: identifying in DNA from the subject one or more variant differentially methylated regions (DMRs), wherein the one or more variant DMRs comprise a somatic mutation; and measuring in the sample or a subsample thereof a level of methylation of the one or more variant DMRs independently of one or more corresponding DMRs not comprising the somatic mutation.Embodiment 5. A method of analyzing DNA in a sample from a subject, comprising: partitioning the DNA into at least first and second subsamples, wherein the first subsample comprises methylated DNA to a greater extent than the second subsample; measuring in the first subsample a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation.Embodiment 6. The method of embodiment 5, wherein the DNA is partitioned based on the presence or absence of 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) nucleic acid bases in the nucleic acids.Embodiment 7. A method of analyzing DNA in a sample from a subject, comprising: subjecting the DNA or a subsample thereof to a conversion procedure that converts a first nucleobase in the DNA and does not convert a second nucleobase, wherein the first nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine different from the first nucleobase, thereby providing a converted sample; measuring in the converted DNA a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein theAtty. Docket No. GH0213WO / 01228-0067-00PCT one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation.Embodiment 7.01. The method of any one of embodiments 1-7, wherein the sample is a first sample obtained at a first time point.Embodiment 7.02. The method of embodiment 7.01, wherein the first sample is a tissue sample, tumor sample, cfDNA sample, or plasma sample.Embodiment 7.03. The method of any one of embodiments 7.01 or 7.02, wherein the first sample is a tumor sample or a tissue sample.Embodiment 7.04. The method of embodiment 7.01-7.03, further comprising sequencing the first sample.Embodiment 7.05. The method of embodiment 7.04, wherein the first sample is sequenced via whole genome sequencing (WGS).Embodiment 7.06. The method of embodiment 7.04, wherein the first sample is sequenced via panel sequencing.Embodiment 7.07. The method of any one of embodiments 7.01-7.06, further comprising obtaining a second sample at second timepoint.Embodiment 7.08. The method of embodiment 7.07, further comprising analyzing the second sample, wherein analyzing the second sample comprises: measuring in the second sample or a subsample thereof a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation that the one or more corresponding DMRs do not comprise.Embodiment 7.09. The method of embodiment 7.07, further comprising analyzing the second sample, wherein analyzing the second sample comprises: identifying in DNA from the subject one or more variant differentially methylated regions (DMRs), wherein the one or more variant DMRs comprise a somatic mutation; and measuring in the sample or a subsample thereof a level of methylation of the one or more variant DMRs independently of one or more corresponding DMRs not comprising the somatic mutation.Embodiment 7.10. The method of embodiment 7.07, further comprising analyzing the second sample, wherein analyzing the second sample comprises: partitioning the DNA into at least first and second subsamples, wherein the first subsample comprises methylated DNA to a greater extent than the second subsample; measuring in the first subsample a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise aAtty. Docket No. GH0213WO / 01228-0067-00PCT somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation.Embodiment 7.11. The method of embodiment 7.07, further comprising analyzing the second sample, wherein analyzing the second sample comprises: subjecting the DNA, or a subsample thereof to a conversion procedure that converts a first nucleobase in the DNA and does not convert a second nucleobase, wherein the first nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine different from the first nucleobase, thereby providing a converted sample; measuring in the converted DNA a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation.Embodiment 7.12. The method of any one of embodiments 7.07-7.11, further comprising sequencing the second sample.Embodiment 7.13. The method of embodiment 7.12, wherein the second sample is sequenced via whole genome sequencing (WGS).Embodiment 7.14. The method of embodiment 7.12, wherein the second sample is sequenced via panel sequencing.Embodiment 7.15. The method of embodiment 7.14, wherein the second sample is sequenced via panel sequencing using a panel that is a subset of the panel used for the first sample and / or is targeted based on the sequencing of the first sample.Embodiment 7.16 The method of embodiment 7.14, wherein the first and second samples are sequenced via panel sequencing and wherein the panel is the same for each sample. Embodiment 7.17 The method of embodiment 7.14 or 7.15, wherein the first and second samples are sequenced via panel sequencing and wherein the panel for the second sample is a subset of the panel for the first.Embodiment 7.18 The method of any one of embodiments 7.14-7.17, wherein the second sample is enriched for target regions of the panel by capture or amplification (e.g., PCR).Embodiment 7.19 The method of embodiment 7.18, wherein the capture or amplification is mutation specific.Embodiment 7.20. The method of any one of embodiments 7.07-7.19, wherein the second sample is a tissue sample.Embodiment 7.21. The method of any one of embodiments 7.07-7.19, wherein the second sample is a tumor sample.Atty. Docket No. GH0213WO / 01228-0067-00PCTEmbodiment 7.22. The method of any one of embodiments 7.07-7.19, wherein the second sample is a cfDNA sample.Embodiment 7.23. The method of any one of embodiments 7.07-7.19, wherein the second sample is a plasma sample.Embodiment 7.24. A method of analyzing DNA in a first sample and a second sample from a subject, comprising: subjecting the DNA from the first sample, or a subsample thereof, to a conversion procedure that converts a first nucleobase in the DNA and does not convert a second nucleobase, wherein the first nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine different from the first nucleobase, thereby providing a converted sample; measuring in the converted DNA a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation; wherein the first sample is a tissue sample or a tumor sample; wherein the first sample is sequenced via whole genome sequencing (WGS); and subjecting the DNA from the second sample, or a subsample thereof, to a conversion procedure that converts a first nucleobase in the DNA and does not convert a second nucleobase, wherein the first nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine different from the first nucleobase, thereby providing a converted sample; measuring in the converted DNA a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation; wherein the second sample is a plasma sample or a cfDNA sample, and wherein the second sample is sequenced via whole genome sequencing (WGS).Embodiment 7.25. A method of analyzing DNA in a first sample and a second sample from a subject, comprising: subjecting the DNA from the first sample, or a subsample thereof, to a conversion procedure that converts a first nucleobase in the DNA and does not convert a second nucleobase, wherein the first nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine different from the first nucleobase, thereby providing a converted sample; measuring in the converted DNA a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise theAtty. Docket No. GH0213WO / 01228-0067-00PCT somatic mutation; wherein the first sample is a tissue sample or a tumor sample; wherein the first sample is sequenced via whole genome sequencing (WGS); and subjecting the DNA from the second sample, or a subsample thereof, to a conversion procedure that converts a first nucleobase in the DNA and does not convert a second nucleobase, wherein the first nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine different from the first nucleobase, thereby providing a converted sample; measuring in the converted DNA a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation; wherein the second sample is a plasma sample or a cfDNA sample, and the second sample is sequenced via panel sequencing.Embodiment 7.26. A method of analyzing DNA in a first sample and a second sample from a subject, comprising: subjecting the DNA from the first sample, or a subsample thereof, to a conversion procedure that converts a first nucleobase in the DNA and does not convert a second nucleobase, wherein the first nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine different from the first nucleobase, thereby providing a converted sample; measuring in the converted DNA a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation; wherein the first sample is a tissue sample or a tumor sample and wherein the first sample is sequenced via panel sequencing; and subjecting the DNA from the second sample, or a subsample thereof, to a conversion procedure that converts a first nucleobase in the DNA and does not convert a second nucleobase, wherein the first nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine different from the first nucleobase, thereby providing a converted sample; measuring in the converted DNA a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation; wherein the second sample is a plasma sample or a cfDNA sample, and the second sample is sequenced via panel sequencing.Embodiment 7.27. The method of embodiment 7.26, wherein the panel sequencing of the first sample and the panel sequencing of the second sample use the same panel.Atty. Docket No. GH0213WO / 01228-0067-00PCTEmbodiment 7.28. The method of embodiment 7.26, wherein the panel sequencing of the second sample uses a panel that is a subset of the panel used with the first sample and / or the panel sequencing of the second sample uses a patient-specific and / or personalized panel. Embodiment 8. The method of any one of embodiments 1-4, 6, or 7.01-7.28, further comprising partitioning the sample based on the presence or absence of 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) nucleic acid bases in the nucleic acids. Embodiment 9. The method of any one of embodiments 1, 4, 5, or 7.01-7.28, further comprising identifying in DNA from the subject one or more variant DMRs.Embodiment 10. The method of the immediately preceding embodiment, wherein the one or more variant DMRs is identified using a comparison to a germline sequence.Embodiment 11. The method of the immediately preceding embodiment, wherein the germline sequence is obtained from the same sample or subsample from the subject. Embodiment 12. The method of any one of embodiments 10-12, wherein the germline sequence is obtained from a sample or subsample obtained at an earlier time from the subject. Embodiment 13. The method of any one of embodiments 10-12, wherein the germline sequence is obtained from a blood sample from the subject.Embodiment 14. The method of any one of embodiments 10-12, wherein the germline sequence is obtained from a plasma sample from the subject.Embodiment 15. The method of any one of embodiments 10-12, wherein the germline sequence is obtained from a buffy coat sample from the subject.Embodiment 16. The method of any one of embodiments 10-12, wherein the germline sequence is obtained from a tumor sample from the subject.Embodiment 17. The method of embodiment 16, wherein the method comprises partitioning the tumor sample and wherein the one or more variant DMRs are identified by sequencing the tumor sample.Embodiment 18. The method of the immediately preceding embodiment, wherein the one or more variant DMRs are identified by comparing the mutations identified in methylation-enriched tumor sequencing to mutations identified in the germline sequence. Embodiment 18.1. The method of embodiment 18, wherein the methylation-enriched tumor sequencing comprises enriching the DNA for sequence-variable target regions comprising the mutations identified in the germline sequence.Embodiment 19. The method of embodiment 18 or 18.1, wherein the one or more variant DMRs comprise one or more mutations identified in the methylation-enriched tumor sequencing that are not identified in the germline sequence.Atty. Docket No. GH0213WO / 01228-0067-00PCTEmbodiment 20. The method of any one of embodiments 1-19, wherein the one or more variant and corresponding DMRs are specific to a cancer and / or a tissue.Embodiment 21. The method of any one of preceding embodiments, wherein the level of methylation is measured using single stranded methylation conversion (SSM). Embodiment 22. The method of any one of embodiments 1-20, wherein the level of methylation is determined based on partitioning of the DNA via methyl-specific enrichment probes.Embodiment 23. The method of any one of embodiments 1-22, wherein the somatic mutation is a passenger mutation.Embodiment 24. The method of any one of embodiments 1-23, wherein the somatic mutation comprises a single nucleotide variant (SNV).Embodiment 25. The method of any one of embodiments 1-23, wherein the somatic mutation comprises an insertion or deletion (InDei).Embodiment 26. The method of any one of embodiments 1-23, wherein the somatic mutation comprises a copy number variation (CNV).Embodiment 27. The method of any one of preceding embodiments, wherein the somatic mutation is not present in healthy cells of the subject.Embodiment 28. The method of any one of preceding embodiments, wherein the somatic mutation is present in cancer cells of the subject.Embodiment 29. The method of any one of preceding embodiments, wherein the somatic mutation is not due to clonal hematopoiesis of indeterminate potential (CHIP). Embodiment 30. The method of embodiment 7, wherein the conversion procedure selectively converts the base pairing specificity of 5-methylcytosines (5mC) or unmethylated cytosines (C) in the sample.Embodiment 31. The method of embodiment 7 or 30, wherein the conversion procedure comprises contacting the DNA in the sample with a deaminase.Embodiment 32. The method of any one of embodiments 7, 30 or 31, wherein the conversion procedure comprises contacting the DNA in the sample with a Tet enzyme and a deaminase.Embodiment 33. The method of embodiment 31 or 32, wherein the deaminase enzyme is APOBEC3A.Embodiment 34. The method of any one of embodiments 7 or 30-33, wherein unmethylated CpGs in the DNA are converted to UpGs.Atty. Docket No. GH0213WO / 01228-0067-00PCTEmbodiment 35. The method of embodiment 31 or 32, wherein the deaminase is a dsDNA deaminase.Embodiment 36. The method of embodiment 31 or 32, wherein the deaminase is a ssDNA deaminase.Embodiment 37. The method of embodiment 31 or 32, wherein the deaminase is a methyl-insensitive deaminase.Embodiment 38. The method of the immediately preceding embodiment, wherein the methyl-insensitive deaminase is A3 A.Embodiment 39. The method of embodiment 31 or 32, wherein the deaminase is a methyl-sensitive deaminase.Embodiment 40. The method of the immediately preceding embodiment, wherein the methyl-sensitive deaminase is modification-sensitive DNA deaminase A (MsddA) or a modification-sensitive DNA deaminase A (MsddA)-like deaminase.Embodiment 41. The method of embodiment 7, wherein the conversion procedure comprises Tet-assisted conversion of nucleic acids with a substituted borane reducing agent, wherein 5hmC nucleic acid bases are protected from conversion, optionally through glucosylation.Embodiment 42. The method of the immediately preceding embodiment, wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, ammonia borane or pyridine borane.Embodiment 43. The method of embodiment 30, wherein the conversion procedure comprises: reacting the nucleic acids with a variant methyltransferase having carboxymethyltransferase activity in the presence of carboxy -5-adenosyl-Z-methionine (CxSAM) substrate, thereby labelling any unmethylated C and rendering it resistant to deaminase action, wherein 5hmC nucleic acid bases are protected from conversion through glucosylation; and contacting the nucleic acids of step (a) with a deaminase enzyme.Embodiment 44. The method of the immediately preceding embodiment, wherein the variant methyltransferase having carboxymethylase activity is a recombinant M.MpelN374K.Embodiment 45. The method of embodiment 7, wherein the conversion procedure selectively converts the base pairing specificity of unmethylated cytosines (C) in the nucleic acids.Embodiment 46. The method of the immediately preceding embodiment, wherein the conversion procedure is bisulfite conversion.Atty. Docket No. GH0213WO / 01228-0067-00PCTEmbodiment 47. The method of any one of preceding embodiments, wherein the methylation status of the nucleic acids in the sample is determined by analyzing the base coverage of cytosines in a reference sequence.Embodiment 48. The method of any one of preceding embodiments, wherein the DNA comprises adapters.Embodiment 49. The method of any one of the preceding embodiments, wherein the DNA comprises barcodes.Embodiment 50. The method of any one of the preceding embodiments, wherein the method further comprises ligating adapters comprising barcodes to the DNA.Embodiment 51. The method of embodiment 50, wherein the method comprises ligating adapters comprising barcodes to the DNA prior to the amplifying.Embodiment 52. The method of embodiment 50, wherein the method comprises ligating adapters comprising barcodes to the DNA prior to the contacting the DNA with the deaminase.Embodiment 53. The method of embodiment 48, wherein the method comprises ligating adapters to the DNA after cutting the DNA.Embodiment 54. The method of any one of embodiments 48-53, wherein the adapters are Y-shaped adapters.Embodiment 55. The method of embodiment 7, further comprising, prior to the conversion procedure subjecting the DNA in the sample to end repair to generate end- repaired DNA molecules, wherein the end repair is performed using deoxynucleotide triphosphates (dNTPs), wherein at least one type of dNTP comprises a modified base, and the at least one dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.Embodiment 56. The method of embodiment 55, wherein the end repair is performed using a DNA polymerase that does not have 5 ’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.Embodiment 57. The method of embodiment 55, wherein the end repair is performed using a DNA polymerase that has 5 ’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.Embodiment 58. The method of any one of embodiments 55 to 57, wherein the at least one type of dNTP which comprises a modified base, wherein the modified base includes a dNTP comprising 4-methylcytosine (4mC), a dNTP comprising 5-methylcytosine (5mC), a dNTP comprising 5-hydroxymethyl-cytosine (5hmC), a dNTP comprising N6-Atty. Docket No. GH0213WO / 01228-0067-00PCT methyladenosine (6mA), a dNTP comprising bromodeoxyuridine (BrdU) and / or a dNTP comprising 8-oxoguanine (8oxoG).Embodiment 59. The method of any one of embodiments 55 to 58, further comprising performing an A-tailing reaction, optionally after a step of subjecting a DNA sample to end repair.Embodiment 60. The method of embodiment 59, wherein the end-repair and the A- tailing reaction are performed in the same reaction mixture, optionally wherein the end-repair and the A-tailing reaction are performed a single tube and / or optionally wherein the endrepair and the A-tailing reaction are performed without an intervening clean-up step.Embodiment 61. The method of embodiment 59, wherein the A-tailing is performed using a DNA polymerase that does not possess 5 ’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase, optionally wherein the DNA polymerase is HemoKlen Taq.Embodiment 62. The method of embodiment 59, wherein the A-tailing is performed using a thermostable DNA polymerase.Embodiment 63. The method of any one of embodiments 7, 30-47, or 55-62, prior to the conversion procedure, performing a methylation-preserving amplification of the DNA of the sample.Embodiment 64. The method of the immediately preceding embodiment, wherein the methylation-preserving amplification is a linear, methylation-preserving amplification. Embodiment 65. The method of embodiment 63 or 64, wherein the methylationpreserving amplification comprises contacting the DNA with a methyltransferase.Embodiment 66. The method of any one of embodiments 63-65, wherein the methylation-preserving amplification comprises one or more of polymerase chain reaction, linear amplification, rolling circle amplification, ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustained sequence-based replication.Embodiment 67. The method of any one of embodiments 63-66, wherein the methylation-preserving amplification comprises thermocycled amplification.Embodiment 68. The method of any one of embodiments 63-67, wherein the methylation-preserving amplification comprises isothermal amplification.Embodiment 69. The method of any one of the preceding embodiments, further comprising sequencing at least a portion of the amplified DNA.Atty. Docket No. GH0213WO / 01228-0067-00PCTEmbodiment 70. The method of any one of the preceding embodiments, further comprising quantifying a level of methylation at one or more differentially methylated regions of the DNA.Embodiment 71. The method of the immediately preceding embodiment, wherein quantifying 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. Embodiment 72. The method of embodiment 69, wherein the sequencing is nextgeneration sequencing (NGS).Embodiment 73. The method of the immediately preceding embodiment, wherein the NGS is pyrosequencing, sequencing-by-synthesis, semiconductor sequencing, sequencing- by-ligation, or sequencing-by-hybridization.Embodiment 74. The method of embodiment 69, wherein the sequencing comprises nanopore-based sequencing or single-molecule real time (SMRT) sequencing.Embodiment 75. The method of any one of the preceding embodiments, further comprising enriching the DNA for a plurality of target regions, optionally prior to the step of sequencing the DNA.Embodiment 76. The method of embodiment 75, wherein the plurality of target regions comprises epigenetic target regions.Embodiment 77. The method of embodiment 76, wherein the epigenetic target regions comprise hypermethylation variable target regions.Embodiment 78. The method of embodiment 76 or 77, wherein the epigenetic target regions comprise hypomethylation variable target regions.Embodiment 79. The method of any one of embodiments 75-79, wherein the plurality of target regions comprises sequence-variable target regions.Embodiment 80. The method of the immediately preceding embodiment, wherein the DNA comprises cell-free DNA.Embodiment 81. The method of any one of embodiments 1-80, wherein the DNA comprises DNA from formalin fixed paraffin embedded samples.Embodiment 82. The method of any one of embodiments 1-81, wherein the subject is an animal.Embodiment 83. The method of the immediately preceding embodiment, wherein the subject is a human.Embodiment 84. The method of any one of embodiments 1-81 wherein the subject has or is at risk of having a cancer.Atty. Docket No. GH0213WO / 01228-0067-00PCTEmbodiment 85. The method of the immediately preceding embodiment, wherein the cancer has low aberrant hypermethylation.Embodiment 86. The method of the immediately preceding embodiment, wherein the cancer is early stage, stage 0 cancer, stage 1 cancer, non-metastatic, or carcinoma in situ. Embodiment 87. The method of any one of embodiments 1-86, further comprising determining the presence or status of a cancer in the subject.Embodiment 88. The method of the immediately preceding embodiment, wherein the cancer is identified based on the somatic mutation.Embodiment 89. The method of any one of embodiments 1-88, further comprising characterizing a cancer or tumor of the subject.Embodiment 90. The method of any one of the preceding embodiments, wherein the sample or subsample thereof is a subsample comprising methylated DNA.Embodiment 91. The method of any one of the preceding embodiments, wherein the sample or subsample thereof is a subsample comprising hypermethylated DNA.Embodiment 92. The method of any one of the preceding embodiments, wherein the level of methylation comprises a count of molecules including the one or more variant DMRs.Embodiment 93. The method of any one of the preceding embodiments, wherein cancer is detected based on a count of molecules including the one or more variant DMRs. Embodiment 94. The method of the immediately preceding embodiment, wherein cancer is detected when a count of molecules including the one or more variant DMRs is one or more in the sample from the subject or a subsample thereof.Embodiment 95. The method of the immediately preceding embodiment, wherein cancer is detected when a count of molecules including the one or more variant DMRs is one or more in a first subsample of the sample from the subject, the first subsample comprising methylated DNA.Embodiment 96. The method of the immediately preceding embodiment, wherein the first subsample comprises methylated DNA to a greater extent than a second subsample of the sample from the subject.Embodiment 97. The method of embodiment 95 or 96, wherein the first and, optionally, the second subsample are obtained by partitioning the sample from the subject.Atty. Docket No. GH0213WO / 01228-0067-00PCTBRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGs.lA-lC illustrate currently available workflows for identifying methylation and somatic variant molecules. Fig. 1 A illustrates a tumor-informed somatic assays. Figure IB illustrates tumor sequencing that is then compared to germline sequencing gained from the huffy coat to determine germline mutations or clonal hematopoiesis of indeterminate potential (CHIP). Figure 1C illustrates plasma sequencing and filtering results against candidate somatic mutations in both plasma and tumors.

[0010] FIGs. 2A-2C illustrate potential workflows for identifying methylation and somatic variant molecules for MRD tracking. Fig. 2A illustrates a methylation-enriched sequencing of tumor tissue DNA. Figure 2B illustrates a tumor and MBD enriched buffy coat sequencing panel workflow. Figure 2C illustrates a methyl-enriched cfDNA seq on panel to saturation.

[0011] FIG. 3 is a schematic diagram of an example of a system suitable for use with some embodiments of the disclosure.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0012] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with such embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.

[0013] 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, reference to “a cell” includes a plurality of cells, and the like.

[0014] 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. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0015] 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; embodiments in the specification that recite “consisting of’ various components are also contemplated as “comprising” or “consisting essentially of’ the recited components; and embodiments in the specification that recite “consisting essentially of’ various components are also contemplated as “consisting of’ or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0016] The section headings used herein are for organizational purposes and are not to be construed as limiting the disclosed subject matter in any way. In the event that any document or other material incorporated by reference contradicts any explicit content of this specification, including definitions, this specification controls.Definitions

[0017] “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 previously 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. cfDNA molecules may occur as DNA fragments.

[0018] 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.

[0019] As used herein, “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., TAtty. Docket No. GH0213WO / 01228-0067-00PCT cells, B cells, NK cells, dendritic cells, and monocytes) and polymorphonuclear (e.g., granulocytes such as neutrophils and eosinophils) white blood cells.

[0020] “Enriching” or “capturing” one or more target nucleic acids or one or more nucleic acids comprising at least one target region refers to preferentially isolating or separating the one or more target nucleic acids or one or more nucleic acids comprising at least one target region from non-target nucleic acids or from nucleic acids that do not comprise at least one target region.

[0021] An “enriched set” or “captured set” of nucleic acids or “enriched” or “captured” nucleic acids refers to nucleic acids that have undergone capture.

[0022] As used herein, a “capture moiety” is a molecule that allows affinity separation of molecules, such as nucleic acids, 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.

[0023] As used herein, a “cell type” is a set of cells having a shared characteristic. For example, cell types can include cells of different origins, differentiation types, different activation types, or any combination of different origins, different differentiation types, and different activation types. Indeed, differentiation status and activation status can overlap and often change together in a given cell, such as an immune cell or a cancer cell. For example, activation of an immune cell may induce differentiation of the cell. In some embodiments, cell types may be distinguished based on characteristics such as one or more cell surface markers, a genetic signature (such as expression (or expression level) of a particular gene or set of genes), and / or an epigenetic signature, such as regions of DNA hypermethylation or hypomethylation.

[0024] As used herein, a “cell cluster” or “cluster” is a plurality of related cell types, e.g., immune cell types, tissue-specific cell types, and / or cancer cell types. In some embodiments, the cell types within a cluster have similar DNA methylation profiles, e.g., in a plurality of hypermethylation variable target regions and / or hypomethylation variable target regions.

[0025] A “converted nucleobase” is a nucleobase having an altered base pairing specificity, wherein the original base pairing specificity of the nucleobase was changed by a procedure. For example, certain procedures convert unmethylated or unmodified cytosine to dihydrouracil, or more generally, at least one modified or unmodified form of cytosine undergoes deamination, resulting in uracil (considered a modified nucleobase in the contextAtty. Docket No. GH0213WO / 01228-0067-00PCT of DNA) or a further modified form of uracil. As used herein, a “converted sample” is a sample comprising DNA comprising at least one converted nucleobase.

[0026] As used herein, an “oligonucleotide probe” refers to a single-stranded nucleic acid molecule (such as a DNA molecule or an RNA molecule) that can hybridize to a complementary nucleic acid sequence (e.g., a target sequence) within a target nucleic acid. An oligonucleotide probe may be 10 to 50 bases in length and may be synthesized as a specified base sequence.

[0027] “Specifically binds” in the context of a primer, a probe, or other oligonucleotide and a target sequence (e.g., a nucleic acid comprising a sequence that is partially or completely complementary to the primer, probe, or other oligonucleotide) means that under appropriate hybridization conditions, the primer, probe, or other oligonucleotide hybridizes to its target sequence, or replicates thereof, to form a stable hybrid, while at the same time formation of stable non-target hybrids is minimized. Thus, a primer, probe, or other oligonucleotide hybridizes to a target sequence or replicate thereof to a sufficiently greater extent than to a non-target sequence, to ultimately enable enrichment 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). An oligonucleotide probe can “preferentially form a substrate for extension with,” e.g., a target nucleic acid comprising a wild-type sequence relative to a target nucleic acid comprising a variant sequence, a target nucleic acid that comprises a converted nucleotide relative to a target nucleic acid that does not comprise the converted nucleotide, or a target nucleic acid that does not comprise a converted nucleotide relative to a target nucleic acid that comprises the converted nucleotide. For example, an oligonucleotide probe that preferentially forms a substrate for extension with a target nucleic acid comprising a wild-type sequence relative to a target nucleic acid comprising a variant sequence may be at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times more likely to bind the target nucleic acid comprising the wild-type sequence relative to the target nucleic acid comprising the variant sequence.

[0028] A “target region” refers to a genomic locus targeted for identification and / or capture, for example, by using probes (e.g., through sequence complementarity). A “target region” orAtty. Docket No. GH0213WO / 01228-0067-00PCT“set of target regions” refers to a plurality of genomic loci targeted for identification and / or capture, for example, by using a set of probes (e.g., through sequence complementarity). A “target region” can comprise regions that share at least one common feature. In some embodiments, a target region is identified by the at least one common feature. For example, a hypermethylation variable target region comprises regions of DNA that are hypermethylated.

[0029] “Sequence-variable target regions” refer to 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) relative to normal cells. A “sequence-variable target region” refers to a set of sequencevariable target regions. In some embodiments, the sequence-variable target regions are target regions that may exhibit changes that affect less than or equal to 50 contiguous nucleotides, e.g., less than or equal to 40, 30, 20, 10, 5, 4, 3, 2, or 1 nucleotides.

[0030] “Epigenetic target regions” refers to target regions that may show sequenceindependent differences in different cell or tissue types (e.g., different types of immune cells) or in abnormal cells, such as neoplastic cells (e.g., tumor cells and cancer cells), relative to normal cells; or that may show sequence-independent differences (i.e., in which there is no change to the nucleotide sequence, e.g., differences in methylation, nucleosome distribution, or other epigenetic features) in DNA, e.g., from different cell types or from subjects having cancer relative to DNA from healthy subjects. Examples of sequence-independent changes include, but are not limited to, changes in methylation (increases or decreases), nucleosome distribution, fragmentation patterns, CCCTC-binding factor (“CTCF”) binding, transcription start sites (e.g., with respect to any one of more of binding of RNA polymerase components, binding of regulatory proteins, fragmentation characteristics, and nucleosomal distribution), and regulatory protein binding regions. Epigenetic target regions thus include, but are not limited to, hypermethylation variable target regions, hypomethylation variable target regions, and fragmentation variable target regions, such as CTCF binding sites and transcription start sites. 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 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 dependAtty. Docket No. GH0213WO / 01228-0067-00PCT on the accuracy of base calls at one or a few individual positions. An “epigenetic target region” is a set of epigenetic target regions.

[0031] 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 differentially methylated region has a detectably higher degree of methylation (e.g., a hypermethylated region) in at least one cell or tissue type, such as at least one immune cell type, relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type, such as other immune cell types, or from the same cell or tissue type from a healthy subject. In some embodiments, a differentially methylated region has a detectably lower degree of methylation (e.g., a hypom ethylated region) in at least one cell or tissue type, such as at least one immune cell type, relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type, such as other immune cell types, or from the same cell or tissue type from a healthy subject. A “corresponding DMR” refers to a DMR from the same locus or covering the same sequence as a first (e.g., variant) DMR.

[0032] A nucleic acid is “produced by a tumor” if it originated from a tumor cell. 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). As used herein, “precancer” or a “precancerous condition” is an abnormality that has the potential to become cancer, wherein the potential to become cancer is greater than the potential if the abnormality was not present, i.e., was normal. Examples of precancer include but are not limited to adenomas, hyperplasias, metaplasias, dysplasias, benign neoplasias (benign tumors), premalignant carcinoma in situ, and polyps. It should be noted that certain types of carcinoma in situ are recognized in the field as cancerous, e.g., Stage 0 cancer, as opposed to premalignant.

[0033] The term “methylation” or “DNA methylation” refers to addition of a methyl group to a nucleobase 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 inAtty. Docket No. GH0213WO / 01228-0067-00PCTN6-methyladenine. In some embodiments, DNA methylation is 5-methylation (modification of the 5thcarbon of the 6-carbon ring of cytosine). 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 3rdcarbon of the 6-carbon ring of cytosine). In some embodiments, 3C methylation comprises addition of a methyl group to the 3C position of the cytosine to generate 3 -methylcytosine (3mC). Methylation can also occur at non CpG sites, for example, methylation can occur at a CpA, CpT, or CpC site. DNA methylation can change the activity of methylated DNA region. For example, when DNA in a promoter region is methylated, transcription of the gene may be repressed. DNA methylation is critical for normal development and abnormality in methylation may disrupt epigenetic regulation. The disruption, e.g., repression, in epigenetic regulation may cause diseases, such as cancer. Promoter methylation in DNA may be indicative of cancer.

[0034] The term “hypermethylation” refers to an increased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules comprising the same genetic information 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.

[0035] The term “hypomethylation” refers to a decreased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules comprising the same genetic information within a population (e.g., 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.

[0036] The terms “agent that recognizes a modified nucleobase in DNA,” such as an “agent that recognizes a modified cytosine in DNA” refers to a molecule or reagent that binds to or detects one or more modified nucleobases in DNA, such as methyl cytosine. A “modified nucleobase” is a nucleobase that comprises a difference in chemical structure from an unmodified nucleobase. In the case of DNA, an unmodified nucleobase is adenine, cytosine, guanine, or thymine. In some embodiments, a modified nucleobase is a modified cytosine. InAtty. Docket No. GH0213WO / 01228-0067-00PCT 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) and antibodies specific for methyl cytosine. In some embodiments, such antibodies bind to 5-methyl cytosine in DNA. In some such embodiments, the DNA may be single-stranded or doublestranded. Suitable agents include agents that recognize modified nucleotides in doublestranded DNA, single-stranded DNA, and both double-stranded and single-stranded DNA.

[0037] The term “epigenetic status” refers to a certain level or extent of a sequenceindependent variable that may be present in a DNA sequence. In some embodiments, the epigenetic status of a DNA sequence refers to the extent or level of methylation, nucleosome distribution, cfDNA fragmentation pattern, CCCTC-binding factor (“CTCF”) binding, transcription start site, or regulatory protein binding region of the sequence. Epigenetic statuses thus include, but are not limited to, hypermethylation, hypomethylation, and the presence of absence of CTCF binding sites or transcription start sites. The epigenetic status of a sequence may be a “reference epigenetic status” that can be used for comparison to the epigenetic status of the corresponding sequence in other DNA molecules. An example of a reference epigenetic status is a status that is prevalent in samples obtained from healthy subjects and is not associated with cancer.

[0038] As used herein, “methylation status” refers to the presence or absence of a methyl group on a DNA nucleobase (e.g. cytosine) at a particular genomic position in a nucleic acid, the degree of methylation of a nucleic acid (e.g., high, low, intermediate, or unmethylated), or the number of nucleotides methylated in a particular nucleic acid molecule. A nucleic acid “in methylated form” means that it comprises a sequence containing a methylated DNA nucleobase, e.g., a methylated cytosine in a CpG dinucleotide.

[0039] 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 a referred to as a cancer or a cancerous tumor.

[0040] 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) orAtty. Docket No. GH0213WO / 01228-0067-00PCT 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 of different molecular barcodes are used such that there is a low probability (e.g., less than about a 10%, less than about a 5%, less than about a 1%, or less than about a 0.1% chance) that any two molecules may have the same endogenous sequence information (e.g., start and / or stop positions, subsequences of one or both ends of a sequence, and / or lengths) and also have the same molecular barcode.

[0041] As used herein, “partitioning” refers to physically separating, sorting, and / or fractionating a mixture of nucleic acid molecules in a sample into a plurality of subsamples or subpopulations of nucleic acids based on a characteristic of the nucleic acid molecules. AAtty. Docket No. GH0213WO / 01228-0067-00PCT sample or population may be partitioned into one or more partitioned subsamples or subpopulations based on a characteristic that is indicative of a genetic or epigenetic change or a disease state. 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. Stated differently, partitioning may include physically partitioning nucleic acid molecules based on the presence or absence of one or more methylated nucleobases. 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.

[0042] 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 partition, or residual partitioned set). In another example, the nucleic acid molecules can be partitioned based on the number of methylated nucleotides - one partitioned set can have nucleic acid molecules with nine methylated nucleotides, and another partitioned set can have unmethylated nucleic acid molecules (zero methylated nucleotides).

[0043] As used herein, a “restriction endonuclease” refers to an enzyme that can cleave a double stranded nucleic acid molecule (such as a double stranded DNA or cDNA) at a specific recognition site (a particular nucleotide sequence recognized by the restriction endonuclease, also referred to herein as a “cleavage site”) in the double stranded DNA or cDNA molecule. Exemplary restriction endonucleases of use herein include, but are not limited to, BspQI, AfUII, BsiHKAI, Btrl, Maell, and Sdul. In some embodiments, theAtty. Docket No. GH0213WO / 01228-0067-00PCT restriction endonuclease is BspQI and claves a double-stranded nucleic acid at a 5’- GCTCTTCN-3’ recognition site.

[0044] As used herein, “sample” means anything capable of being analyzed by the methods and / or systems disclosed herein.

[0045] As used herein, “sequencing” refers to any of a number of technologies used to determine the sequence (e.g., the identity and order of monomer units) of a biomolecule, e.g., a nucleic acid such as DNA or RNA. Examples of sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co-amplification at lower denaturation temperature-PCR (COLD- PCR), multiplex PCR, sequencing by reversible dye terminator, paired-end sequencing, near- term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverseterminator sequencing, long-read 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.

[0046] As used herein, “next-generation sequencing” or “NGS” refers to sequencing technologies having increased throughput as compared to traditional Sanger- and capillary electrophoresis-based approaches, for example, with the ability to generate hundreds of thousands of relatively small sequence reads at a time. Some examples of next-generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. In some embodiments, next-generation sequencing includes the use of instruments capable of sequencing single molecules. Example of commercially available instruments for performing next-generation sequencing include, but are not limited to, NextSeq, HiSeq, NovaSeq, MiSeq, Ion PGM and Ion GeneStudio S5.

[0047] 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.Atty. Docket No. GH0213WO / 01228-0067-00PCTAnimals 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 autoimmune disease.

[0048] As used herein, the phrase “non-complementary restriction endonuclease cleavage site” refers to a restriction endonuclease cleavage site in a first strand of a double-stranded nucleic acid in which the second strand comprises at least one nucleotide that is not complementary to the corresponding nucleotide of the first strand. Non-complementary restriction endonuclease cleavage sites can comprise 1, 2, 3, 4, 5, 6, or more than 6 nucleotides that are not complementary to one another in this fashion. For example, in some embodiments of a double-stranded nucleic acid comprising 5’ and 3’ adapters on both strands, a 5’ adapter and a 3’ adapter comprise “non-complementary restriction endonuclease cleavage sites” when the cleavage site in the 5’ adapter comprises at least one nucleotide that is not complementary to the paired nucleotide of the 3’ adapter when the nucleic acids are aligned antiparallel to one another. Restriction endonuclease cleavage sites of use herein include cleavage sites that are recognized by a restriction endonuclease that cleaves a doublestranded nucleic acid comprising the cleavage site. Thus, in some embodiments, non- complementary restriction endonuclease cleavage sites, such as non-complementary restriction cleavage sites in otherwise complementary sense and antisense strands of a nucleic acid, may comprise cleavage sites that are recognized by different restriction endonucleases. Non-complementary restriction endonuclease cleavage sites can thus comprise a sense strand cleavage site that is longer or shorter than the cleavage site in the antisense strand.

[0049] As used herein, “substantially” refers to a range of numerical values (e.g., values within 5-10% of the specified vale) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). For example, “substantially” can include values greater than or equal to 90% of a specified value.Atty. Docket No. GH0213WO / 01228-0067-00PCTSubstantially does not require 100% (such as exactly 100% of the target nucleic acids of a population of target nucleic acids), but can include an amount greater than or equal to 90%, such as an amount greater than or equal to any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% (such as 90%-100% of the target nucleic acids of a population of target nucleic acids).

[0050] “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.

[0051] “ Germline mutation” and “germline variation” are used interchangeably and refer to an inherited mutation (i.e., not one arising post-conception). Germline mutations may be the only mutations that can be passed on to the offspring and may be present in every somatic cell and germline cell in the offspring. As used herein, the term “germline sequence” refers to the genetic sequences from the germline (the haploid gametes and those diploid cells from which they are formed).

[0052] “ Somatic mutation” and “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.

[0053] As used herein, a “passenger mutation” or “hitchhiker mutation” refers to a mutation which does not alter fitness but occurred in a cell that coincidentally or subsequently acquired a driver mutation, and are therefore found in every cell with that driver mutation. Conversely, a “driver mutation” refers to a mutation that confer a fitness advantage to somatic cells in their microenvironment, thereby driving the cell lineage to cancer.

[0054] 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.

[0055] 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.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0056] 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. 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.

[0057] A “X1 / / / / / / X2 mutation” in a specified polypeptide as used herein, where Xi and X2 are amino acids and nnn is a position in an amino acid sequence, refers to a substitution in the polypeptide of amino acid Xi present 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 X1 / / / / / / X2 mutation may, but does not necessarily, comprise additional differences from the wild-type sequence, including but not limited to truncations and deletions as well as other substitutions. For example, a “T1372S mutation” in TET2 refers to a substitution in a TET2 enzyme of the threonine present at position 1372 of the full- length wild-type human TET2 enzyme with a serine. Position 1372 of wild-type human TET2 aligns to position 258 and 248, respectively, of the truncated TET2 sequences disclosed as SEQ ID NOs: 23 and 24 of US Patent 10,961,525. Similarly, a “V1900X2 mutation” where X2 is A, C, G, I, or P in TET2 refers to a substitution in a TET2 enzyme of the valine present at position 1900 of the full-length wild-type human TET2 enzyme with an alanine, cysteine, glycine, isoleucine, or proline.

[0058] “ Or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise.Exemplary MethodsOverview

[0059] Cancer formation and progression may arise from both genetic modification and epigenetic features of DNA. However, assaying specific changes related to cancer that occur at very low frequencies, such as in early-stage cancers and pre-cancers, requires ultra-deep sequencing and / or enrichment, which can substantially increase assay costs. Some methods are available that allow for non-desired sequence depletion in RNA-seq workflows (such as for depletion of ribosomal RNA in a sample, See, e.g., US Patent Application No. 2015 / 0299767 Al). However, such methods are not designed to deplete targeted abundant wildtype sequences of a given locus to enrich for sequence variants of the same locus inAtty. Docket No. GH0213WO / 01228-0067-00PCT detection assays (such as assays that can detect sequence variants in a liquid biopsy sample from a subject).

[0060] Tumor-informed somatic assays (as shown in Figure 1 A) are subject to significant technical and biological noise, which can be mostly mitigated through increased assay costs (deep tumor, normal, plasma sequencing). As depicted in Figure 1 A, unique reads from tumor sequence are indicated by hatched bars. Candidate tumor somatic mutations are indicated by circles. Biological noise can be the result of CHIP, but this can be overcome by sequencing buffy coat, although this increases cost of sequencing. Each column of reads in Fig. 1 A corresponds to the same locus as the column in the same position in Fig. IB. As shown in Figure IB, tumor sequencing is then compared to germline sequencing gained from the buffy coat to identify germline mutations or CHIP, which may require 0.3 - 1.1 billion reads and large amounts of reagents to filter somatic mutations (open bars represent individual reads of buffy coat sequencing, and open circles represent mutations found therein). Candidate tumor somatic mutations that are found in the buffy coat sequence reads can be classified as due to heterozygous or homozygous germline mutations or CHIP depending on prevalence, while candidate tumor somatic mutations that are not found in the buffy coat sequence reads can be classified as filtered tumor somatic mutations. When sequencing cells from the bloodstream, there can also be relatively high noise background from other tissues and / or cells shedding into bloodstream that have methylation states matching cancer-associated tumor molecules. Figure 1C shows that plasma sequencing and filtering results against candidate somatic mutations in both plasma and tumors is time consuming and error-prone, e.g., due to PCR or sequencing error. Figure 1C illustrates that filtered somatic tumor mutations (e.g., as identified in Figure 1 A) can be compared against mutations identified through plasma sequencing to identify candidate somatic mutations. However, a substantial depth of sequencing (e.g., 0.3-1.1 billion reads) is required to overcome technical noise from mutations introduced from PCR and / or sequencing error (indicated by the arrow) and to ensure that a single observed molecule bearing a mutation from filtered list is indeed from a tumor. Candidate tumor somatic mutations that are also found in the plasma sequence reads can be classified as filtered plasma somatic mutations.

[0061] Accordingly, the present disclosure provides methods for analyzing DNA in a sample from a subject, comprising measuring in the sample or a subsample thereof a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation that the one or more corresponding DMRs do not comprise.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0062] The present disclosure also provides methods for analyzing DNA in a sample from a subject, comprising: identifying in DNA from the subject one or more variant differentially methylated regions (DMRs), wherein the one or more variant DMRs comprise a somatic mutation; and measuring in the sample or a subsample thereof a level of methylation of the one or more variant DMRs independently of one or more corresponding DMRs not comprising the somatic mutation.

[0063] The present disclosure provides methods for analyzing DNA in a sample from a subject, comprising: partitioning the DNA into at least first and second subsamples, wherein the first subsample comprises methylated DNA to a greater extent than the second subsample; measuring in the first subsample a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation.

[0064] The present disclosure provides methods for analyzing DNA in a sample from a subject, comprising: subjecting the DNA or a subsample thereof to a conversion procedure that converts a first nucleobase in the DNA and does not convert a second nucleobase, wherein the first nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine different from the first nucleobase, thereby providing a converted sample; measuring in the converted DNA a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation.

[0065] Presently available methods including tumor-informed somatic assays or tumor- informed methylation-only assays are subject to significant technical and biological noise and mitigating such noise increases assay costs. The present disclosure provides methods that meet the need for tracking of tumor molecules to improve performance of MRD and monitoring tests, while keeping technical and biological noise to a minimum, and reduced assay costs.

[0066] In some embodiments, the sample or subsample thereof is a subsample comprising methylated DNA. In some embodiments, the sample or subsample thereof is a subsample comprising hypermethylated DNA. For example, a subsample coprising hypermethylated DNA can be obtained based on binding to a methylated DNA-binding agent, e.g., as described with respect to partitioning elsewhere herein.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0067] 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), and / or levels or abundance or proportions of one or more metabolites or metabolite signatures, and / or levels or abundance or proportions of one or more lipid molecules or lipid signatures, and / or the levels or proportion or abundance of one or more proteins and / or nucleic acids associated extracellular vesicles (e.g., exosomes). 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.Differentially Methylated Regions (DMRs)

[0068] In some embodiments, the disclosed methods comprise identifying in DNA from the subject one or more variant Differentially Methylated Regions (DMRs).

[0069] In some embodiments, the one or more variant DMRs is identified using a comparison to a germline sequence. Germline mutations, unlike somatic mutations, may be the only mutations that can be passed on to the offspring and may be present in essentially every somatic cell and germline cell in the offspring (with the limited possible exception of a subpopulation of somatic cells that coincidentally have acquired a further mutation or reversion in the same locus).

[0070] In some embodiments, the germline sequence is obtained from the same sample or subsample from the subject. In some embodiments, the germline sequence is obtained from a sample or subsample obtained at an earlier time from the subject. In some embodiments, the germline sequence is obtained from a blood sample from the subject. In some embodiments, the germline sequence is obtained from a plasma sample from the subject. In someAtty. Docket No. GH0213WO / 01228-0067-00PCT embodiments, the germline sequence is obtained from a buffy coat sample from the subject. In some embodiments, the germline sequence is obtained from a tumor sample from the subject.

[0071] In some embodiments, the method comprises partitioning the tumor sample and wherein the one or more variant DMRs are identified by sequencing the tumor sample. In some embodiments, the one or more variant DMRs are identified by comparing the mutations identified in methylation-enriched tumor sequencing to mutations identified in the germline sequence. In some embodiments, the one or more variant DMRs comprise one or more mutations identified in the methylation-enriched tumor sequencing that are not identified in the germline sequence. In some embodiments, the one or more variant and corresponding DMRs are specific to a cancer and / or a tissue.

[0072] In some embodiments, the level of methylation is measured using single stranded methylation conversion (SSM). In some embodiments, the level of methylation is determined based on partitioning of the DNA via methyl-specific enrichment probes. In some embodiments, the level of methylation comprises a count of molecules including the one or more variant DMRs. In some embodiments, cancer is detected based on a count of molecules including the one or more variant DMRs. In some embodiments, cancer is detected when a count of molecules including the one or more variant DMRs is one or more in the sample from the subject or a subsample thereof, e.g., in a first subsample that comprises methylated DNA, e.g., to a greater extent than a second subsample. Such first and, optionally, second subsamples may be obtained by partitioning as described elsewhere herein.Mutations

[0073] In some embodiments, the present disclosure provides methods for analyzing DNA in a sample from a subject, wherein the one or more variant DMRs comprise a somatic mutation that the one or more corresponding DMRs do not comprise.

[0074] In some embodiments, the somatic mutation is a passenger mutation.

[0075] In some embodiments, the somatic mutation comprises a single nucleotide variant (SNV). In some embodiments, the somatic mutation comprises an insertion or deletion (InDei). In some embodiments, the somatic mutation comprises a copy number variation (CNV). In some embodiments, the somatic mutation is not present in healthy cells of the subject. In some embodiments, the somatic mutation is present in cancer cells of the subject. In some embodiments, the somatic mutation is not due to clonal hematopoiesis of indeterminate potential (CHIP).Atty. Docket No. GH0213WO / 01228-0067-00PCTConverting DNA

[0076] The methods disclosed herein can comprise converting DNA, wherein the conversion procedure selectively converts the base pairing specificity of 5-methylcytosines (5mC) or unmethylated cytosines I in the sample.

[0077] In some embodiments, the conversion procedure comprises contacting the DNA in the sample with a deaminase. In some embodiments, the conversion procedure comprises contacting the DNA in the sample with a Tet enzyme and a deaminase. In some embodiments, the deaminase enzyme is APOBEC3A. In some embodiments, unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, the deaminase is a dsDNA deaminase. In some embodiments, the deaminase is a ssDNA deaminase. In some embodiments, the deaminase is a methyl -insensitive deaminase. In some embodiments, wherein the methyl-insensitive deaminase is A3 A. In some embodiments, the deaminase is a methyl-sensitive deaminase. In some embodiments, the methyl-sensitive deaminase is modification-sensitive DNA deaminase A (MsddA) or a modification-sensitive DNA deaminase A (MsddA)-like deaminase. In some embodiments, the conversion procedure comprises Tet-assisted conversion of nucleic acids with a substituted borane reducing agent, wherein 5hmC nucleic acid bases are protected from conversion, optionally through glucosylation. In some embodiments, the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, ammonia borane or pyridine borane.

[0078] In some embodiments, the deaminase (e.g., the methyl-sensitive deaminase or the methyl-insensitive deaminase) comprises any one or more of the following deaminases or a truncated version thereof, such as any of the truncated versions disclosed in Vaisvila et al., Mol Cell. 2024 Mar 7;84(5):854-866.e7. doi: 10.1016 / j.molcel.2024.01.027. Epub 2024 Feb 22, available on w w ert.com at / molecular-cell / fulltext / S 1097-2765(24)00094-7 (“Vaisvila 2024”): MsddA, AshDaOl, MGYPDa21, PpDa03, SbDaOl, BlDaOl, PpDaO4, CsDaOl, MGYPDa22, FIDaOl, MGYPDa24, AaDaO2, MmgDaOl, PbDaOl, BcDaO2, LsfDaOl, SmgDaOl, XcDaOl, KsDaOl, PwDaOl, CaDaOl, SrDaOl, NgDaOl, NsDaOl, SzDaOl, SpDaOl, AdDaOl, MGYPDa23, WWTPDaO7, PdDaOl, MGYPDa25, MGYPDa26, DaDaOl, EcDaOl, EcDaO2, NgDaO2, PaDaOl, AsDaOl, HgmDaOl, MsDaO2, XinDaOl, XjaDaOl, RhDaOl, MGYPDaO4, MGYPDa05, BaDaOl, WWTPDaO4, PbDaO2, CrDaOl, MGYPDal5, MGYPDal6, MGYPDal7, BaDaO2, VsDaOl, MGYPDal8, MGYPDal9, HmDaO6, MmgDaO2, HgmDaO2, CgmDaOl, FbiDaOl, PvmDaOl, MGYPDa408, MGYPDa687, MGYPDa917, MGYPDa624, DddA, StsDaOl, LbsDaOl, BpDaO2, AmDa03, MsDaOl, KsDaO2, MGYPDa829, PaDaO2, RaDaOl, BadTF3,HmDa01, HmDaO2, HmDa03, AmDaOl,Atty. Docket No. GH0213WO / 01228-0067-00PCTSjDaOl, MGYPDaOl, SqDaOl, TeDaOl, StsDaO3, SaDaO2, PpDaO2, EcDaO4, MGYPDaO2, MGYPDaO3, BcDaOl, IfDaOl, PcDaOl, StsDaO4,AmDaO4, AbDaO2, WWTPDaO5, WWTPDaO6, PeDaOl, SaDaO3, HgDaOl, AbcDaOl, HmDaO4, AmDaO2, AcDaOl, MGYPDal3, LbDaOl, CbDaOl, HcDaOl, MGYPDaO6, CseDaOl, AvDaOl, LbDaO2, MGYPDaO7, FbDaOl, IfDaO2, RsDaOl, NoDaO 1, PfDaOl, ScDaO3, PsDaOl, PvDaOl, CdDaOl, AzDaOl, BdDaOl, MGYPDaO8, MGYPDaO9, AoDaOl, MGYPDalO, MGYPDal l, MGYPDal2, MGYPDal4, SsdA, gp317, xpl2da, APOBEC3A, KcDaOl, TuDaOl, BsDaOl, PpDaOl, SaDaOl, CpDaOl, EcDaO3, ScDaOl, BpDaOl, ScDaO2, StsDaO2, OTT-1508, NpDaOl, BmDaOl, BsDaO2, PIDaOl, BbDaOl, OlDaOl, WcDaOl, BbDaO2, PrDaOl, VRDaO2, VRDaO3, VRDaO4, VRDaO5, VRDaO6, AbDaOl, AaDaOl, WWTPDaOl, WWTPDaO2, WWTPDaO3, SoCaDaOl, SoCaDa02, SoCaDa03, SoCaDa04, SoCaDa05, SoCaDa06, SoCaDa07, SoCaDa08, or SoCaDa09. In some embodiments, the deaminase (e.g., the methyl-sensitive deaminase or the methyl-insensitive deaminase) comprises a mutant deaminase or an alternatively truncated deaminase. In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of any one or more of the mutant deaminases or alternatively truncated deaminases of Table S8 of the Supplemental information of Vaisvila et al., Mol Cell. 2024 Mar 7;84(5):854-866.e7. doi: 10.1016 / j.molcel.2024.01.027. Epub 2024 Feb 22, available on www.ceH.cc- at / molecular- cell / fulltext / S 1097-2765(24)00094-7 (“Vaisvila 2024”): C_terminus_MGYPDa829_onto_StsDa01; Cdl l_MGYPDa829; d21_HcDa01; dl6_HmDaO2; dl8_MGYPDal7; MGYPDal7_extN16; MGYPDal8_extN28;PaDaO2_extN35; Cd4_PeDa01_extN21; Cd4_PeDa01; PeDa01_extN21; or RhDa01_extN10.

[0079] In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of C_terminus_MGYPDa829_onto_StsDa01 of Table S8 of the Supplemental information of Vaisvila 2024.

[0080] In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of Cdl l_MGYPDa829 of Table S8 of the Supplemental information of Vaisvila 2024.

[0081] In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of d21_HcDa01 of Table S8 of the Supplemental information of Vaisvila 2024.

[0082] In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of dl6_HmDaO2 of Table S8 of the Supplemental information of Vaisvila 2024.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0083] In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of dl8_MGYPDal7 of Table S8 of the Supplemental information of Vaisvila 2024.

[0084] In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of MGYPDal7_extN16 of Table S8 of the Supplemental information of Vaisvila 2024.

[0085] In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of MGYPDal8_extN28 of Table S8 of Table S8 of the Supplemental information of Vaisvila 2024.

[0086] In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of PaDaO2_extN35 of Table S8 of the Supplemental information of Vaisvila 2024.

[0087] In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of Cd4_PeDa01_extN21 of Table S8 of the Supplemental information of Vaisvila 2024.

[0088] In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of Cd4_PeDa01 of Table S8 of the Supplemental information of Vaisvila 2024.

[0089] In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of PeDa01_extN21 of Table S8 of the Supplemental information of Vaisvila 2024.

[0090] In some embodiments, the mutant deaminase or alternatively truncated deaminase comprises the amino acid sequence of RhDaOl extNIO of Table S8 of the Supplemental information of Vaisvila 2024.

[0091] In some embodiments, the methyl-sensitive deaminase comprises MsddA. In some embodiments, the methyl-sensitive deaminase comprises any one or more of MsddA, AshDaOl, MGYPDa21, PpDa03, SbDaOl, BlDaOl, PpDaO4, CsDaOl, MGYPDa22, FIDaOl, MGYPDa24, AaDaO2, MmgDaOl, PbDaOl, BcDaO2, LsfDaOl, SmgDaOl, XcDaOl, KsDaOl, PwDaOl, CaDaOl, SrDaOl, NgDaOl, NsDaOl, SzDaOl, SpDaOl, AdDaOl, MGYPDa23, WWTPDaO7, PdDaOl, MGYPDa25, MGYPDa26, DaDaOl, EcDaOl, EcDaO2, NgDaO2, PaDaOl, AsDaOl, HgmDaOl, MsDaO2, XinDaOl, XjaDaOl, RhDaOl, MGYPDaO4, MGYPDa05, BaDaOl, WWTPDaO4, PbDaO2, CrDaOl, MGYPDal5, MGYPDal6, MGYPDal7, BaDaO2, VsDaOl, MGYPDal8, MGYPDal9, HmDaO6, MmgDaO2, HgmDaO2, CgmDaOl, FbiDaOl, PvmDaOl, SjDaOl, HmDaO2, DddA, RaDaOl,Atty. Docket No. GH0213WO / 01228-0067-00PCTMGYPDa624, AvDaOl, CbDaOl, PfDaOl, NoDaO 1, or SsdA. In some embodiments, the amino acid sequence of the methyl -sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MsddA (Accession: MGYP001011792517; database: MGnify).

[0092] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of XcDaOl (Accession: B0RYS5; database: UniProt).

[0093] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of KsDaOl (Accession: E4NEH0; database: UniProt).

[0094] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of PwDaOl (Accession: UPI0001B0E918; database: UniParc).

[0095] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of CaDaOl (Accession: C7QJN3; database: UniProt).

[0096] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SrDaOl (Accession: D6Z8J1; database: UniProt).

[0097] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of NgDaOl (Accession: G3Z1X9; database: UniProt).

[0098] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of NsDaOl (Accession: D3A5B8; database: UniProt).

[0099] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SzDaOl (Accession: G2GKN7; database: UniProt).

[0100] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SpDaOl (Accession: D6X696; database: UniProt).

[0101] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of AdDaOl (Accession: WP 203730777.1; database: GenBank).Aty. Docket No. GH0213WO / 01228-0067-00PCT

[0102] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDa23 (Accession: MGYP000056170311; database: MGnify).

[0103] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of WWTPDaO7 (Accession: tig00001818-10-11279730_497; database: GenBank).

[0104] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of PdDaOl (Accession: JH605467.1; database: GenBank).

[0105] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDa25 (Accession: MGYP001677015708; database: MGnify).

[0106] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDa26 (Accession: MGYP003599500301; database: MGnify).

[0107] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of AshDaOl (Accession: A0A7H8HBT9; database: UniProt).

[0108] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDa21 (Accession: MGYP001278432191; database: MGnify).

[0109] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of PpDa03 (Accession: A0A3S0YAN6; database: UniProt).

[0110] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SbDaOl (Accession: A0A7G6KCF9; database: UniProt).Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0111] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of BlDaOl (Accession: A0A6P2WMK2; database: UniProt).

[0112] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of PpDaO4 (Accession: A0A5M9IK01; database: UniProt).

[0113] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of CsDaOl (Accession: A0A3E1NUV0; database: UniProt).

[0114] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDa22 (Accession: MGYP001462196871; database: MGnify).

[0115] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of FIDaOl (Accession: A0A0Q4RZG0; database: UniProt).

[0116] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDa24 (Accession: MGYP000620945751; database: MGnify)).

[0117] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of AaDaO2 (Accession: A0A2S8AG27; database: UniProt).

[0118] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MmgDaOl (Accession: JANFCG010307071; database: GenBank).

[0119] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of PbDaOl (Accession: MCL1918637.1; database: GenBank).

[0120] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of BcDaO2 (Accession: KVH32961.1; database: GenBank).Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0121] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of LsfDaOl (Accession: UHQ21442.1; database: GenBank).

[0122] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SmgDaOl (Accession: JAHZIM010172802.1; database: GenBank).

[0123] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of DaDaOl (Accession: tr|A0A2T4Z6L8|A0A2T4Z6L8_9BACL; database: UniProt).

[0124] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of EcDaOl (Accession: WP 001289064.1; database: GenBank).

[0125] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of EcDaO2 (Accession: ABG02915.1; database: GenBank).

[0126] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of NgDaO2 (Accession: WP_003703542.1; database: GenBank).

[0127] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of PaDaOl (Accession: WP 006660219.1; database: GenBank).

[0128] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of AsDaOl (Accession: WP 005802165.1; database: GenBank).

[0129] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of HgmDaOl (Accession: JAEZY0010000802.1; database: GenBank).

[0130] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MsDaO2 (Accession: tr|A0A2G2M3J8|A0A2G2M3J8_9GAMM; database: UniProt).Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0131] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of XinDaOl (Accession: tr|A0AlN6MQY7|A0AlN6MQY7_9GAMM; database: UniProt).

[0132] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of XjaDaOl (Accession: tr|A0AlI5CFXl |A0AH5CFXl_9GAMM; database: UniProt).

[0133] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of RhDaOl (Accession: tr|A0AlV4SQ45|A0AlV4SQ45_RUMHU; database: UniProt).

[0134] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDaO4 (Accession: MGYP001129217467; database: MGnify).

[0135] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDa05 (Accession: MGYP001094202578; database: MGnify).

[0136] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of BaDaOl (Accession: tr|A0A410KXF4|A0A410KXF4_9BACI; database: UniProt).

[0137] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of WWTPDaO4 (Accession: tig00000754- 10-8122160_273; database: GenBank).

[0138] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of PbDaO2 (Accession: A0A356KTR0; database: UniProt).

[0139] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity toAtty. Docket No. GH0213WO / 01228-0067-00PCT the amino acid sequence of CrDaOl (Accession: tr|AOAlS8L869|AOAlS8L869_9CLOT; database: UniProt).

[0140] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDal5 (Accession: MGYP001492404292; database: MGnify).

[0141] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDal6 (Accession: MGYP001076701719; database: MGnify).

[0142] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDal7 (Accession: MGYP001100966096; database: MGnify).

[0143] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of BaDaO2 (Accession: tr|A0A6B3W113|A0A6B3W113_9BACI; database: UniProt).

[0144] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of VsDaOl (Accession: tr|A0AlN6M042|A0AlN6M042_9VIBR; database: UniProt).

[0145] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDal8 (Accession: MGYP000969250293; database: MGnify).

[0146] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MGYPDal9 (Accession: MGYP000654575191; database: MGnify).

[0147] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of HmDaO6 (Accession: 3300043543_Ga0453689_0082580_8_1099; database: IMG / M hot metagenome bin).Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0148] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of MmgDaO2 (Accession: JANFCOO 10000966.1; database: GenBank).

[0149] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of HgmDaO2 (Accession: JAEWLH010000049.1; database: GenBank).

[0150] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of CgmDaOl (Accession: JAEEGH011632547; database: GenBank).

[0151] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of FbiDaOl (Accession: JAJXVD010000193.1; database:GenBank).

[0152] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of PvmDaOl (Accession: WFLX011519007; database: GenBank).

[0153] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SjDaOl (Accession: tr|A0A7G8SQM7|A0A7G8SQM7_9GAMM; database: UniProt).

[0154] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of HmDaO2 (Accession: 3300044995_Ga0484949_008810_2_589; database: IMG / M hot metagenome bin).

[0155] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of DddA (Accession: P0DUH5; database: UniProt).

[0156] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of RaDaOl (Accession: A0A373WC03; database: UniProt).

[0157] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity toAtty. Docket No. GH0213WO / 01228-0067-00PCT the amino acid sequence of MGYPDa624 (Accession: MGYP001011623624; database: MGnify).

[0158] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of AvDaOl (Accession: tr|A0A448PMLl|A0A448PMLl_ACTVI; database: UniProt).

[0159] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of CbDaOl (Accession: NLK69555.1; database: GenBank).

[0160] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of PfDaOl (Accession: tr|A0A0F4TG59|A0A0F4TG59_PSEFL; database: UniProt).

[0161] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence ofNoDaOl (Accession: WP 223985731.1; database: GenBank).

[0162] In some embodiments, the amino acid sequence of the methyl-sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SsdA (Accession: WP 046719483.1; database: GenBank).

[0163] In some embodiments, the methyl-sensitive deaminase comprises the APOBEC3 A protein containing a substitution mutation at Y130, such as Y130L, Y130W, or Y130H. In some embodiments, the methyl-sensitive deaminase comprises the APOBEC3 A protein (Accession: AKE33285.1; database: GenBank) containing a substitution mutation at Y132, such as Y132P or Y132H, e.g., in addition to a substitution at Y130, e.g., Y132H and either Y130V or Y130W. In some embodiments, the methyl -sensitive deaminase comprises the APOBEC3A protein containing a substitution mutation at Y130 and Y132. In some embodiments, the Y130 substitution mutation is Y130L. In some embodiments, the amino acid sequence of the methyl -sensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the APOBEC3 A protein (Accession: AKE33285.1; database: GenBank) and contains the Y130L mutation. In some embodiments, the Y130 substitution mutation is Y130W. In some embodiments, the Y132 substitution mutation is Y132P. In some embodiments, the methyl-sensitive deaminase has substitution mutations Y130V and Y132H substitution mutation. In some embodiments, the methylsensitive deaminase has substitution mutations Y130W and Y132H. In some embodiments,Atty. Docket No. GH0213WO / 01228-0067-00PCT the amino acid sequence of the methyl-sensitive deaminase has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of APOBEC3 A protein (Accession: AKE33285.1; database: GenBank) and contains the Y130L mutation. In some embodiments, the amino acid sequence of the methyl-sensitive deaminase has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of APOBEC3A protein (Accession: AKE33285.1; database: GenBank) and contains the Y130W mutation. In some embodiments, the amino acid sequence of the methyl-sensitive deaminase has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of APOBEC3A protein (Accession: AKE33285.1; database: GenBank) and contains the Y132P mutation. In some embodiments, the amino acid sequence of the methyl -sensitive deaminase has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of APOBEC3 A protein (Accession: AKE33285.1; database: GenBank) and contains the Y130V and Y132H mutations. In some embodiments, the amino acid sequence of the methyl-sensitive deaminase has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of APOBEC3A protein (Accession: AKE33285.1; database: GenBank) and contains the Y130W and Y132H.

[0164] In some embodiments, the conversion procedure comprises: reacting the nucleic acids with a variant methyltransferase having carboxymethyltransferase activity in the presence of carboxy-S-adenosyl-L-methionine (CxSAM) substrate, thereby labelling any unmethylated C and rendering it resistant to deaminase action, wherein 5hmC nucleic acid bases are protected from conversion through glucosylation; and contacting the nucleic acids of step (a) with a deaminase enzyme. In some embodiments, the variant methyltransferase having carboxymethylase activity is a recombinant M.Mpel N374K.

[0165] In some embodiments, the conversion procedure selectively converts the base pairing specificity of unmethylated cytosinl(C) in the nucleic acids. In some embodiments, the conversion procedure is bisulfite conversion. In some embodiments, the methylation status of the nucleic acids in the sample is determined by analyzing the base coverage of cytosines in a reference sequence.

[0166] The methods disclosed herein can comprise contacting DNA in a sample with a deaminase, thereby providing a converted sample in which unmethylated CpGs in the DNA are converted to UpGs. This step can be referred to as, or be included in, a conversion procedure, such as any of the conversion procedures described elsewhere herein. DNA in the converted sample that comprises methylated CpGs or conversion products of methylatedAtty. Docket No. GH0213WO / 01228-0067-00PCTCpGs is then selectively amplified. In some embodiments, the selectively amplified DNA is sequenced, and a level of methylation at one or more differentially methylated regions of the DNA is quantified. Such embodiments may also comprise a step of end-repair prior to the sequencing.

[0167] Table 1 summarizes exemplary methods of deamination with the type of modified bases detectable with these methods. These are described in more detail below.Table Error! Unknown switch argument. - Exemplary deamination methods

[0168] As outlined below, there are various methods of detecting and / or identifying modified nucleosides that rely on a conversion procedure that changes the base-pairing specificity of a nucleoside, based on the modification status of the nucleosides. These changes of base-pairing specificity can then be detected, and thus the modification status of the nucleoside inferred, by sequencing.

[0169] In some embodiments, the conversion procedure used in the methods of the disclosure is one that changes the base pairing specificity of an unmodified nucleoside (e.g. cytosine), but does not change the base pairing specificity of the corresponding modified nucleoside (e.g. methylated cytosine, such as 5hmC and / or 5mC) or does not change the base pairing specificity of any modified nucleoside (e.g. modified cytosine, adenosine, guanosine and thymidine (or uracil)). In methods that require denaturation for conversion, failure to denature a DNA molecule will result in non-conversion of all bases in the DNA molecule. As biological changes in methylation are predominantly concerted to a localized regions of interest, these non-random (localized) non-conversion events can appear as false negatives (non-methylated regions). Random non-conversion methods can maximally affect a low percent of bases within a region, and thus the specificity of methylation change detection can be maximized (reduce false positives) by placing a threshold on percentage of bases within a region that are methylated / non-methylated. Hence, in some cases, a conversion procedure that does not involve denaturation is preferred.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0170] The skilled person can select a suitable method according to their needs, incllg which nucleoside modifications are to be detected and / or identified and which type of modified base is used in the end repair reaction.

[0171] The methods described herein could in principle use any suitable enzymatic conversion procedure that changes the base-pairing specificity of the modified cytosine and thereby allows the modified base to be distinguished from the corresponding unmodified cytosine and / or other types of modification when sequenced. For example, any enzymatic conversion procedure could be used allowing 5-methylcytosine (m5c or 5-mC or 5mC) to be distinguished from unmodified cytosine. In particular embodiments, the conversion procedure converts unmodified (e.g., unmethylated) cytosines to uracils using a deaminase.

[0172] In some embodiments, the conversion procedure which converts unmodified cytosines comprises APOBEC-coupled epigenetic (ACE) conversion. In ACE conversion, an AID / APOBEC family DNA deaminase enzyme such as APOBEC3 A (A3 A) is used to deaminate an unmodified cytosine and 5mC without deaminating 5hmC, 5fC, or 5-caC. Sequencing of ACE-converted DNA identifies positions that are read as cytosine as being 5hmC, 5fC, or 5-caC positions. Meanwhile, positions that are read as T are identified as being T, unmodified C, or 5mC. Performing ACE conversion as described herein thus facilitates distinguishing positions containing 5hmC from positions containing 5mC or unmodified C using the sequence reads obtained from the first subsample. In some embodiments, the end repair reaction can be performed with dNTPs, wherein at least one type of dNTP comprises a 5hmC, and regions synthesized during the end repair reaction can be identified as those regions comprising 5hmC (via C being called at these positions) at non- CpG positions. For an exemplary description of ACE conversion, see, e.g., Schutsky et al., Nature Biotechnology 2018; 36: 1083-1090.

[0173] In some embodiments, the conversion procedure comprises enzymatic conversion of a nucleobase, 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 / 2019.12.20.884692vl. For example, TET2 and T4-PGT or 5-hydroxymethylcytosine carbamoyltransferase (described in Yang et al., Bio-protocol, 2023; 12(17): e4496) can be used to convert 5mC and 5hmC into substrates 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.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0174] In some embodiments, the conversion procedure comprises enzymatic conversion of the a nucleobase using a non-specific, modification-sensitive double-stranded DNA deaminase, e.g., as in SEM-seq. See, e.g., Vaisvila et al., Mol Cell. 2024 Mar 7;84(5):854- 866. e7. doi: 10.1016 / j.molcel.2024.01.027. Epub 2024 Feb 22, available on www.ceH.com at / molecular-cell / fulltext / S1097-2765(24)00094-7 (“Vaisvila 2024”). SEM-Seq employs a nonspecific, modification-sensitive double-stranded DNA deaminase (MsddA) in a nondestructive single-enzyme 5-methylctyosine sequencing (SEM-seq) method that deaminates unmodified cytosines. Accordingly, SEM-seq does not require the TET2 and T4- PGT or 5 -hydroxymethyl cytosine carbamoyltransferase 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 (5-caC). In SEM-seq, unmodified cytosines in the DNA are deaminated to uracil and is read as “T” during sequencing. Modified cytosines (e.g., 5mC) are not converted and are read as “C” during sequencing. Cytosines that are read as thymines are identified as unmodified (e.g., unmethylated) cytosines or as thymines in the DNA. Performing SEM-seq conversion thus facilitates identifying positions containing 5mC using the sequence reads obtained. In some embodiments, the conversion procedure comprises enzymatic conversion of unmodified cytosine using MsddA or a modification-sensitive DNA deaminase A (MsddA)-like deaminase. For an exemplary description of MsddA and MsddA-like deaminases, see, e.g., Vaisvila et al., Mol Cell. 2024 Mar 7;84(5):854-866.e7. doi: 10.1016 / j.molcel.2024.01.027. Epub 2024 Feb 22, available on www;ceiLcom at / molecular-cell / fulltext / S1097- 2765(24)00094-7 (“Vaisvila 2024”), 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 2024, such as analysis of deamination of C in E. coli or lambda dem- DNA, deamination of 5mC in XP12 phage DNA, deamination of 5hmC in a C-hydroxymethylated adenovirus PCR fragment or fully C-hydroxymethylated T4147 phage DNA, and deamination of 5gmC in alpha-glucosyltransferase knockout (AGT-) T4 phage DNA). Deamination can be performed by contacting substrate DNA with deaminase and analyzed using NGS as follows: 50 ng of unmodified E. coli C2566 genomic DNA can be combined with the control DNAs (about 1 ng of Lambda, XP12, and T4147, and 0.1 ng of the 5hmC Adenovirus PCR fragment), sheared to about 300 bp and ligated to pyrrolo-dC adapters with 1 uL of in vitro synthesized deaminase (e.g., synthesized using the PURExpress In VitroAtty. Docket No. GH0213WO / 01228-0067-00PCTProtein Synthesis kit (NEB, Ipswich, MA) following manufacturer’s recommendations with 100-400 ng of PCR fragment template DNA containing codon-optimized deaminase coding sequence and T7 promoter and terminator). Exemplary deamination reaction conditions are 50 mM Bis-Tris pH 6.0, 0.1% Triton X-100 for 1 hour at 37 degrees C. After the deamination reaction, 1 uL of Thermolabile Proteinase K (NEB, Ipswich, MA) can be added and incubated for 30 min at 37 degrees C and then the Proteinase K can be heat inactivated at 60 degrees C for 10 minutes. The deaminated product can then be used for library amplification using the NEBNext Q5U Master Mix (New England Biolabs, Ipswich, MA, USA) with 5mMof NEBNext Unique Dual Index Primers. The resulting library can be purified using IX NEBNext Sample Purification Beads according to the manufacturer’s instructions and the purified library can be analyzed and 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.

[0175] Exemplary cytosine deaminases for use herein include APOBEC enzymes, for example, APOBEC3 A. Generally, AID / APOBEC family DNA deaminase enzymes such as APOBEC3A (A3 A) are used to deaminate (unprotected) unmodified cytosine and 5mC. For an exemplary description of APOBEC enzymes, see, e.g., Gajula et al., Nucleic Acids Res. 2014 Sep;42(15):9964-75 and Schutsky etal., Nucleic Acids Res. 2017 Jul 27;45(13):7655- 7665. For an exemplary description of APOBEC conversion, see, e.g., Schutsky et al., Nature Biotechnology 2018; 36: 1083-1090.

[0176] Optionally, the conversion procedure further includes enzymatic protection of 5hmCs, such as by glucosylation of the 5hmCs (e.g., using PGT) or by carbamoylation of the 5hmCs (e.g., using 5-hydroxymethylcytosine carbamoyltransferase), in the DNA prior to the deamination of unprotected modified cytosines. In this method, 5hmC can be protected from conversion, for example through glucosylation using P-glucosyl transferase (PGT), forming (5-glucosylhydroxymethylcytosine) 5ghmC, or through carbamoylation using 5- hydroxymethylcytosine carbamoyltransferase, forming 5cmC. This is described, for example, in Yu et al., Cell 2012; 149: 1368-80, and in Yang et al., Bio-protocol, 2023; 12(17): e4496. Glucosylation or carbamoylation of 5hmC can reduce or eliminate deamination of 5hmC by a deaminase such as APOBEC3A.

[0177] As discussed above, in some embodiments, a TET protein can be used to convert 5mC and optionally 5hmC (but not unmodified C) into substrates (e.g., 5caC) that cannot beAtty. Docket No. GH0213WO / 01228-0067-00PCT deaminated by a deaminase, and then a deaminase (e.g., AP0BEC3A) 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 VI 900 TET mutant, such as a VI 900 A, V1900C, V1900G, VI 9001, or V1900P TEI 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. 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 APOBEC3 A. Maximizing formation of 5-caC thus reduces the risk of false calls in which a base is identified as unmethylated because it underwent deamination even though it was methylated (or hydroxymethylated) in the original sample. Accordingly, in some embodiments, the TET enzyme comprises a mutation that increases formation of 5-caC. Exemplary mutations are set forth above. “A mutation that increases formation of 5-caC” means that the TET enzyme having the mutation produces more 5-caC than a TET enzyme that lacks the mutation but is otherwise identical. 5-caC production can be measured as described, e.g., in Liu et al., Nat Chem Biol 13: 181-187 (2017) (see Online Methods section, TET reactions in vitro subsection, “driving” conditions). Any variants and / or mutants described in Liu et al. (2017) can be used in the disclosed methods as appropriate.Ligation to Adapters

[0178] In some embodiments, the DNA comprises adapters. In some embodiments, the DNA comprises barcodes. In some embodiments, the methods comprise ligating adapters comprising barcodes to the DNA prior to the amplifying. In some embodiments, the adaptersAtty. Docket No. GH0213WO / 01228-0067-00PCT are Y-shaped adapter. In some embodiments, the method comprises ligating adapters to the DNA after cutting the DNA.

[0179] 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. In some embodiments, once the DNA has been end- repaired it can be subjected to blunt-end ligation with blunt-ended adapters, in cases where A-tailing is not performed, or sticky end ligation with T-tailed adapters, when A tailing is performed. 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). In some embodiments, the ligation step can take place before or after the conversion step. In general, “conversion step” or “conversion procedure” refers to any step or procedure that changes the base pairing specificity of one or more nucleotides.

[0180] In some embodiments, the conversion step comprises contacting DNA (e.g., DNA in a sample) with a deaminase. In some embodiments, the method comprises ligating adapters comprising barcodes to the DNA prior to the contacting the DNA with the deaminase. In some embodiments, the conversion step of contacting DNA (e.g., DNA in a sample) with a deaminase provides a converted sample in which unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, the ligation step is performed after the conversion step. In some embodiments, the ligation step occurs before contacting the DNA with a deaminase. In some embodiments, the ligation step occurs before selectively amplifying the DNA in the converted sample. In some embodiments, the ligation step occurs after contacting the DNA with a deaminase and before selectively amplifying the DNA in the converted sample. In some embodiments, adapters are ligated to end-repaired DNA molecules or the adapters are ligated to the DNA molecule or a plurality of DNA molecules. In some such embodiments, the ligation reaction also seals nicks present in the end-repaired DNA.

[0181] 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.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0182] In some embodiments, the ligase used in ligation reactions can act on both single strand DNA nicks and double stranded DNA ends. In some cases, the ligase is T4 DNA ligase or T3 DNA ligase. Adapters can include nucleic acid primer binding sites to permit amplification of a sample DNA molecule flanked by adapters at both ends, and / or a sequencing primer binding site, including primer binding sites for sequencing applications, such as various next generation sequencing (NGS) applications. Adapters can include a sequence for hybridizing to a solid support, e.g., a flow cell sequence. Adapters can also include binding sites for capture probes, such as an oligonucleotide attached to a flow cell support or the like. Adapters can also include sample indexes and / or molecular barcodes. These are typically positioned relative to amplification primer and sequencing primer binding sites, such that the sample index and / or molecular barcode is included in amplicons and sequencing reads of a given DNA molecule. Adapters of the same or different sequence can be linked to the respective ends of a sample DNA molecule. In some cases, adapters of the same or different sequence are linked to the respective ends of the DNA molecule except that the sample index and / or molecular barcode differs in its sequence. In some embodiments, the adapter is a Y-shaped adapter in which one end is blunt ended or tailed as described herein, for joining to a nucleic acid molecule, which is also blunt ended or tailed with one or more complementary nucleotides to those in the tail of the adapter. In another exemplary embodiment, an adapter is a bell-shaped adapter that includes a blunt or tailed end for joining to a DNA molecule to be analyzed. Other exemplary adapters include T-tailed, C-tailed or hairpin shaped adapters. For example, a hairpin shaped adaptor 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 adaptors 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 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.

[0183] 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 or DNA in a converted sample prior toAtty. Docket No. GH0213WO / 01228-0067-00PCT amplification. In some embodiments, adapters are ligated to the DNA in the sample prior to contacting the DNA with a deaminase. In some embodiments, adapters are ligated to the DNA in a sample or DNA in a converted sample prior to cutting DNA (e.g., DNA comprising an AP site) using an AP lyase. 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.

[0184] 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.

[0185] 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 is used, this can be referred to as library prep-PCR or LP-PCR), before, or after an amplification step. In some embodiments, adapters are added by other approaches, such as ligation. In some such methods, first adapters are added to the 3’ ends of the nucleic acids by ligation, which may include ligation to single-stranded DNA. In some embodiments, prior to any partitioning or capturing steps, first adapters are added to the nucleic acids by ligation, which may include ligation to single-stranded DNA (e.g., to the 3’ ends thereof). In some embodiments, the capture probes can be isolated after partitioning and ligation. For example, the hypomethylated partition can be ligated with adapters and a portion of the ligated hypomethylated partition can then be used to generate the capture probes for rearrangements. The adapter can be used as a priming site for second-strand synthesis, e.g., using a universal primer and a DNA polymerase. A second adapter can then be ligated to at least the 3’ end of the second strand of the now double-stranded molecule. In some embodiments, the firstAtty. Docket No. GH0213WO / 01228-0067-00PCT 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.

[0186] In some embodiments, the single-stranded DNA library preparation is performed in a one-step combined phosphorylation / ligation reaction, e.g., as described in Troll et al., BMC Genomics, 20: 1023 (2019), available at https: / / doi.org / 10.1186 / sl2864-019-6355-0. This method, 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.

[0187] 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 random nucleotides of the splint adapter can be annealed to the single-stranded templateAtty. Docket No. GH0213WO / 01228-0067-00PCT 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.

[0188] 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.

[0189] 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.

[0190] In some embodiments, following attachment of adapters, the DNA or a sub sample 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.

[0191] 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.Molecular Tagging

[0192] In some embodiments, the DNA molecules of the sample may be tagged with sample indexes and / or molecular barcodes (referred to generally as “tags”), which may be present in the adapters. In some embodiments, the DNA molecules of the sample comprise barcodes, e.g., in the adapters. In some embodiments, the DNA comprises barcodes. Tags can be molecules, such as nucleic acids, containing information that indicates a feature of the molecule with which the tag is associated; tags, such as tags contained in adapters, can beAtty. Docket No. GH0213WO / 01228-0067-00PCT attached to sample DNA molecules, e.g., by ligation, such as during library preparation. For example, DNA molecules can bear a sample tag or sample index (which distinguishes molecules in one sample from those in a different sample), a partition tag (which distinguishes molecules in one partition from those in a different partition) and / or a molecular tag / molecular barcode (which distinguishes different molecules from one another (in both unique and non-unique tagging scenarios).

[0193] Tagging strategies can be divided into unique tagging and non-unique tagging strategies. In unique tagging, all or substantially all of the molecules in a sample bear a different tag, so that reads can be assigned to original molecules based on tag information alone. Tags used in such methods are sometimes referred to as “unique tags”. In non-unique tagging, different molecules in the same sample can bear the same tag, so that other information in addition to tag information is used to assign a sequence read to an original molecule. Such information may include start and stop coordinate, coordinate to which the molecule maps, start or stop coordinate alone, etc. Tags used in such methods are sometimes referred to as “non-unique tags”. Accordingly, it is not necessary to uniquely tag every molecule in a sample. It suffices to uniquely tag molecules falling within an identifiable class within a sample. Thus, molecules in different identifiable families can bear the same tag without loss of information about the identity of the tagged molecule.

[0194] In certain embodiments, a tag can comprise one or a combination of barcodes. As used herein, the term “barcode” refers to a nucleic acid molecule having a particular nucleotide sequence, or to the nucleotide sequence, itself, depending on context. A barcode can have, for example, between 10 and 100 nucleotides. A collection of barcodes can have degenerate sequences or can have sequences having a certain Hamming distance, as desired for the specific purpose. So, for example, a molecular barcode can be comprised of one barcode or a combination of two barcodes, each attached to different ends of a molecule. Additionally or alternatively, for different partitions and / or samples, different sets of molecular barcodes, molecular tags, or molecular indexes can be used such that the barcodes serve as a molecular tag through their individual sequences and also serve to identify the partition and / or sample to which they correspond based the set of which they are a member.

[0195] 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 beAtty. Docket No. GH0213WO / 01228-0067-00PCT incorporated into or otherwise joined to adapters. Tags can be incorporated by ligation, overlap extension PCR among other methods. Tags can be used to label the individual polynucleotide population partitions so as to correlate the tag (or tags) with a specific partition. Alternatively, tags can be used in embodiments of the disclosure that do not employ a partitioning step. In some embodiments, a single tag can be used to label a specific partition. In some embodiments, multiple different tags can be used to label a specific partition. In embodiments employing multiple different tags to label a specific partition, the set of tags used to label one partition can be readily differentiated for the set of tags used to label other partitions. In some embodiments, the tags may have additional functions, for example the tags can be used to index sample sources or used as unique molecular identifiers (which can be used to improve the quality of sequencing data by differentiating sequencing errors from mutations, for example as in Kinde et al., Proc Nat’ 1 Acad Sci USA 108: 9530- 9535 (2011), Kou et al., PLoS ONE, 11 : e0146638 (2016)) or used as non-unique molecule identifiers, for example as described in US Pat. No. 9,598,731. Similarly, in some embodiments, the tags may have additional functions, for example the tags can be used to index sample sources or used as non-unique molecular identifiers (which can be used to improve the quality of sequencing data by differentiating sequencing errors from mutations).

[0196] 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 sample DNA molecule. In other embodiments, one or more rounds of amplification cycles (e.g., PCR amplification) may be applied to introduce sample indexes to a nucleic acid molecule using conventional nucleic acid amplification methods. The amplifications may be conducted in one or more reaction mixtures (e.g., a plurality of microwells in an array). Molecular barcodes and / or sample indexes may be introduced simultaneously, or in any sequential order. In some embodiments, molecular barcodes and / or sample indexes are introduced prior to and / or after any conversion procedure. In the case of molecular barcodes and / or sample indexes being introduced through amplification processes, the conversion step will occur before the molecular barcodes and / or sample indexes are introduced. In some embodiments, molecular barcodes and / or sample indexes are introduced prior to and / or after sequence capturing steps, if present, are performed. In some embodiments, only the molecular barcodes are introduced prior to probe capturing and the sample indexes are introduced after sequence capturing steps are performed. In some embodiments, both the molecular barcodes and the sample indexes are introduced prior toAtty. Docket No. GH0213WO / 01228-0067-00PCT performing probe-based 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).

[0197] In some embodiments, the tags may be located at one end or at both ends of the sample DNA molecule. In some embodiments, tags are predetermined or random or semirandom sequence oligonucleotides. 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 DNA molecules randomly or non-randomly.

[0198] In some embodiments, each sample or partition (discussed below) is uniquely tagged with a sample index or a combination of sample indexes. In some embodiments, each nucleic acid molecule of a sample or sub-sample is uniquely tagged with a molecular barcode or a combination of molecular barcodes. In other embodiments, a plurality of molecular barcodes may be used such that molecular barcodes are not necessarily unique to one another in the plurality (e.g., non-unique molecular barcodes). In these embodiments, molecular barcodes are generally attached (e.g., by ligation as part of an adapter) to individual molecules such that the combination of the molecular barcode and the sequence it may be attached to creates a unique sequence that may be individually tracked. Detection of non-unique molecular barcodes in combination with endogenous sequence information (e.g., the beginning (start) and / or end (stop) genomic location / position corresponding to the sequence of the original DNA molecule in the sample, start and stop genomic positions corresponding to the sequence of the original DNA 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, subsequences of sequence reads at one or both ends, length of sequence reads, and / or length of the original DNA molecule in the sample) typically allows for the assignment of a unique identity to a particular molecule. In some embodiments, beginning region comprises the first 1, first 2, the first 5, the first 10, the first 15, the first 20, the first 25, the first 30 or at least the first 30 base positions at the 5' end of the sequencing read that align to the reference sequence. In some embodiments, the end region comprises the last 1, last 2, the last 5, the last 10, the last 15, the last 20, the last 25, the last 30 or at least the last 30 base positions at the 3' end of the sequencing read that align to the reference sequence. The length, or number of base pairs, of an individual sequence read are also optionally used to assign a unique identityAtty. Docket No. GH0213WO / 01228-0067-00PCT 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.

[0199] 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). 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).

[0200] In some embodiments, molecular barcodes are introduced at an expected ratio of a set of identifiers (e.g., a combination of unique or non-unique molecular barcodes) to molecules in a sample. One example format uses from about 2 to about 1,000,000 different molecular barcode sequences, or from about 5 to about 150 different molecular barcode sequences, or from about 20 to about 50 different molecular barcode sequences, ligated to both ends of a target molecule. Alternatively, from about 25 to about 1,000,000 different molecular barcode sequences may be used. For example, 20-50 x 20-50 molecular barcode sequences (i.e., one of the 20-50 different molecular barcode sequences can be attached to each end of the target molecule) can be used. Such numbers of identifiers are typically sufficient for different molecules having the same start and stop points to have a high probability (e.g., at least 94%, 99.5%, 99.99%, or 99.999%) of receiving different combinations of identifiers. In some embodiments, about 80%, about 90%, about 95%, or about 99% of molecules have the same combinations of molecular barcodes. For example, in a sample of about 5 ng to 30 ng of cell free DNA, one expects around 3000 molecules to map to a particular nucleotide coordinate, and between about 3 and 10 molecules having any start coordinate to share the same stop coordinate. Accordingly, about 50 to about 50,000 different tags (e.g., between about 6 and 220 barcode combinations) can suffice to uniquely tag all such molecules. To uniquely tag allAtty. Docket No. GH0213WO / 01228-0067-00PCT3000 molecules mapping across a nucleotide coordinate, about 1 million to about 20 million different tags would be required.

[0201] In some embodiments, the assignment of unique or non-unique molecular barcodes in reactions is performed using methods and systems described for example, U.S. Patent Application Nos. 20010053519, 20030152490, and 20110160078, and U.S. Patent Nos. 6,582,908, 7,537,898, 9,598,731, and 9,902,992, each of which is 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. Tags can be linked to sample nucleic acids randomly or non-randomly.

[0202] In some embodiments, the assignment of unique molecular barcodes in reactions is performed using methods and systems described in Lim et al., Communications Biology . (2025)8: 1098, e.g., SPIDER-seq. In some such embodiments, amplicons are tagged with a pair of two unique molecular barcodes using primers that contain a barcode. Successive daughter strands synthesized through each round of PCR amplification are grouped into clusters (e.g., peer-to peer networks, as illustrated in Fig. 1c of Lim et al.) based on a chain of common unique barcodes between immediate parent and daughter strands. That is, strand synthesis events (with a synthesized strand as a template) involve copying one barcode from the template and include one new barcode from the primer, so each daughter strand shares a unique barcode with its parent. By clustering strands in this way, a consensus can be generated that reduces errors.

[0203] 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, 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.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0204] 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.

[0205] 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.

[0206] 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) 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 anAtty. Docket No. GH0213WO / 01228-0067-00PCT 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.End repair and A-tailing

[0207] In some embodiments, the disclosed methods comprise subjecting the DNA in the sample to end repair to generate end-repaired DNA molecules. In some embodiments, the disclosed methods comprise prior to the conversion procedure subjecting the DNA in the sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using deoxynucleotide triphosphates (dNTPs), wherein at least one type of dNTP comprises a modified base, and the at least one dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations. In some embodiments, the end repair is performed before contacting the DNA in the sample with a deaminase. In some embodiments, the end repair is performed using deoxynucleotide triphosphates (dNTPs). In some embodiments, at least one type of dNTP comprises a modified base, and the at least one dNTP comprising a modified base is incorporated into repaired regions of the end-repaired DNA molecules at one or more locations.

[0208] End repair refers to methods for repairing DNA by the conversion of non-blunt ended DNA into blunt ended DNA. Sequencing workflows typically use end repair to make ends of DNA molecules compatible with adapters, which are subsequently ligated onto the DNA. Fragmented and / or damaged DNA (e.g. cfDNA or DNA from FFPE samples) often contain non-blunt ends, which contain 3’overhangs and / or 5’overhangs. A 3’overhang refers to the 3’ end of a DNA strand which extends beyond the 5 ’end of the paired strand, resulting in one or more unpaired nucleotides at the 3 ’end of the DNA strand. Conversely, a 5 ’overhang refers to the 5’ end of a DNA strand which extends beyond the 3 ’end of the paired strand, resulting in one or more unpaired nucleotides at the 5 ’end of the DNA strand.

[0209] In some embodiments, the end repair is performed using a DNA polymerase that does not have 5 ’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase. In some embodiments, the end repair is performed using a DNA polymerase that has 5 ’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0210] The process of end repair involves the conversion of double-stranded DNA with 3 ’overhangs and / or 5 ’overhangs to double-stranded DNA without overhangs. This can be done using one or more enzymes such as T4 DNA polymerase and / or Klenow fragment. The 3’ to 5’ exonuclease activity of these enzymes removes the 3 ’ends at 3 ’overhangs and the 5’ to 3’ polymerase activity of these enzymes extends the 3’ ends at 5’ overhangs to remove the 5’ overhang, thereby generating a blunt-ended DNA molecule. In order to fill in these 5’ overhangs, end repair is conducted in the presence of dATP, dCTP, dGTP and dTTP. End repair can also include a second step, which involves the addition of a phosphate group to the 5' ends of DNA, by an enzyme such as polynucleotide kinase. This makes the 5’ends of the end-repaired DNA molecules compatible with the subsequent action of DNA polymerases and DNA ligases.

[0211] In some embodiments, the methods disclosed herein further comprise performing an A-tailing reaction, optionally after a step of subjecting a DNA sample to end repair. As used herein, the term “A-tailing” refers to the addition of a single deoxyadenosine residue to the end of a blunt-ended double-stranded DNA fragment to form a 3' deoxy adenosine single-base overhang. Such A tailing reactions are conducted with polymerases which have the ability to add a non-templated’ A to the 3' end of a blunt, double-stranded DNA molecule. Polymerases capable of A-tailing typically do not possess 3 ’-5’ exonuclease activity. When A-tailing is performed as a separate reaction to end repair, it is typically conducted in the presence of dATP, but the absence of dCTP, dTTP and dGTP. A-tailed fragments are not compatible for self-ligation (i.e., self-circularizatian and concantenation of the DNA), but they are compat’ble with 3' T-overhangs, which can be used on adapters. Methods comprising end repair, A-tailing and ligation to adap’ers with 3' T-overhangs can result in higher efficiency ligation, compared to blunt ended ligation, as blunt ligation can lead to self-ligation of the adapters and / or DNA molecules.

[0212] In some embodiments, the end-repair and the A-tailing reaction are performed in the same reaction mixture, optionally wherein the end-repair and the A-tailing reaction are performed a single tube and / or optionally wherein the end-repair and the A-tailing reaction are performed without an intervening clean-up step. In some embodiments, the A-tailing is performed using a DNA polymerase that does not possess 5 ’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase, optionally wherein the DNA polymerase is HemoKlen Taq. In some embodiments, the A-tailing is performed using a thermostable DNA polymerase.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0213] In some embodiments, the methods disclosed herein comprise end repair of the DNA molecules followed by blunt end ligation of adapters. In other embodiments, the methods disclosed herein comprise end repair of the DNA molecules followed by A-tailing and sticky- end ligation of T-tailed adapters. When the methods disclosed herein comprise an A-tailing step, it may be performed separately from the end repair with an intervening reaction cleanup step or it may be performed in the same reaction as the end repair (e.g. using NEBNext® Ultra™ II End Repair / dA-Tailing Module (E7546)). In some embodiments, the reaction clean-up step removes unincorporated dNTPs. In instances wherein the A-tailing reaction is performed in the same reaction as end repair, the sticky-end ligation may be performed with a mixture of T-tailed adapters and C-tailed adapters.

[0214] End repair and A tailing reactions can have varying impacts on the composition of the DNA molecule, dependent on the exact workflow and reaction components used. These reactions can lead to the synthesis of regions at the 3 ’ends of DNA strands, but also the synthesis of internal regions through nick translation and through gap filling followed by ligation.

[0215] In some embodiments, end repair can lead to 3 ’fill in with unmethylated cytosines, which may not reflect the true methylation status of that position in the DNA molecule prior to the generation of the 5 ’overhang. In nicked DNA, polymerases which contain 5’ to 3’ exonuclease activity and / or strand displacement activity can lead to the synthesis of internal regions of the DNA molecule through nick translation. If the end repair reaction is conducted with non-methylated deoxy cytidine triphosphate (dCTP), the synthesized regions will incorporate the non-methylated dCTP, potentially at positions which initially comprised methylated cytosines. In gapped DNA, both the DNA polymerases used in end repair and A tailing can lead to the generation of synthesized regions. The gaps can be filled in with DNA polymerases used in the end repair reaction, regardless of whether they possess 5’ to 3’ exonuclease activity or strand displacement activity. After this gap filling, a nick will still exist between the synthesized region and the region of the original DNA molecule 3’ of the gap. The A-tailing enzymes may then introduce further synthesized regions through nick translation, as described for the nicked DNA. This synthesized region may extend to the 3 ’end of the DNA molecule.

[0216] In some embodiments, the end-repair and the A-tailing reactions are performed in a single tube. In such cases, the A tailing reaction can be performed at a higher temperature than the end repair. Optionally, end repair is performed at ambient temperature (e.g. 15-35°C) and A tailing is performed at a temperature over 60°C, including e.g., about 60°C-75°C. TheAtty. Docket No. GH0213WO / 01228-0067-00PCTA tailing reaction can be performed using a thermostable polymerase (e.g. Taq DNA polymerase, Tfl DNA polymerase, Bst DNA Polymerase, Large Fragment or Tth DNA polymerase) and the method further comprises increasing temperature of the sample after the end repair to inactivate the polymerase used in end repair (e.g. T4 DNA polymerase or Klenow fragment). In some embodiments the A-tailing is performed using a DNA polymerase that: (i) does not possess 5 ’-3’ exonuclease activity; and / or (ii) is not a strand displacing DNA polymerase. These properties reduce the ability of the DNA polymerase to extend from nick. This reduces the level of synthesis which may occur during the end repair and A-tailing reactions thus reducing the proportion of sequencing data that may be filtered out as potentially containing artifactual data. Accordingly, in some embodiments, the A- tailing is performed using a DNA polymerase that cannot extend from a nick in the DNA such as HemoKlen Taq. In other embodiments, the A-tailing is performed using Taq DNA polymerase. In other embodiments, the A-tailing is performed using Tfl polymerase, Bst DNA Polymerase, Large Fragment or Tth polymerase.

[0217] In order to reduce the level of synthesized regions, the end repair reaction can be performed using DNA polymerases can be used which lack 5 ’to 3’ exonuclease activity and / or strand displacement activity (e.g. T4 DNA polymerase or Klenow fragment).

[0218] In nicked DNA and gapped DNA, nick translation is reduced in end repair through the use of polymerases which lack 5 ’to 3’ exonuclease activity and / or strand displacement activity. The separation of the end repair and A tailing reaction by a reaction clean-up means that only dATP (not dCTP, dTTP or dGTP) is present in the A tailing reaction. This means that efficient nick translation cannot occur in the A tailing reaction because the three of the four nucleotide components are not present in the reaction mixture. In gapped DNA, the gaps can be filled in with DNA polymerases used in the end repair reaction, regardless of whether they possess 5’ to 3’ exonuclease activity or strand displacement activity. These filled gaps thereby generate synthesized regions.

[0219] In some embodiments, the end-repair is performed with a polymerase which lacks 5 ’to 3’ exonuclease activity and / or strand displacement activity. In some cases, the polymerase used in the end repair reaction may be Q5® High-Fidelity DNA Polymerase, Q5U® Hot Start High-Fidelity DNA Polymerase, Phusion® High-Fidelity DNA Polymerase, Hemo Klen 7bt / , phi29 DNA Polymerase, T7 DNA Polymerase, DNA Polymerase I (E. coli), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”) or T4 DNA Polymerase. In some embodiments, the polymerase used in the end repair is T4 DNA Polymerase or Klenow fragment. In some embodiments, the end repair is performed with a DNAAtty. Docket No. GH0213WO / 01228-0067-00PCT polymerase which has 5 ’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

[0220] In some embodiments, the methods disclosed herein comprise an A tailing reaction after the end repair and before the ligation reaction, wherein the end repair and A tailing reactions are separated by a reaction cleanup. The A tailing reaction is typically performed in the presence of dATP, but in the absence of dCTP, dTTP and dGTP. In some embodiments, the A tailing reaction is performed using Klenow Fragment’ lacking 3'-5' exonuclease activity.

[0221] In some embodiments, a dNTP that comprises a modified base is used in end repair, which may be any modified base wherein the presence or the absence of the modification can be detected by a type of sequencing. In some embodiments, a dNTP comprising a modified base can be used in a combined end repair and A-tailing reaction. In some embodiments, the modified base is incorporated in the synthesized regions at both CpG sites and CpH sites (i.e. CpA, CpC and CpT sites). While methylation of cytosines in non-CpG contexts has been described, it is thought to comprise 0.02% of total methyl -cytosine in differentiated somatic cells (Jang et al. Genes (Basel). 2017 Jun; 8(6): 148). The presence of methylated cytosine in non-CpG contexts in the end-repaired DNA can therefore be interpreted as being introduced during the end repair and / or A-tailing reactions. Using a dNTP comprising a modified base can therefore be used to effectively label the synthesized regions of the end repaired DNA.

[0222] In some embodiments, a dNTP that comprises a modified base may comprise any modified base wherein the presence or the absence of the modification can be detected by a type of sequencing. The modified base may be 5-caryboxylcytosine (5-caC), 4- methylcytosine (4mC), 5 -methylcytosine (5mC), 5 -hydroxymethyl -cytosine (5hmC), N6- methyladenosine (6mA), bromodeoxyuridine (BrdU), 5-fluorodeoxyuridine (FldU), 5- iododeoxyuridine (IdU), 5-ethynyldeoxyuridine (EdU) and / or 8-oxoguanine (8oxoG).

[0223] When a dNTP comprising a modified base is used, it may be used in place of the equivalent unmodified base in the end repair reaction. For instance, if a dCTP comprising 5mC is used in the end repair reaction, there may be no dCTP comprising an unmodified cytosine. This would ensure that dCTPs incorporated into the DNA molecule during the end repair reaction contain 5mC. In some embodiments, multiple types of dNTP comprising a modified base are used in the end repair. For example, dATP comprising 6mA and dCTP comprising 5mC can be used in the end repair reaction in place of dATP comprising unmodified adenine and dCTP comprising unmodified cytosine. The use of multiple types of dNTP comprising a modified base is advantageous because it provides increased resolution inAtty. Docket No. GH0213WO / 01228-0067-00PCT defining the regions of the end-repaired DNA molecule which have been synthesized during the end repair reaction. This is because, in this example, the end of a synthesized region can be defined as the first unmodified adenine or unmodified cytosine after a stretch of containing 6mAs and / or 5mCs, rather than relying on the detection of solely an unmodified adenine or solely an unmodified cytosine.

[0224] The sequencing method used will depend on the type of modified base used in the end-repair reaction such that the specific modification can be detected. Exemplary conversion-based methods are described above alongside the base modification which they can detect. Moreover, nanopore-based sequencing can be used to detect 5-caC, 4mC, 5mC, 5hmC, 6mA, BrdU, FldU, IdU, and EdU, and single-molecule real time (SMRT) sequencing from Pacific Biosciences can be used to detect 5-caC, 4mC, 5mC, 5hmC, 6mA, and 8oxoG.

[0225] The disclosed methods use at least one type of dNTP which comprises a modified base (e.g. a methylated deoxycytidine triphosphate, such as deoxycytidine triphosphate comprising 5-methylcytosine (5mC) and / or 5 -hydroxymethyl -cytosine (5hmC)) in the end repair reaction. In particular embodiments, the dNTP is 5mC. During end repair reactions, the methylated deoxy cytidine triphosphate will be incorporated into the synthesized regions regardless of the sequence context. This will result in methylated cytosines in non-CpG positions (i.e., methylated cytosines in a CpH context), which are very rare in nature. These methylated non-CpG cytosines can therefore be used as labels for identifying synthesized regions in the end repaired DNA molecule. Similarly, other types of dNTP which comprise a modified base uncommon or absent in nature can be used. The identification of such modified bases can be performed using sequencing, and regions comprising these modifications can be interpreted as defining regions which were synthesized in the end repair reaction.

[0226] In other embodiments of the disclosed methods, the modified base is a methylated cytosine, such as 5mC or 5hmC. In some embodiments, the methylated cytosine is 5mCpH (where H = A, T, or C). In other embodiments, the modified base is other than 5mC or 5hmC. In some embodiments wherein the modified base is other than 5mC or 5hmC, a repaired region is defined as (i) the sequence between two non-modified bases spanning a modified base, wherein the bases are of the same identity to the modified bases present in the at least one type of dNTP; and / or (ii) the sequence between a non-modified base and the end of a sequence read, wherein there is no additional non-modified bases between the non-modified base and the end of the sequence read, where the non-modified bases are the same identity as the modified base present in the at least one type of dNTP. In some embodiments wherein theAtty. Docket No. GH0213WO / 01228-0067-00PCT modified base is a methylated cytosine, such as 5mC or 5hmC, a repaired region is defined as (i) the sequence between two non-methylated cytosines which span one or more methylated CpH cytosines; and / or (ii) the sequence between a methylated CpH cytosine and an end of a sequence read, wherein the methylated CpH cytosine is the CpH cytosine most distant from the end of the sequence read, or a subsequence thereof comprising one or more methylated CpH cytosines.Partitioning

[0227] In some instances, the DNA is partitioned into two or more partitions (sub-samples). In some embodiments, each partition is differentially tagged. Tagged partitions can then be pooled together for collective sample prep and / or sequencing. The partitioning-tagging- pooling steps can occur more than once, with each round of partitioning occurring based on a different characteristics, and tagged using differential tags that are distinguished from other partitions and partitioning means. In some embodiments, the DNA is partitioned based on the presence or absence of 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) nucleic acid bases in the nucleic acids.

[0228] Examples of characteristics that can be used for partitioning include sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and / or proteins that bind to DNA. Resulting partitions can include one or more of the following nucleic acid forms: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), shorter DNA fragments and longer DNA fragments. In some embodiments, partitioning based on a cytosine modification (e.g., cytosine methylation) or methylation generally is performed and is optionally combined with at least one additional partitioning step, which may be based on any of the foregoing characteristics or forms of DNA. In some embodiments, a heterogeneous population of nucleic acids is partitioned into nucleic acids with one or more base modifications and without the one or more base modifications. Examples of base modifications are described elsewhere herein. Alternatively or additionally, a heterogeneous population of nucleic acids can be partitioned into nucleic acid molecules associated with nucleosomes and nucleic acid molecules devoid of nucleosomes. Alternatively or additionally, a heterogeneous population of nucleic acids may be partitioned into singlestranded DNA (ssDNA) and double-stranded DNA (dsDNA). Alternatively, or additionally, a heterogeneous population of nucleic acids may be partitioned based on nucleic acid length (e.g., molecules of up to 160 bp and molecules having a length of greater than 160 bp).

[0229] 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 oneAtty. Docket No. GH0213WO / 01228-0067-00PCT partition is subjected to an end repair and sequencing procedure described herein. In some embodiments at least one partition is not subjected to the end repair and sequencing procedure described herein. In cases where the method comprises a conversion procedure, corresponding sequences from the converted and non-converted partitions can be compared to identify single nucleotides that have undergone conversion and therefore identify corresponding modified nucleosides in the initial sample.

[0230] In some embodiments, partition tagging comprises tagging molecules in each partition with a partition tag. After re-combining partitions (e.g., to reduce the number of sequencing runs needed and avoid unnecessary cost) and sequencing molecules, the partition tags identify the source partition. In another embodiment, different partitions are tagged with different sets of molecular tags, e.g., comprised of a pair of barcodes. In this way, each molecular barcode indicates the source partition as well as being useful to distinguish molecules within a partition. For example, a first set of 35 barcodes can be used to tag molecules in a first partition, while a second set of 35 barcodes can be used tag molecules in a second partition.

[0231] In some embodiments, after partitioning and tagging with partition tags, the molecules may be pooled for sequencing in a single run. In some embodiments, a sample tag is added to the molecules, e.g., in a step subsequent to addition of partition tags and pooling. Sample tags can facilitate pooling material generated from multiple samples for sequencing in a single sequencing run.

[0232] Alternatively, in some embodiments, partition tags may be correlated to the sample as well as the partition. As a simple example, a first tag can indicate a first partition of a first sample; a second tag can indicate a second partition of the first sample; a third tag can indicate a first partition of a second sample; and a fourth tag can indicate a second partition of the second sample.

[0233] While tags may be attached to molecules already partitioned based on one or more characteristics, the final tagged molecules in the library may no longer possess that characteristic. For example, while single stranded DNA molecules may be partitioned and tagged, the final tagged molecules in the library are likely to be double stranded. Similarly, while DNA may be subject to partition based on different levels of methylation, in the final library, tagged molecules derived from these molecules are likely to be unmethylated. Accordingly, the tag attached to a molecule in the library typically indicates the characteristic of the “parent molecule” from which the ultimate tagged molecule is derived, not necessarily to characteristic of the tagged molecule, itself.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0234] As an example, barcodes 1, 2, 3, 4, etc. are used to tag and label molecules in the first partition; barcodes A, B, C, D, etc. are used to tag and label molecules in the second partition; and barcodes a, b, c, d, etc. are used to tag and label molecules in the third partition. Differentially tagged partitions can be pooled prior to sequencing. Differentially tagged partitions can be separately sequenced or sequenced together concurrently, e.g., in the same flow cell of an Illumina sequencer.

[0235] After sequencing, analysis of reads can be performed on a partition-by-partition level, as well as a whole DNA population level. Tags are used to sort reads from different partitions. Analysis can include in silico analysis to determine genetic and epigenetic variation (one or more of methylation, chromatin structure, etc.) using sequence information, genomic coordinates length, coverage, and / or copy number. In some embodiments, higher coverage can correlate with higher nucleosome occupancy in genomic region while lower coverage can correlate with lower nucleosome occupancy or a nucleosome depleted region (NDR).

[0236] Disclosed methods herein comprise analyzing DNA in a sample. In some embodiments described herein, the disclosed methods comprise partitioning DNA. In such methods, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically partitioned based on one or more characteristics of the DNA. This approach can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated. In some embodiments, a first subsample or aliquot of a sample is subjected to steps for making capture probes as described elsewhere herein and a second subsample or aliquot of a sample is subjected to partitioning. In some embodiments, a sample or subsample or aliquot thereof is subjected to partitioning and differential tagging, followed by a capture step using capture probes for rearranged sequences and optionally additional capture probes, e.g., for sequence-variable and / or epigenetic target regions.

[0237] 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.

[0238] In some embodiments, the partitioning comprises contacting the DNA with an agent that recognizes a modification associated with (e.g., in) the DNA. In some embodiments, theAtty. Docket No. GH0213WO / 01228-0067-00PCT agent that recognizes the modification is an antibody. In some embodiments, the agent is immobilized on a solid support. In some embodiments, the partitioning comprises immunoprecipitation, e.g., using the antibody agent, such as an antibody, immobilized on solid support.

[0239] In some embodiments, the modification is methylation, and in some such embodiments, the partitioning comprises partitioning on the basis of methylation level. In some such embodiments, the agent is a methyl binding reagent. In some embodiments, the methyl binding reagent specifically recognizes 5-methylcytosine. In some such embodiments, the agent is a hydroxymethyl binding reagent. In some embodiments, the methyl binding reagent specifically recognizes 5 -hydroxymethylcytosine, biotinylated 5- hydroxymethylcytosine, glucosylated 5-hydroxymethylcytosine, or sulfonylated 5- hydroxymethylcytosine. In some embodiments, the partitioning comprises partitioning on the basis of binding to a protein comprising contacting the sample comprising the DNA with a binding reagent specific for the protein. In some such embodiments, binding reagent specifically binds a methylated protein, an acetylated protein, such as a methylated or acetylated histone. In some embodiments, the binding reagent specifically binds an unmethylated or unacetylated protein epitope.

[0240] In some embodiments, the modification is hydroxymethylation, and in some such embodiments, the partitioning comprises partitioning on the basis of hydroxymethylation level. In some such embodiments, the agent is a hydroxymethyl binding reagent, such as an antibody. In some embodiments, the hydroxymethyl binding reagent (e.g., antibody) specifically recognizes 5-hydroxymethylcytosine (5-hmC). In some embodiments, a modification such as hydroxymethylation is labeled (e.g., biotinylated, glucosylated, or sulfonated) before being contacted with an agent that recognizes the labeled form of the modification. For example, 5-hmC can be enzymatically glucosylated and then partitioned based on binding to J-binding protein 1. Exemplary methods of labeling and / or partitioning 5- hmC are provided, e.g., in Song et al., Nat. Biotech. 29:68-72 (2010); Ko et al., Nature 468:839-843 (2010); and Robertson et al., Nucleic Acids Res. 39:e55 (2011).

[0241] Where immunoprecipitation is used and involves an antibody that recognizes singlestranded DNA, the DNA may be converted to double-stranded form by complementary strand synthesis before a subsequent step. Such synthesis may use an adapter as a primer binding site, or can use random priming.

[0242] Partitioning nucleic acid molecules in a sample can increase a rare signal, e.g., by enriching rare nucleic acid molecules that are more prevalent in one partition of the sample.Atty. Docket No. GH0213WO / 01228-0067-00PCTFor example, a genetic variation present in hypermethylated DNA but less (or not) present in hypomethylated DNA can be more easily detected by partitioning a sample into hypermethylated and hypomethylated nucleic acid molecules. By analyzing multiple partitions of a sample, a multi-dimensional analysis of a single molecule can be performed and hence, greater sensitivity can be achieved. Partitioning may include physically partitioning nucleic acid molecules into partitions or subsamples based on the presence or absence of one or more methylated nucleobases. A sample may be partitioned into partitions or subsamples based on a characteristic that is indicative of differential gene expression or a disease state. A sample may be partitioned based on a characteristic, or combination thereof that provides a difference in signal between a normal and diseased state during analysis of nucleic acids, e.g., cell free DNA (cfDNA), non-cfDNA, tumor DNA, circulating tumor DNA (ctDNA) and cell free nucleic acids (cfNA).

[0243] In some embodiments, hypermethylation and / or hypomethylation variable epigenetic target regions are analyzed to determine whether they show differential methylation characteristic of tumor cells or cells of a type that does not normally contribute to the DNA sample being analyzed (such as cfDNA), and / or particular immune cell types.

[0244] In some instances, heterogeneous DNA in a sample is partitioned into two or more partitions (e.g., at least 3, 4, 5, 6 or 7 partitions). In some embodiments, each partition is differentially tagged. Tagged partitions can then be pooled together for collective sample prep and / or sequencing. The partitioning-tagging-pooling steps can occur more than once, with each round of partitioning occurring based on a different characteristic (examples provided herein), and tagged using differential tags that are distinguished from other partitions and partitioning means. In other instances, the differentially tagged partitions are separately sequenced.

[0245] In some embodiments, sequence reads from differentially tagged and pooled DNA are obtained and analyzed in silico. After sequencing, analysis of reads can be performed on a partition-by-partition level, as well as a whole DNA population level. Tags are used to sort reads from different partitions. Analysis to detect genetic variants can be performed on a partition-by-partition level, as well as whole nucleic acid population level. For example, analysis can include in silico analysis to determine genetic variants, such as copy number variations (CNVs), single nucleotide variations (SNVs), insertions / deletions (indels), and / or fusions in nucleic acids in each partition. In some instances, in silico analysis can include analysis to determine epigenetic variation (one or more of methylation, chromatin structure, etc.). Analysis can include in silico using sequence information, genomic coordinates length,Atty. Docket No. GH0213WO / 01228-0067-00PCT coverage, and / or copy number. For example, coverage of sequence reads can be used to determine nucleosome positioning in chromatin. Tags are used to sort reads from different partitions. Higher coverage can correlate with higher nucleosome occupancy in genomic region while lower coverage can correlate with lower nucleosome occupancy or nucleosome depleted region (NDR).

[0246] The agents used to partition populations of nucleic acids within a sample can be affinity agents, such as antibodies with the desired specificity, natural binding partners or variants thereof (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68-72 (2011)), or artificial peptides selected e.g., by phage display to have specificity to a given target. In some embodiments, the agent used in the partitioning is an agent that recognizes a modified nucleobase. In some embodiments, the modified nucleobase recognized by the agent is a modified cytosine, such as a methylcytosine (e.g., 5- methylcytosine). In some embodiments, the modified nucleobase recognized by the agent is a product of a procedure that affects the first nucleobase in the DNA differently from the second nucleobase in the DNA of the sample. In some embodiments, the modified nucleobase may be a “converted nucleobase,” meaning that its base pairing specificity was changed by a procedure. For example, certain procedures convert unmethylated or unmodified cytosine to dihydrouracil, or more generally, at least one modified or unmodified form of cytosine undergoes deamination, resulting in uracil (considered a modified nucleobase in the context of DNA) or a further modified form of uracil. Examples of partitioning agents include antibodies, such as antibodies that recognize a modified nucleobase, which may be a modified cytosine, such as a methylcytosine (e.g., 5- methylcytosine). In some embodiments, the partitioning agent is an antibody that recognizes a modified cytosine other than 5-methylcytosine, such as 5-carboxylcytosine (5-caC). Alternative partitioning agents include methyl binding domain (MBDs) and methyl binding proteins (MBPs) as described herein, including proteins such as MeCP2, MBD2, and antibodies preferentially binding to 5-methylcytosine. Where an antibody is used to immunoprecipitate methylated DNA, the methylated DNA may be recovered in singlestranded form. In such embodiments, a second strand can be synthesized. Hypermethylated (and optionally intermediately methylated) subsamples may then be contacted with a methylation sensitive nuclease that does not cleave hemi-methylated DNA, such as Hpall, BstUI, or Hin6i. Alternatively or in addition, hypomethylated (and optionally intermediately methylated) subsamples may then be contacted with a methylation dependent nuclease that cleaves hemi-methylated DNA.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0247] Additional, non-limiting examples of partitioning agents are histone binding proteins which can separate nucleic acids bound to histones from free or unbound nucleic acids. Examples of histone binding proteins that can be used in the methods disclosed herein include RBBP4, RbAp48 and SANT domain peptides.

[0248] 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.

[0249] 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).

[0250] In some embodiments, methylation levels can be determined using partitioning, modification-sensitive conversion such as direct detection during sequencing, methylationsensitive restriction enzyme digestion, methylation-dependent restriction enzyme digestion, or any other suitable approach. For example, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically partitioned based on one or more characteristics of the DNA. For example, a methylated DNA binding protein (e.g., an MBD such as 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.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0251] 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.

[0252] When using MeDIP or MethylMiner®Methylated DNA Enrichment Kit (ThermoFisher Scientific) various levels of methylation can be partitioned using sequential elutions. For example, a hypomethylated partition (no methylation) can be separated from a methylated partition by contacting the nucleic acid population with the MBD from the kit, which is attached to magnetic beads. The beads are used to separate out the methylated nucleic acids from the non- methylated nucleic acids. Subsequently, one or more elution steps are performed sequentially to elute nucleic acids having different levels of methylation. For example, a first set of methylated nucleic acids can be eluted at a salt concentration of 160 mM or higher, e.g., at least 150 mM, at least 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or 2000 mM. After such methylated nucleic acids are eluted, magnetic separation is once again used to separate higher level of methylated nucleic acids from those with lower level of methylation. The elution and magnetic separation steps can be repeated to create various partitions such as a hypomethylated partition (enriched in nucleic acids comprising no methylation), a methylated partition (enriched in nucleic acids comprising low levels of methylation), and a hyper methylated partition (enriched in nucleic acids comprising high levels of methylation).

[0253] 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).Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0254] 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.

[0255] For further details regarding portioning nucleic acid samples based on characteristics such as methylation, see WO2018 / 119452, which is incorporated herein by reference.

[0256] 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.

[0257] Nucleic acid molecules can be partitioned based on DNA-protein binding. Protein- DNA complexes can be partitioned based on a specific property of a protein. Examples of such properties include various epitopes, modifications (e.g., histone methylation or acetylation) or enzymatic activity. Examples of proteins which may bind to DNA and serve as a basis for fractionation may include, but are not limited to, protein A and protein G. Any suitable method can be used to partition the nucleic acid molecules based on protein bound regions. Examples of methods used to partition nucleic acid molecules based on protein bound regions include, but are not limited to, SDS-PAGE, chromatin-immuno-precipitation (ChIP), heparin chromatography, and asymmetrical field flow fractionation (AF4).

[0258] In some embodiments, the partitioning comprises contacting the DNA with a methylation sensitive restriction enzyme (MSRE) and / or a methylation dependent restriction enzyme (MDRE). Following the treatment of the DNA with a MSRE or a MDRE, the DNA may be partitioned based on size to generate hypermethylated (longest DNA molecules following MSRE treatment and shortest DNA fragments following MDRE treatment), intermediate (intermediate length DNA molecules following MSRE or MDRE treatment), and hypomethylated (shortest DNA molecules following MSRE treatment and longest DNA fragments following MDRE treatment) subsamples.

[0259] 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, anAtty. Docket No. GH0213WO / 01228-0067-00PCTMBD 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.

[0260] 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.

[0261] Examples of agents that recognize a modified nucleobase contemplated herein include, but are not limited to:

[0262] (a) MeCP2 is a protein that preferentially binds to 5-methyl-cytosine over unmodified cytosine.

[0263] (b) RPL26, PRP8 and the DNA mismatch repair protein MHS6 preferentially bind to 5- hydroxymethyl-cytosine over unmodified cytosine.

[0264] (c) FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3 preferably bind to 5-formyl- cytosine over unmodified cytosine (lurlaro et al., Genome Biol. 14: R119 (2013)).

[0265] (d) Antibodies specific to one or more methylated or modified nucleobases or conversion products thereof, such as 5mC, 5-caC, or DHU.

[0266] 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. GH0213WO / 01228-0067-00PCT

[0267] 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 singlestranded 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.

[0268] In some embodiments, the partitions of DNA are desalted and concentrated in preparation for enzymatic steps of library preparation.

[0269] Sequences that comprise aberrantly high copy numbers may tend to be hypermethylated. Accordingly, in some embodiments, the DNA contacted with capture probes specific for members of an epigenetic target region 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.

[0270] 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.

[0271] Disclosed methods herein can comprise analyzing DNA in a sample. In some embodiments described herein, the disclosed methods comprise partitioning DNA. In such methods, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically partitioned based on one or more characteristics of the DNA. This approach can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated. In some embodiments, a first subsample or aliquot of a sample is subjected to steps for making capture probes as described elsewhere herein and a second subsample or aliquot of a sample is subjected to partitioning. In some embodiments, a sample or subsample or aliquot thereof is subjected to partitioning and differential tagging, followed by a captureAtty. Docket No. GH0213WO / 01228-0067-00PCT step using capture probes for rearranged sequences and optionally additional capture probes, e.g., for sequence-variable and / or epigenetic target regions.

[0272] 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.Amplification

[0273] In some embodiments, the DNA is amplified. In some embodiments, the DNA can be subjected to a plurality of distinct amplification reactions. For example, DNA in a converted sample, as described herein, can be selectively amplified.

[0274] In some embodiments, the adapters are added to the DNA to yield DNA flanked by adapters. DNA flanked by adapters, as described herein, can be selectively amplified and, optionally, be subjected to methylation-preserving amplification. In some embodiments, the adapters are added to the DNA before methylation-preserving amplification, before contacting the DNA in the sample with a deaminase, and / or before selective amplification of the DNA in the converted sample. In some embodiments, the adapters are added to the DNA after methylation-preserving amplification, after contacting the DNA in the sample with a deaminase, and / or after selective amplification of the DNA in the converted sample.

[0275] In some embodiments, DNA can be amplified by methylation-preserving amplification. In some embodiments, the methylation-preserving amplification can occur before the selective amplification of the DNA in a converted sample. In some embodiments, the methylation-preserving amplification can occur after the selective amplification of the DNA in a converted sample.

[0276] 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, including 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 primersAtty. Docket No. GH0213WO / 01228-0067-00PCT 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 (HD A), loop-mediated isothermal amplification (LAMP) (Notomi et al., Nuc. Acids Res., 28, e63, 2000), rollingcircle 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. In some embodiments, the methylation-preserving amplification comprises thermocycled amplification. In some embodiments, the methylationpreserving amplification comprises isothermal amplification.

[0277] In some embodiments, methylation-preserving amplification comprises amplification performed in the presence of a methyltransferase. Methylating agents of use in methylationpreserving amplification methods described herein are known to those of ordinary skill in the art, and can include, for example, any suitable methyltransferase. In some embodiments, the methylating agent is DNMT1. DNMT1 is the most abundant DNA methyltransferase in mammalian cells and predominantly methylates hemimethylated CpG di-nucleotides in the mammalian genome. For example, DNA molecules replicated using PCR amplification with DNMT1 incubation will maintain their methylation status post-amplification, for use in further analyses, such as those described herein (such as an epigenetic base conversion step and / or an enrichment step).

[0278] Additional methylating agents useful herein include the mammalian methyltransferases, DNMT3a and DNMT3b, the plant methyltransferases, MET1, and CMT3. In some embodiments, DNMT1 or another suitable methyltransferase is used with a methyl donor and may be used with or without cofactors known to those of ordinary skill in the art. DNMT1 works in vitro at 95% efficiency without a cofactor; however, DNMT1 may be used with a cofactor such as NP95(Uhrfl), such as described in Bashtrykov PI, et al. “The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism.” J Biol Chem. 2014. In some embodiments, DNMT1 is used at a concentration of about 50-10000 U / mL, such as about 50- 2000, about 50-5000, about 2500-7500, or about 5000-10000 U / mL. In some embodiments,Atty. Docket No. GH0213WO / 01228-0067-00PCTDNMT1 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.

[0279] In some embodiments, an amplification of the DNA in a sample comprises amplifying rolling-circle amplification (RCA). In some embodiments, RCA comprises circularizing a DNA template (e.g., DNA in the converted sample). In some embodiments, RCA comprises copying the circularized DNA template using a rolling circle polymerase to generate a plurality of circularized DNA templates. In some embodiments, the rolling circle polymerase is a phi29 DNA polymerase. Exemplary methods of RCA are provided, e.g., in Lou et al., Proc. Natl. Acad. Sci. 110 (49) 19872-19877 (2013). In some embodiments, the RCA occurs prior to a step of sequencing the DNA.

[0280] This may be an additional amplification step subsequent to an earlier amplification step, such as amplification as described elsewhere herein. In some embodiments, amplification of adapted DNA comprises RCA, e.g., as described above. In some embodiments, RCA comprises copying the circularized DNA template using a rolling circle polymerase to generate a plurality of circularized DNA templates. In some embodiments, the rolling circle polymerase is a phi29 DNA polymerase.

[0281] In some embodiments, sequencing DNA that was amplified using RCA (e.g., as described elsewhere herein) provides sequence reads comprising multiple copies of the sequence of an original sample molecule or converted molecule and the copies are used to determine a consensus sequence of the original sample molecule or converted moleculeSequencing

[0282] In some embodiments, the one or more variant DMRs and the one or more corresponding DMRs are sequenced. In some embodiments, the one or more variant DMRs and the one or more corresponding DMRs are sequenced via whole-genome sequencing (WGS). In some embodiments, the methods described herein further comprise sequencing at least a portion of the amplified DNA.

[0283] In general, sample nucleic acids 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-Atty. Docket No. GH0213WO / 01228-0067-00PCT 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, panel sequencing, 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 means of processing multiple sample sets substantially simultaneously. Sample processing unit can also include multiple sample chambers to enable processing of multiple runs simultaneously.

[0284] 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.

[0285] 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, et al., “Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis,” FlOOOResearch, 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,Atty. Docket No. GH0213WO / 01228-0067-00PCT 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 adaptors 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.

[0286] 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., FlOOOResearch, 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) 63 639-648). Accordingly, in some embodiments, the sequencing comprises SMRT sequencing. In such embodiments, the end repair may be performed using dNTPs, which comprise 5-caC, 4mC, 5mC, 5hmC, 6mA, and / or 8oxoG.

[0287] 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 through 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 aAtty. Docket No. GH0213WO / 01228-0067-00PCT 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.

[0288] One example of a nanopore-based single molecule sequencing system is that commercialized by Oxford Nanopore Technologies (ONT). (Weirather JL, et al., FlOOOResearch, 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 adaptor. Therefore, the DNA template passes through the nanopore, followed by a hairpin and finally the complement. The raw read can be split into two “ID” reads (“template” and “complement”) by removing the adaptor. The consensus sequence of two “ID” reads is a “2D” read with a higher accuracy.

[0289] 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) 63 639-648; Kutyavin, Biochemistry (2008), 47, 51, 13666-1367; Muller 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. In such embodiments, the end repair may be performed using dNTPs, which comprise 5-caC, 4mC, 5mC, 5hmC, 6mA, BrdU, FldU, IdU, and / or EdU.

[0290] 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 adaptors at both ends (Fullgrabe, 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 its 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 anyAtty. Docket No. GH0213WO / 01228-0067-00PCT 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 (Pl 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.

[0291] 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.

[0292] 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, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the genome. Sequence coverage can be performed on, for example, at least 5, 10, 20, 70, 100,Atty. Docket No. GH0213WO / 01228-0067-00PCT200 or 500 different genes, or up to, for example, 5000, 2500, 1000, 500 or 100 different genes.

[0293] Simultaneous sequencing reactions may be performed using multiplex sequencing. In some embodiments, 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 embodiments, 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 1000-50000 or 1000-10000 or 1000-20000 reads per locus (base).

[0294] In some embodiments, sequencing DNA that was amplified using RCA (e.g., as described elsewhere herein) provides sequence reads comprising multiple copies of the sequence of an original sample molecule or converted molecule and the copies are used to determine a consensus sequence of the original sample molecule or converted molecule.

[0295] In general, sequencing of epigenetic target regions, e.g. to analyze a modified nucleoside 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.

[0296] In some embodiments, the methods described herein further comprise quantifying a level of methylation at one or more differentially methylated regions of the DNA. In some embodiments, quantifying 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. In some embodiments, the sequencing is next-generation sequencing (NGS). In some embodiments, the NGS is pyrosequencing, sequencing-by-synthesis, semiconductor sequencing, sequencing-by-ligation, or sequencing-by-hybridization. In some embodiments, the sequencing comprises nanopore-based sequencing or single-molecule real time (SMRT) sequencing.

[0297] In some embodiments, the methods described herein further comprise panel sequencing. Panel sequencing comprises targeted sequencing of one or more sets of targetAtty. Docket No. GH0213WO / 01228-0067-00PCT regions, such as any of the sets of target regions described elsewhere herein, e.g., sequencevariable target regions and epigenetic target regions, e.g., sequence-variable target regions and hypermethylation variable target regions, or sequence-variable target regions and hypomethylation variable target regions. Targeted sequencing may comprise, e.g., capturing or amplifying the target regions.

[0298] In some embodiments, first and second samples are sequenced via panel sequencing and the panel is the same for each. In some embodiments, first and second samples are sequenced via panel sequencing and the panel for the second sample is a subset of the panel for the first sample. Subset panels can be targeted, e.g., personalized or made patient-specific based on observations from the sequencing results of the first sample of which target regions are informative (e.g., which target regions include a variant DMR). In some embodiments, first and second samples are sequenced via panel sequencing and the panel for the second sample can be enriched for the panel by capture or PCR. Capture or PCR can be mutation specific, e.g., specific for one or more regions comprising a variant DMR or one or more regions found to comprise a variant DMR in the first sample. In certain embodiments comprising a conversion procedure, probes can account for methylation status.Enriching

[0299] In some embodiments, the methods described herein further comprise enriching the DNA for a plurality of target regions, optionally prior to the step of sequencing the DNA. In some embodiments, the plurality of target regions comprises epigenetic target region. In some embodiments, the plurality of target regions comprises epigenetic target regions. In some embodiments, the epigenetic target regions comprise hypermethylation variable target regions. In some embodiments, the epigenetic target regions comprise hypomethylation variable target regions. In some embodiments, the plurality of target regions comprise sequence-variable target regions. In some embodiments, the sequence-variable target regions comprise mutations identified in a germline sequence.

[0300] DNA molecules in a sample can be subject to a capture step, in which molecules having target sequences are captured for subsequent analysis. In some embodiments, methods disclosed herein comprise a step of capturing one or more sets of target regions of DNA, such as cfDNA. In some embodiments, the capture step (also referred to herein as an “enriching” or “enrichment” step) is performed prior to a step of subjecting the end-repaired DNA molecules to sequencing, prior to a step of selectively amplifying DNA in a converted sample, prior to a step of subjecting a DNA sample to sequencing, and / or prior to a step of subjecting a DNA sample comprising a plurality of DNA molecules to sequencing. CaptureAtty. Docket No. GH0213WO / 01228-0067-00PCT 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 biotin or the other examples noted below. The probes can have sequences selected to tile across a panel of regions, such as genes. Such bait sets are combined with a sample under conditions that allow hybridization of the target molecules with the baits. Then, captured molecules are isolated using the capture moiety. For example, a biotin capture moiety by bead-based streptavidin. Such methods are further described in, for example, U.S. patent 9,850,523, issuing December 26, 2017, which is incorporated herein by reference.

[0301] Capture moieties include, without limitation, biotin, avidin, streptavidin, a nucleic acid comprising a particular nucleotide sequence, a hapten recognized by an antibody, and magnetically attractable particles. 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 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.

[0302] A panel of regions targeted for enrichment can be selected such that they do not contain regions known to include the base modification used in the end repair reaction. When the end repair is performed with dNTPs comprising 5mC or 5hmC, a panel of regions targeted for enrichment may be selected such that they do not contain CpH dinucleotides which are known to be naturally methylated in the subject (e.g. humans). Such CpH dinucleotides can be identified through the use of publicly available resources (e.g. MethBank3.0: a database of DNA methylomes across a variety of species Nucleic Acids Res 2018). Such an approach has the advantage that any detected methylated CpH dinucleotides can unambiguously be attributed to regions synthesized in the end repair.

[0303] In some embodiments, capturing comprises contacting the DNA to be captured with a set of target-specific probes. The set of target-specific probes may have any of the features described herein for sets of target-specific probes, including but not limited to in the embodiments set forth above and the sections relating to probes below. Capturing may be performed on one or more subsamples prepared during methods disclosed herein. In someAtty. Docket No. GH0213WO / 01228-0067-00PCT embodiments, DNA is captured from at least the first subsample or the second subsample, e.g., at least the first subsample and the second subsample. In some embodiments, the subsamples are differentially tagged (e.g., as described herein) and then pooled before undergoing capture.

[0304] 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.

[0305] In some embodiments, a method described herein comprises capturing cfDNA obtained from a subject for a plurality of sets of target regions. The target regions comprise epigenetic target regions, which may show differences in methylation levels and / or fragmentation patterns depending on whether they originated from a tumor or from healthy cells. The target regions also comprise sequence-variable target regions, which may show differences in sequence depending on whether they originated from a tumor or from healthy cells. The capturing step produces a captured set of cfDNA molecules, and the cfDNA molecules corresponding to the sequence-variable target region are captured at a greater capture yield in the captured set of cfDNA molecules than cfDNA molecules corresponding to the epigenetic target region. For additional discussion of capturing steps, capture yields, and related aspects, see W02020 / 160414, which is incorporated herein by reference for all purposes.

[0306] In some embodiments, a method described herein comprises contacting cfDNA obtained from a subject 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 at a greater capture yield than cfDNA corresponding to the epigenetic target region.Sequencing Methods with Partitioning

[0307] As a variation on grouping sequencing reads of the same original molecule by molecular barcodes, a sample can be partitioned into aliquots as described in PCT / US2025 / 035226, incorporated by reference herein. Partitioning can be used either for individual samples or pooled samples, in which nucleic acids from different samples are distinguished by sample indexes. Partitioning preferably occurs before any amplification of original sample nucleic acid molecules so that amplicons of the same original molecule are not partitioned from each other. Partitioning reduces the number of instances of nucleic acid molecules having the same start and stop points in an individual aliquot relative to the sampleAtty. Docket No. GH0213WO / 01228-0067-00PCT before partitioning. Preferably the number of instances of nucleic acid molecules having the same start and stop points is reduced such that at least 75%, 80%, 90%, 95% or 99% of nucleic acid molecules in each aliquot have unique start and stop sequences.

[0308] The number of partitions depends on the characteristics of a population of nucleic acid molecules to be partitioned. These characteristics include the mean, median and mode of nucleic acid molecules having the same start and stop points, the maximum number of instances of nucleic acid molecules having the same start and stop points, and the overall distribution of instances of nucleic acid molecules having the same start and stop points.

[0309] For it to be statistically probable that an aliquot contains no instances of multiple nucleic acid molecules with the same start and stop points then the number of partitions should be equal to or greater (e.g., at least lx, 2x, 5 x or lOx) than the maximum number of instances of the same start and stop points in the sample before partition. Eight or sixteen partitions can sometimes be suitable.

[0310] With or without additional processing steps in separated partitions, the partitioned nucleic acid molecules can be labelled with partition indexes, such that nucleic acid molecules in the same aliquot receive the same partition index and nucleic acid molecules in at least some, and sometimes all of the different aliquots receive different partition indexes. Thus, linkage of sample molecules to partition indexes does not require random assortment of the partition indexes to the sample molecules. Partition indexes can be linked to sample molecules as primer components or by ligation, e.g., as a component of a further adapter. Preferably a partition index is included in one or both members of a pair of primers suitable for amplification of nucleic acid molecules in an aliquot. For example, such a primer pair can have 3’ regions complementary to adapter sequences flanking sample nucleic acid molecules, with one or both of the primers having a 5’ tail region including a partition index. If partition indexes are included in both members of a primer pair, the partition indexes can be the same or different from each other. After hybridization of such primers to adapter sequences, an amplification can conducted thereby covalently attaching partition indexes to sample nucleic acids.

[0311] An index is a short nucleic acid (e.g., less than 500, 100, 50, 20, 15, 10 or 5 nucleotides long), used to label nucleic acid molecules, for example to distinguish nucleic acids from different samples (a sample index), or nucleic acid molecules in different aliquots of the sample (partition indexes). The particular code stored by an index can be referred to as a designation of an index. Indexes are typically provided as sets of multiple different individual indexes for distinguishing samples or aliquots of a sample. That is, differentAtty. Docket No. GH0213WO / 01228-0067-00PCT samples receive different sample indexes from a set of sample indexes, and different aliquots receive different partition indexes.

[0312] In general, the distinction between a set of sample indexes and a set of partition indexes lies in the stages at which they added, the number of different indexes in the set, how the indexes are linked to samples nucleic acids, and the molecules they are used to distinguish rather than in indexes themselves. In principle, a set of sample indexes could be used as a set of partition indexes and vice versa. Preferably the code designations of a set of sample and partition indexes are mutually exclusive with one another.

[0313] After incorporation of partition indexes, further processing steps can be conducted on the aliquots separately or aliquots differentially labelled with partition indexes can be pooled and further processing steps performed on the pooled aliquots. Alternatively, the methods can be performed without use of partition indexes, in which case, all further processing steps are performed on separate aliquots so that it is known which sequencing reads originate from which aliquots. The methods can also be performed with some aliquots pooled and some kept separate from one another. The methods can also be performed with aliquots grouped in subpools, in which the aliquots within a subpool have different partition indexes from one another but aliquots in different subpools can have the same partition indexes as any of the other subpools. The different subpools are then kept separate from one another in subsequent processing whereas the aliquots within a subpooled are processed together. Sequencing reads can be traced back to the aliquot of origin based on a combination of the partition index present in a sequencing read and knowledge of the subpool from which it originated.

[0314] Further processing steps can include further amplification, affinity-enrichment for DNA molecules from selected genomic regions, sequencing and analysis of sequence reads. When partition indexes are used, sequencing is preferably performed after pooling of aliquots into a single vessel. Thus, nucleic acid molecules from the previously separate aliquots and different samples are sequenced together. When partition indexes are not used, sequencing is preferably performed keeping nucleic acid molecules from the different aliquots separate.

[0315] Sequencing reads from a sample are grouped to their molecule of origin by aliquot of origin determined by partition index or otherwise as described above, and a measure of sequence identity or similarity between sequencing reads. This measure can be start and stop points, which can be determined, for example, after alignment of sequencing reads with a reference sequence, length of sequencing reads, or minimum sequence similarity between reads (e.g., at least 95 or 99% identity after maximal alignment). If samples are pooled, sequence reads can be traced to a sample of origin from a sample index in the sequencingAtty. Docket No. GH0213WO / 01228-0067-00PCT read. Ian information, when determined, can also be used in grouping sequencing reads. Grouping of sequencing reads by molecule of origin permits distinction of genuine genetic or epigenetic variation from amplification and sequencing errors as further described below.

[0316] Methylation analysis can involve methylation-based separation of nucleic acid molecules. In some embodiments, methylation-based separation of nucleic acid molecules is performed by contacting the nucleic acid molecules with an agent that recognizes methylated DNA, such as 5 -methylcytosine. In particular embodiments, the agent is a methyl binding reagent. In particular embodiments, the methyl binding reagent is a methyl binding domain (MBD) protein or an antibody. In some embodiments, the methyl binding reagent specifically recognizes 5-methylcytosine. For example, methylated fragments in a DNA sample can be separated via methylated DNA immunoprecipitation (MeDIP), or methylated fragments can be separated from unmethylated fragments using methyl binding domain proteins (e.g., MethylMinder Methylated DNA Enrichment Kit (ThermoFisher Scientific).

[0317] One application of partition methods is analysis of methylation state of nucleic acids. Methylation analysis can comprise subjecting parent nucleic acids or amplification products thereof to a procedure that affects a first nucleobase in the nucleic acid differently from a second nucleobase, for example wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity. In some embodiments, the procedure that affects a first nucleobase of the nucleic acid differently from a second nucleobase of the nucleic acid is a methylationsensitive conversion. In particular embodiments, the methylation-sensitive conversion is bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, APOBEC-coupled epigenetic (ACE) conversion, enzymatic methyl-seq (EM- seq) conversion, single-enzyme 5-methylcytosine sequencing (SEM-seq) conversion, or direct methylation sequencing (DM-seq).

[0318] Comparison of sequencing reads from treated and control groups indicates which cytosines were subject of modification. Splitting into groups for analysis of DNA modification is preferably performed after partitioning of samples or combined samples into aliquots so members of the same pairs of duplex strands are present in the same aliquot. Conversion also preferably precedes amplification. Conversion can occur before or after enrichment. If conversion occurs before enrichment, probes must be modified to hybridize with modified bases (e.g., U / T in place of C). Thus, a preferred order of steps is to attachAtty. Docket No. GH0213WO / 01228-0067-00PCT sample indexes to different samples, pool the different samples, partition the pooled samples, conversion of portions of the partitioned samples, amplification, enrichment and sequencing.

[0319] Methylation analysis can alternatively involve methylation-based separation of nucleic acid molecules. In some embodiments, methylation-based separation of nucleic acid molecules is performed by contacting the nucleic acid molecules with an agent that recognizes methylated DNA, such as 5-methylcytosine. In particular embodiments, the agent is a methyl binding reagent. In particular embodiments, the methyl binding reagent is a methyl binding domain (MBD) protein (e.g., see WO2018119452) or an antibody. In some embodiments, the methyl binding reagent specifically recognizes 5-methylcytosine. For example, methylated fragments in a DNA sample can be separated via methylated DNA immunoprecipitation (MeDIP), or methylated fragments can be separated from unmethylated fragments using methyl binding domain proteins (e.g., MethylMinder Methylated DNA Enrichment Kit (ThermoFisher Scientific). These types of methods separate DNA fragments having a high methyl C content from those with a low methyl C content before sequencing.

[0320] In one format, MBD separation is performed on individual samples, resulting in two portions for each sample, one having high methyl C content, the other lower methyl C content. The portions are then labelled with sample indexes. The portions are then pooled, high methyl content portions being pooled together, and low methyl content portions being pooled together. The two pools are then partitioned. Amplification and enrichment are performed in the separate partitions followed by attachment of partitions indexes. The partitions are then combined for sequencing. In another format, after ligation of sample indexes all portions are combined in the same pool instead of splitting into high and low methyl content pools. In another form, samples indexes are attached to samples before MBD separation. Thus, high and low methyl portions after MBD separation have the same sample index and are kept separate by pooling into two pools one with high methyl content, the other low methyl content. The two pools are separately portioned. The partitions are subject to amplification and enrichment followed by incorporation of partition indexes. The partitions are then combined for sequencing.

[0321] In some embodiments, sequencing of different aliquots is performed in different flow cells or different regions or lanes of the same flow cell. Different aliquots can be tracked using aliquot-specific partition indices (“Variation #1”) or tracked using partition indices and separate sequencing (“Variation #2”). In both variations, 96 samples, for example, are each ligated to a different sample index, and subsequently mixed and aliquoted into 96 wells. The particular numbers of samples and partitions are provided as an example. In Variation #1,Atty. Docket No. GH0213WO / 01228-0067-00PCT each well receives a different partition index via PCR with labelled primers (i.e., the partition indices are aliquot-specific), and aliquots are subsequently pooled into a single pool prior to sequencing. The deconvolution of sequencing reads to original molecules is performed using the partition index, start / stop positions, and (for sample demultiplexing) the sample index. In Variation #2, each column of wells receives the same partition index whereas partition indices vary across each row, such that partition indices are aliquot-specific only with respect to a subset of the aliquots (and not all aliquots). In this variation, each row of aliquots is pooled (i.e., the pooling is amongst aliquots differentially labelled with partition indices), and each subset pool is sequenced separately). For example, each of the eight subset pools can be loaded onto a separate lane of a flow cell comprising eight lanes (or loaded on different flow cells or different sequencing instruments). The deconvolution of sequencing reads to original molecules is performed using the partition index, the separate sequencing, start / stop positions, and (for sample demultiplexing) the sample index.

[0322] In some embodiment, the methods do not necessarily involve an initial step of sample mixing before partitioning. Mixing or pooling nucleic acids from different samples after initial processing steps advantageously allows different samples to be subjected to different processing steps (e.g. different enrichment reactions). For example, in one embodiment, each of 96 samples is partitioned into eight aliquots, i.e. one column of wells per sample. The particular numbers of samples and partitions are provided as an example. Partition indices are introduced via PCR, wherein four different partition indices are used, such that two aliquots of each sample receive the same partition index. Aliquots of the same sample that have been differentially labelled with partition indices are then pooled such that two subset pools are generated per sample, which in turn means that two enrichment reactions are performed per sample (the enrichment reactions are performed on the subset pool). The two different subset pools deriving from the same sample are sequenced separately (e.g. in different lanes), and subsequent deconvolution of sequencing reads to original molecules is performed using the partition index, the separate sequencing, and start / stop positions. The partition indices may not be sample-specific (the partition indices are the same across rows), so the method can use tagging with sample indices before sample multiplexing. Alternatively, partition indices can be sample-specific, e.g. each column of wells can receive a different set of four partition indices; in such a case the ligation of separate indices for sample demultiplexing is not required. Demultiplexing by sample of origin is based on the sample index or the samplespecific partition index.Atty. Docket No. GH0213WO / 01228-0067-00PCTAdditional features of certain disclosed methodsSamples

[0323] A sample can be any biological sample isolated from a subject. A sample can be a bodily sample. Samples can include body tissues, such as known or suspected solid tumors, whole blood, buffy coat, PBMCs, platelets, serum, plasma, stool, red blood cells, white blood cells or leukocytes, 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, urine. Samples are preferably body fluids, particularly blood and fractions thereof, and 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. Thus, preferred body fluids for analysis include body fluids comprising cells, such as whole blood, buffy coat separated from whole blood, PBMCs separated from whole blood, a leukapheresis sample, and / or plasma or serum.

[0324] In some embodiments, a population of nucleic acids (such as a population of cell-free nucleic acids, such as cell-free DNA or cell-free RNA) is obtained from a serum, plasma, or blood sample (such as a buffy coat sample or any other sample comprising cells or a blood sample (e.g., a whole blood sample, a leukapheresis sample, or a PBMC 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.

[0325] 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. 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.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0326] In some embodiments, the sample is a first sample obtained at a first time point.

[0327] In some embodiments, the first sample is a tissue sample, tumor sample, cfDNA sample, or plasma sample. In some embodiments, the method further comprises sequencing the first sample. In some embodiments, the first sample is sequenced via whole genome sequencing (WGS) or panel sequencing. In some embodiments, the first sample is a tumor sample or a tissue sample.

[0328] In some embodiments, the method further comprises obtaining a second sample at second timepoint. In some embodiments, the method further comprises sequencing the second sample. In some embodiments, the second sample is sequenced via whole genome sequencing (WGS) or panel sequencing. In some embodiments, the second sample is sequenced via panel sequencing and wherein the panel is targeted based on the sequencing of the first sample. In some embodiments, the second sample is a tissue sample, tumor sample, cfDNA sample, or plasma sample. In some embodiments, panel sequencing comprises capturing one or more sets of target regions, such as any of the sets of target regions described elsewhere herein, e.g., sequence-variable target regions and epigenetic target regions, e.g., sequence-variable target regions and hypermethylation variable target regions, or sequence-variable target regions and hypomethylation variable target regions

[0329] In some embodiments, the sample comprises plasma. The volume of plasma obtained can depend on the desired read depth for sequenced regions. Exemplary volumes are 0.4-40 mL, 5-20 mL, 10-20 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 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.

[0330] 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.

[0331] 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 ofAty. Docket No. GH0213WO / 01228-0067-00PCT 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.

[0332] 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 sampled PBMCs may be 1 to 10 mL. In some embodiments, the sample volume is 0.1-0.5 mL of PBMCs, such as 0.3 mL of PBMCs, per 10 mL whole blood.

[0333] 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.

[0334] A sample can comprise various amount of nucleic acid that contains genome equivalents. For example, a sample of about 30 ng cDNA can contain about 10,000 (104) haploid human genome equivalents. Similarly, a sample of about 100 ng of cDNA can contain about 30,000 haploid human genome equivalents.

[0335] A sample can comprise nucleic acids from different sources, e.g., from cells of the same subject or from cells of different subjects. A sample can comprise nucleic acids carrying mutations. For example, a sample can comprise cDNA carrying germline mutations and / or somatic mutations. Germline mutations refer to mutations existing in germline nucleic acids of a subject. Somatic mutations refer to mutations originating in somatic cells of a subject, e.g., precancer cells or cancer cells. A sample can comprise cDNA 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.

[0336] Exemplary amounts of nucleic acids (e.g., cDNA prepared from RNA from a sample comprising cells or a blood sample (e.g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample) in a sample before amplification range from about 1 fg to about 1 pg, e.g., 1 pg to 200 ng, 1 ng to 100 ng, 10 ng to 1000 ng. For example, theAtty. Docket No. GH0213WO / 01228-0067-00PCT 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 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 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 nucleic acid molecules. The method can comprise obtaining 1 femtogram (fg) to 200 ng.

[0337] Nucleic acids can be isolated from cells or bodily fluids, which may comprise cells. Cells can be lysed and cellular nucleic acids processed. 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 C 1 DNA, or DNA or protein for hybridization and / or ligation, may be added throughout the reaction to optimize certain aspects of the procedure such as yield.

[0338] After such processing, samples can include various forms of nucleic acid including double stranded cDNA, single stranded cDNA, and single stranded RNA. In some embodiments, single stranded cDNA and RNA can be converted to double stranded forms so they are included in subsequent processing and analysis steps.

[0339] cDNA molecules can be linked to adapters at either one end or both ends. Typically, double-stranded molecules are blunt ended by treatment with a polymerase with a 5'-3' polymerase and a 3 '-5' exonuclease (or proof-reading function), in the presence of all four standard nucleotides. Klenow large fragment, T4, Vent, or Deep Vent polymerases are examples of suitable polymerase. The blunt ended cDNA molecules can be ligated with at least partially double stranded adapter (e.g., a Y shaped or bell-shaped adapter).Alternatively, complementary nucleotides can be added to blunt ends of sample nucleic acids and adapters to facilitate ligation. Contemplated herein are both blunt end ligation and sticky end ligation. In blunt end ligation, both the nucleic acid molecules and the adapter tags have blunt ends. In sticky-end ligation, typically, the nucleic acid molecules bear an “A” overhang and the adapters bear a “T” overhang.Subjects

[0340] In some embodiments, the population of target nucleic acids (e.g., cfDNA or cfRNA, or DNA or RNA from a sample comprising cells or a blood sample (e.g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample)) is obtained from aAtty. Docket No. GH0213WO / 01228-0067-00PCT subject having a cancer or a precancer, an infection, transplant rejection, or other disease directly or indirectly affecting the immune system. In some embodiments, the population of target nucleic acids is obtained from a subject suspected of having a cancer or a precancer, an infection, transplant rejection, or other disease directly or indirectly affecting the immune system. In some embodiments, the population of target nucleic acids is obtained from a subject having a tumor. In some embodiments, the population of target nucleic acids is obtained from a subject suspected of having a tumor. In some embodiments, the population of target nucleic acids is obtained from a subject having neoplasia. In some embodiments, the population of target nucleic acids is obtained from a subject suspected of having neoplasia. In some embodiments, the population of target nucleic acids 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 or 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.Target regions

[0341] In some embodiments, certain genomic regions of interest are detected and / or enriched. The genomic regions of interest may comprise one or more target region. In some embodiments, target regions comprise variations that are not prevalent in DNA from healthy subjects or not prevalent in DNA obtained from healthy tissue regions. In some embodiments, target regions comprise variations present in healthy cells but not normally present in the sample type, such as a blood sample. In some embodiments, the variations are present in aberrant cells (e.g., hyperplastic, metaplastic, or neoplastic cells). Exemplary target regions include sequence-variable target regions, epigenetic target regions.

[0342] In some embodiments, a first target region is detected, comprising at least epigenetic target regions. In some embodiments, the epigenetic target regions detected in a first subsample comprise hypermethylation variable target regions. In some embodiments, theAtty. Docket No. GH0213WO / 01228-0067-00PCT hypermethylation variable target regions are CpG-containing regions that are unmethylated or have low methylation in DNA (such as cfDNA) from healthy subjects (e.g., below-average methylation relative to bulk cfDNA). In some embodiments, the hypermethylation variable target regions show type-specific hypermethylation in healthy DNA (such as cfDNA) from one or more related cell or tissue types. Without wishing to be bound by any particular theory, the presence of cancer cells may increase the shedding of DNA into the bloodstream (e.g., from the cancer and / or the surrounding tissue). 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.

[0343] In some embodiments, the methods herein comprise detecting a second captured target region from a sample or second subsample, comprising at least epigenetic target regions. In some embodiments, the second epigenetic target region comprises hypomethylation variable target regions. In some embodiments, the hypomethylation variable target regions are CpG-containing regions that are methylated or have high methylation in DNA (such as cfDNA) from healthy subjects (e.g., above-average methylation relative to bulk cfDNA). 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.

[0344] Additionally, target regions may comprise DNA corresponding to a sequence-variable target region.Epigenetic target regions

[0345] In some embodiments, a target region is or comprises an epigenetic target region. Epigenetic target regions may comprise one or more types of target regions likely to differentiate DNA from neoplastic (e.g., tumor or cancer) cells and from healthy cells, e.g., non-neoplastic circulating cells. Exemplary types of such regions are discussed in detail herein. The epigenetic target region may also comprise one or more control regions, e.g., as described herein.

[0346] In some embodiments, the epigenetic target region has 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 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,Atty. Docket No. GH0213WO / 01228-0067-00PCT1-1.5 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 has a footprint of at least 20 Mbp.Hypermethylation and hypomethylation variable target regions

[0347] In some embodiments, an epigenetic target region comprises a hypermethylation variable target region. In some embodiments, the hypermethylation variable target regions are differentially or exclusively hypermethylated in one or more related cell or tissue types. 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 even higher methylation in cfDNA from a diseased cell of 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. Hypermethylation variable target regions may comprise DMRs. 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 500 or 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.

[0348] Table 2: Hypermethylated target regions with aberrantly high copy number in colon cancer or precancerAtty. Docket No. GH0213WO / 01228-0067-00PCT

[0349] In some embodiments, hypermethylation variable target regions refer to regions where an increase in the level of observed methylation, e.g., in a cfDNA sample, indicates an increased likelihood that a sample (e.g., of cfDNA) contains DNA produced by neoplastic cells, such as tumor or cancer cells. For example, hypermethylation of promoters of tumor suppressor genes has been observed repeatedly. See, e.g., Kang et al., Genome Biol. 18:53 (2017) and references cited therein. In another example, as discussed above, hypermethylation variable target regions can include regions that do not necessarily differ in methylation in cancerous tissue relative to DNA from healthy tissue of the same type, but do differ in methylation (e.g., have more methylation) relative to cfDNA that is typical in healthy subjects. Where, for example, the presence of a cancer results in increased cell death such as apoptosis of cells of the tissue type corresponding to the cancer, such a cancer can be detected at least in part using such hypermethylation variable target regions.

[0350] An extensive discussion of methylation variable target regions in colorectal cancer is provided in Lam et al., Biochim Biophys Acta. 1866: 106-20 (2016). These include VIM, SEPT9, ITGA4, OSM4, GATA4 and NDRG4. An exemplary set of hypermethylation variable target regions based on colorectal cancer (CRC) studies is provided in Table 3. Many of these genes likely have relevance to cancers beyond colorectal cancer; for example, TP53 is widely recognized as a critically important tumor suppressor and hypermethylation-based inactivation of this gene may be a common oncogenic mechanism.

[0351] Table 3. Exemplary Hypermethylation Target Regions based on CRC studies.Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0352] In some embodiments, genomic regions targeted for sequencing comprise a plurality of loci listed in Table 3, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 3. In some embodiments, genomic regions are captured using probes. For example, 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, or in the promoter region of the gene. In some embodiments, the one or more probes bind within 300 bp of the transcription start site of a gene in Table 3, e.g., within 200 or 100 bp.

[0353] Methylation variable target regions in various types of lung cancer are discussed in detail, e.g., in Ooki et al., Clin. Cancer Res. 23:7141-52 (2017); Belinksy, Annu. Rev. Physiol. 77:453-74 (2015); Hulbert et al., Clin. Cancer Res. 23: 1998-2005 (2017); Shi et al., BMC Genomics 18:901 (2017); Schneider et al., BMC Cancer. 11 : 102 (2011); Lissa et al., Transl Lung Cancer Res 5(5):492-504 (2016); Skvortsova et al., Br. J. Cancer. 94(10): 1492- 1495 (2006); Kim et al., Cancer Res. 61 :3419-3424 (2001); Furonaka et al., Pathology International 55:303-309 (2005); Gomes et al., Rev. Port. Pneumol. 20:20-30 (2014); Kim et al., Oncogene. 20: 1765-70 (2001); Hopkins-Donaldson et al., Cell Death Differ. 10:356-64 (2003); Kikuchi et al., Clin. Cancer Res. 11 :2954-61 (2005); Heller et al., Oncogene 25:959- 968 (2006); Licchesi et al., Carcinogenesis. 29:895-904 (2008); Guo et al., Clin. Cancer Res. 10:7917-24 (2004); Palmisano et al., Cancer Res. 63:4620-4625 (2003); and Toyooka et al., Cancer Res. 61 :4556-4560, (2001).Atty. Docket No. GH0213WO / 01228-0067-00PCT

[0354] An exemplary set of hypermethylation variable target regions based on lung cancer studies is provided in Table 4. Many of these genes likely have relevance to cancers beyond lung cancer; for example, Casp8 (Caspase 8) is a key enzyme in programmed cell death and hypermethylation-based inactivation of this gene may be a common oncogenic mechanism not limited to lung cancer. Additionally, a number of genes appear in both Tables 1 and 2, indicating generality.Table 4. Exemplary Hypermethylation Target Regions based on Lung Cancer studies

[0355] Any of the foregoing embodiments concerning target regions identified in Table 4 may be combined with any of the embodiments described above concerning target regions identified in Table 3. In some embodiments, genomic regions targeted for sequencingAtty. Docket No. GH0213WO / 01228-0067-00PCT comprise a plurality of loci listed in Table 3 or Table 4, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 3 or Table 4.

[0356] Additional hypermethylation target regions may be obtained, e.g., from the Cancer Genome Atlas. Kang et al., Genome Biology 18:53 (2017), describe construction of a probabilistic method called CancerLocator using hypermethylation target regions from breast, colon, kidney, liver, and lung. In some embodiments, the hypermethylation target regions can be specific to one or more types of cancer. Accordingly, in some embodiments, the hypermethylation target regions include 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.

[0357] In some embodiments, an epigenetic target region comprises a hypomethylation variable target region. In some embodiments, the hypomethylation variable target regions are exclusively hypomethylated in one or more related cell or tissue types. 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 related cell or tissue types.

[0358] In some embodiments, where different epigenetic target regions are captured, the epigenetic target regions comprise hypermethylation and / or hypomethylation variable target regions.

[0359] Further exemplary hypermethylation variable target regions and hypomethylation variable target regions useful for distinguishing between various cell types have been identified by analyzing DNA obtained from various cell types via whole gnome bisulfite sequencing, as described, e.g., in Scott, C.A., Duryea, J.D., MacKay, H. etal., “Identification of cell type-specific methylation signals in bulk whole genome bisulfite sequencing data,” Genome Biol 21, 156 (2020) (doi.org / 10.1186 / sl3059-020-02065-5). Whole-genome bisulfite sequencing data is available from the Blueprint consortium, available on the internet at dcc.blueprint-epigenome.eu.CTCF binding regions

[0360] In some embodiments, an epigenetic target region comprises CTCF binding regions. CTCF is a DNA-binding protein that contributes to chromatin organization and often colocalizes with cohesin. Perturbation of CTCF binding sites has been reported in a variety of different cancers. See, e.g., Katainen et al., Nature Genetics, doi: 10.1038 / ng.3335, published online 8 June 2015; Guo et al., Nat. Commun. 9: 1520 (2018). CTCF binding results in recognizable patterns in cfDNA that can be detected by sequencing, e.g., through fragment length analysis. Thus, perturbations of CTCF binding result in variation in the fragmentationAtty. Docket No. GH0213WO / 01228-0067-00PCT patterns of cfDNA. As such, CTCF binding sites are a type of fragmentation variable target region.

[0361] There are many known CTCF binding sites. See, e.g., the CTCFBSDB (CTCF Binding Site Database), available on the Internet at insulatordb.uthsc.edu / ; Cuddapah et al., Genome Res. 19:24-32 (2009); Martin et al., Nat. Struct. Mol. Biol. 18:708-14 (2011); Rhee et al., Cell. 147: 1408-19 (2011), each of which are incorporated by reference. Exemplary CTCF binding sites are at nucleotides 56014955-56016161 on chromosome 8 and nucleotides 95359169-95360473 on chromosome 13.

[0362] In some embodiments, the CTCF binding regions comprise at least 10, 20, 50, 100, 200, or 500 CTCF binding regions, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 CTCF binding regions, 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, at least some of the CTCF sites can be methylated or unmethylated, wherein the methylation state is correlated with the whether or not the cell is a cancer cell. In some embodiments, the epigenetic target region comprises at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, at least 1000 bp upstream and downstream regions of the CTCF binding sites.Transcription start sites

[0363] In some embodiments, an epigenetic target region comprises variable transcription start sites. Transcription start sites may show perturbations in neoplastic cells. For example, nucleosome organization at various transcription start sites in healthy cells of the hematopoietic lineage — which contributes substantially to cfDNA in healthy individuals — may differ from nucleosome organization at those transcription start sites in neoplastic cells. This results in different cfDNA patterns that can be detected by sequencing, as discussed generally in Snyder et al., Cell 164:57-68 (2016); WO 2018 / 009723; and US20170211143 Al. In another example, transcription start sites may not necessarily differ epigenetically in cancerous tissue relative to DNA from healthy tissue of the same type, but do differ epigenetically (e.g., with respect to nucleosome organization) relative to DNA that is typical in healthy subjects. Perturbations of transcription start sites also result in variation in the fragmentation patterns of cfDNA. As such, transcription start sites are also a type of fragmentation variable target regions.

[0364] Human transcriptional start sites are available from DBTSS (DataBase of Human Transcription Start Sites), available on the Internet at dbtss.hgc.jp and described in Yamashita et al., Nucleic Acids Res. 34(Database issue): D86-D89 (2006), which is incorporated hereinAtty. Docket No. GH0213WO / 01228-0067-00PCT by reference. In some embodiments, the transcriptional start sites comprise 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, at least some of the transcription start sites can be methylated or unmethylated, wherein the methylation state is correlated with whether or not the cell is a cancer cell. In some embodiments, the epigenetic target region comprises at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, at least 1000 bp upstream and downstream regions of the transcription start sites.Focal amplifications

[0365] 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 and may comprise one or more of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAFI.Methylation control regions

[0366] It can be useful to include control regions to facilitate data validation. In some embodiments, the epigenetic target region includes control regions that are expected to be methylated or unmethylated in essentially all samples, regardless of whether the DNA is derived from a cancer cell or a normal cell. In some embodiments, the epigenetic target region includes control hypomethylated regions that are expected to be hypomethylated in essentially all samples. In some embodiments, the epigenetic target region includes control hypermethylated regions that are expected to be hypermethylated in essentially all samples.Sequence-variable target regions

[0367] In some embodiments, a target region is or comprises a sequence-variable target region. Sequence-variable target regions may comprise one or more types of target regions likely to differentiate DNA from neoplastic (e.g., tumor or cancer) cells and from healthy cells, e.g., non-neoplastic circulating cells. Exemplary types of such regions are discussed in detail herein. The sequence-variable target region may also comprise one or more control regions, e.g., as described herein. In some embodiments, a sequence-variable target region comprises a plurality of regions known to undergo somatic mutations in cancer. In some aspects, the sequence-variable target region targets a plurality of different genes or genomic regions (“panel”) selected such that a determined proportion of subjects having a cancerAtty. Docket No. GH0213WO / 01228-0067-00PCT 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. 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.

[0368] Examples of listings of genomic locations of interest may be found in, e.g., Table 5 and Table 6 herein. In some embodiments, a sequence-variable target region comprises portions 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 5. In some embodiments, a sequence-variable target region comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 of the SNVs of Table 5. In some embodiments, a sequence-variable target region comprises portions of at least 1, at least 2, at least 3, at least 4, at least 5, or 6 of the fusions of Table 5. In some embodiments, a sequence-variable target region comprises at least portions of at least 1, at least 2, or 3 of the indels of Table 5. In some embodiments, a sequence-variable target region comprises portions 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 6. In some embodiments, a sequence-variable target region comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the SNVs of Table 6. In some embodiments, a sequence-variable target region comprises portions of at least 1, at least 2, at least 3, at least 4, at least 5, or 6 of the fusions of Table 6. In some embodiments, a sequence-variable target region comprises at least portions 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 6. Each of these genomic locations of interest may be identified as a backbone region or hot-spot region for a given panel. Table 7 shows an example listing of hot-spot genomic locations of interest. In some embodiments, a sequence-variable target region comprises portions 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 atAtty. Docket No. GH0213WO / 01228-0067-00PCT least 20 of the genes of Table 7. Each hot-spot genomic region is listed with the associated gene, chromosome on which it resides, the start and stop position of the genome representing the gene’s locus, the length of the gene’s locus in base pairs, the exons covered by the gene, and the critical feature (e.g., type of mutation) of a given genomic region of interest.Table 5Table 6Aty. Docket No. GH0213WO / 01228-0067-00PCTTable 7Aty. Docket No. GH0213WO / 01228-0067-00PCTAtty. Docket No. GH0213WO / 01228-0067-00PCT

[0369] Examples of listings of target regions of interest may also be found in WO 2020 / 160414, e.g., at Table 4. Additional examples include loci disclosed in Gale et al., PLoS One 13: eO 194630 (2018), incorporated herein by reference, which describes a panel of 35 cancer-related gene targets: AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESRI, 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. In some embodiments, the sequencevariable target region comprises target regions from at least 10, 20, 30, or 35 cancer-related genes, such as the cancer-related genes listed herein and in WO 2020 / 160414.

[0370] In some embodiments, the sequence-variable target region 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 has a footprint in the range of 100-2000 kbp, e.g., 100-200 kbp, 200-300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900-1,000 kbp, 1-1.5 Mbp or 1.5-2 Mbp. In some embodiments, the sequence-variable target region has a footprint of at least 2 Mbp.Collections of target-specific probes

[0371] In some embodiments, a collection of target-specific probes is used in methods described herein. In some embodiments, the collection of target-specific probes comprises target-binding probes specific for a sequence-variable target region and target-binding probes specific for an epigenetic target region. In some embodiments, the capture yield of the targetbinding probes specific for the sequence-variable target region is higher (e.g., at least 2-fold higher) than the capture yield of the target-binding probes specific for the epigenetic target region. In some embodiments, the collection of target-specific probes is configured to have a capture yield specific for the sequence-variable target region higher (e.g., at least 2-fold higher) than its capture yield specific for the epigenetic target region.

[0372] In some embodiments, the capture yield of the target-binding probes specific for the sequence-variable target region 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. In some embodiments, the capture yield of the target-binding probes specific for the sequence-variable target region 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-Atty. Docket No. GH0213WO / 01228-0067-00PCT 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.

[0373] In some embodiments, the collection of target-specific probes is configured to have a capture yield specific for the sequence-variable target region 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. In some embodiments, the collection of target-specific probes is configured to have a capture yield specific for the sequence-variable target region 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.

[0374] The collection of probes can be configured to provide higher capture yields for the sequence-variable target region 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.

[0375] In some embodiments, the target-specific probes specific for the sequence-variable target region are present at a higher concentration than the target-specific probes specific for the epigenetic target region. In some embodiments, concentration of the target-binding probes specific for the sequence-variable target region 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-binding probes specific for the epigenetic target region. In some embodiments, the concentration of the target-binding probes specific for the sequencevariable target region 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. In such embodiments, concentration may refer to the average mass per volume concentration of individual probes in each set.

[0376] In some embodiments, the target-specific probes specific for the sequence-variable target region have a higher affinity for their targets than the target-specific probes specific for the epigenetic target region. Affinity can be modulated in any way known to those skilled in the art, including by using different probe chemistries. For example, certain nucleotideAtty. Docket No. GH0213WO / 01228-0067-00PCT modifications, such as cytosine 5-methylation (in certain sequence contexts), modifications that pr...

Claims

Attv. Docket No. GH0213WO / 01228-0067-00PCTWhat is claimed is:

1. A method of analyzing DNA in a sample from a subject, comprising: measuring in the sample or a subsample thereof a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation that the one or more corresponding DMRs do not comprise.

2. The method of claim 1, wherein the one or more variant DMRs and the one or more corresponding DMRs are sequenced.

3. The method of claim 1 or 2, wherein the one or more variant DMRs and the one or more corresponding DMRs are sequenced via whole-genome sequencing (WGS).

4. A method of analyzing DNA in a sample from a subject, comprising: identifying in DNA from the subject one or more variant differentially methylated regions (DMRs), wherein the one or more variant DMRs comprise a somatic mutation; and measuring in the sample or a subsample thereof a level of methylation of the one or more variant DMRs independently of one or more corresponding DMRs not comprising the somatic mutation.

5. A method of analyzing DNA in a sample from a subject, comprising: partitioning the DNA into at least first and second subsamples, wherein the first subsample comprises methylated DNA to a greater extent than the second subsample; measuring in the first subsample a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation.

6. The method of claim 5, wherein the DNA is partitioned based on the presence or absence of 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) nucleic acid bases in the nucleic acids.

7. A method of analyzing DNA in a sample from a subject, comprising: subjecting the DNA or a subsample thereof to a conversion procedure that converts a first nucleobase in the DNA and does not convert a second nucleobase, wherein the firstAttv. Docket No. GH0213WO / 01228-0067-00PCT nucleobase is a modified or unmodified cytosine, the second nucleobase is a modified or unmodified cytosine different from the first nucleobase, thereby providing a converted sample; measuring in the converted DNA a level of methylation of one or more variant differentially methylated regions (DMRs) independently of one or more corresponding DMRs, wherein the one or more variant DMRs comprise a somatic mutation and the one or more corresponding DMRs do not comprise the somatic mutation.

8. The method of any one of claims 1-4 or 6, further comprising partitioning the sample based on the presence or absence of 5 -methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) nucleic acid bases in the nucleic acids.

9. The method of any one of claim 1, 4, or 5, further comprising identifying in DNA from the subject one or more variant DMRs.

10. The method of the immediately preceding claim, wherein the one or more variant DMRs is identified using a comparison to a germline sequence.

11. The method of the immediately preceding claim, wherein the germline sequence is obtained from the same sample or subsample from the subject.

12. The method of any one of claims 10-12, wherein the germline sequence is obtained from a sample or subsample obtained at an earlier time from the subject.

13. The method of any one of claims 10-12, wherein the germline sequence is obtained from a blood sample from the subject.

14. The method of any one of claims 10-12, wherein the germline sequence is obtained from a plasma sample from the subject.

15. The method of any one of claims 10-12, wherein the germline sequence is obtained from a buffy coat sample from the subject.

16. The method of any one of claims 10-12, wherein the germline sequence is obtained from a tumor sample from the subject.

17. The method of claim 16, wherein the method comprises partitioning the tumor sample and wherein the one or more variant DMRs are identified by sequencing the tumor sample.Attv. Docket No. GH0213WO / 01228-0067-00PCT18. The method of the immediately preceding claim, wherein the one or more variant DMRs are identified by comparing the mutations identified in methylation-enriched tumor sequencing to mutations identified in the germline sequence.

19. The method of the immediately preceding claim, wherein the one or more variant DMRs comprise one or more mutations identified in the methylation-enriched tumor sequencing that are not identified in the germline sequence.

20. The method of any one of claims 1-19, wherein the one or more variant and corresponding DMRs are specific to a cancer and / or a tissue.

21. The method of any one of preceding claims, wherein the level of methylation is measured using single stranded methylation conversion (SSM).

22. The method of any one of claims 1-20, wherein the level of methylation is determined based on partitioning of the DNA via methyl-specific enrichment probes.

23. The method of any one of claims 1-22, wherein the somatic mutation is a passenger mutation.

24. The method of any one of claims 1-23, wherein the somatic mutation comprises a single nucleotide variant (SNV).

25. The method of any one of claims 1-23, wherein the somatic mutation comprises an insertion or deletion (InDei).

26. The method of any one of claims 1-23, wherein the somatic mutation comprises a copy number variation (CNV).

27. The method of any one of preceding claims, wherein the somatic mutation is not present in healthy cells of the subject.

28. The method of any one of preceding claims, wherein the somatic mutation is present in cancer cells of the subject.

29. The method of any one of preceding claims, wherein the somatic mutation is not due to clonal hematopoiesis of indeterminate potential (CHIP).Attv. Docket No. GH0213WO / 01228-0067-00PCT30. The method of claim 7, wherein the conversion procedure selectively converts the base pairing specificity of 5-methylcytosines (5mC) or unmethylated cytosines (C) in the sample.

31. The method of claim 7 or 30, wherein the conversion procedure comprises contacting the DNA in the sample with a deaminase.

32. The method of any one of claims 7, 30 or 31, wherein the conversion procedure comprises contacting the DNA in the sample with a Tet enzyme and a deaminase.

33. The method of claim 31 or 32, wherein the deaminase enzyme is APOBEC3A.

34. The method of any one of claims 7 or 30-33, wherein unmethylated CpGs in the DNA are converted to UpGs.

35. The method of claim 31 or 32, wherein the deaminase is a dsDNA deaminase.

36. The method of claim 31 or 32, wherein the deaminase is a ssDNA deaminase.

37. The method of claim 31 or 32, wherein the deaminase is a methyl-insensitive deaminase.

38. The method of the immediately preceding claim, wherein the methyl -insensitive deaminase is A3 A.

39. The method of claim 31 or 32, wherein the deaminase is a methyl-sensitive deaminase.

40. The method of the immediately preceding claim, wherein the methyl-sensitive deaminase is a modification-sensitive DNA deaminase A (MsddA) or a modification-sensitive DNA deaminase A (MsddA)-like deaminase.

41. The method of claim 7, wherein the conversion procedure comprises Tet-assisted conversion of nucleic acids with a substituted borane reducing agent, wherein 5hmC nucleic acid bases are protected from conversion, optionally through glucosylation.

42. The method of the immediately preceding claim, wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, ammonia borane or pyridine borane.

43. The method of claim 30, wherein the conversion procedure comprises:(a) reacting the nucleic acids with a variant methyltransferase having carboxymethyltransferase activity in the presence of carboxy - -adenosyl-Z-Attv. Docket No. GH0213WO / 01228-0067-00PCT methionine (CxSAM) substrate, thereby labelling any unmethylated C and rendering it resistant to deaminase action, wherein 5hmC nucleic acid bases are protected from conversion through glucosylation; and(b) contacting the nucleic acids of step (a) with a deaminase enzyme.

44. The method of the immediately preceding claim, wherein the variant methyltransferase having carboxymethylase activity is a recombinant M.Mpel N374K.

45. The method of claim 7, wherein the conversion procedure selectively converts the base pairing specificity of unmethylated cytosines (C) in the nucleic acids.

46. The method of the immediately preceding claim, wherein the conversion procedure is bisulfite conversion.

47. The method of any one of preceding claims, wherein the methylation status of the nucleic acids in the sample is determined by analyzing the base coverage of cytosines in a reference sequence.

48. The method of any one of preceding claims, wherein the DNA comprises adapters.

49. The method of any one of the preceding claims, wherein the DNA comprises barcodes.

50. The method of any one of the preceding claims, wherein the method further comprises ligating adapters comprising barcodes to the DNA.

51. The method of claim 50, wherein the method comprises ligating adapters comprising barcodes to the DNA prior to the amplifying.

52. The method of claim 50, wherein the method comprises ligating adapters comprising barcodes to the DNA prior to the contacting the DNA with the deaminase.

53. The method of claim 48, wherein the method comprises ligating adapters to the DNA after cutting the DNA.

54. The method of any one of claims 48-53, wherein the adapters are Y-shaped adapters.

55. The method of claim 7, further comprising, prior to the conversion procedure subjecting the DNA in the sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using deoxynucleotide triphosphates (dNTPs), wherein at least one type of dNTP comprises a modified base, and the at least one dNTP comprisingAttv. Docket No. GH0213WO / 01228-0067-00PCT a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

56. The method of claim 55, wherein the end repair is performed using a DNA polymerase that does not have 5 ’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.

57. The method of claim 55, wherein the end repair is performed using a DNA polymerase that has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

58. The method of any one of claims 55 to 57, wherein the at least one type of dNTP which comprises a modified base, wherein the modified base includes a dNTP comprising 4- methylcytosine (4mC), a dNTP comprising 5-methylcytosine (5mC), a dNTP comprising 5-hydroxymethyl-cytosine (5hmC), a dNTP comprising N6-methyladenosine (6mA), a dNTP comprising bromodeoxyuridine (BrdU) and / or a dNTP comprising 8-oxoguanine (8oxoG).

59. The method of any one of claims 55 to 58, further comprising performing an A-tailing reaction, optionally after a step of subjecting a DNA sample to end repair.

60. The method of claim 59, wherein the end-repair and the A-tailing reaction are performed in the same reaction mixture, optionally wherein the end-repair and the A-tailing reaction are performed a single tube and / or optionally wherein the end-repair and the A-tailing reaction are performed without an intervening clean-up step.

61. The method of claim 59, wherein the A-tailing is performed using a DNA polymerase that does not possess 5 ’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase, optionally wherein the DNA polymerase is HemoKlen Taq.

62. The method of claim 59, wherein the A-tailing is performed using a thermostable DNA polymerase.

63. The method of any one of claims 7, 30-46, or 55-62, further comprising, prior to the conversion procedure, performing a methylation-preserving amplification of the DNA of the sample.

64. The method of the immediately preceding claim, wherein the methylation-preserving amplification is a linear, methylation-preserving amplification.Attv. Docket No. GH0213WO / 01228-0067-00PCT65. The method of claim 63 or 64, wherein the methylation-preserving amplification comprises contacting the DNA with a methyltransferase.

66. The method of any one of claims 63-65, wherein the methylation-preserving amplification comprises one or more of polymerase chain reaction, linear amplification, rolling circle amplification, ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustained sequence-based replication.

67. The method of any one of claims 63-66, wherein the methylation-preserving amplification comprises thermocycled amplification.

68. The method of any one of claims 63-67, wherein the methylation-preserving amplification comprises isothermal amplification.

69. The method of any one of the preceding claims, further comprising sequencing at least a portion of the amplified DNA.

70. The method of any one of the preceding claims, further comprising quantifying a level of methylation at one or more differentially methylated regions of the DNA.

71. The method of the immediately preceding claim, wherein quantifying 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.

72. The method of claim 69, wherein the sequencing is next-generation sequencing (NGS).

73. The method of the immediately preceding claim, wherein the NGS is pyrosequencing, sequencing-by -synthesis, semiconductor sequencing, sequencing-by-ligation, or sequencing-by -hybridization.

74. The method of claim 69, wherein the sequencing comprises nanopore-based sequencing or single-molecule real time (SMRT) sequencing.

75. The method of any one of the preceding claims, further comprising enriching the DNA for a plurality of target regions, optionally prior to the step of sequencing the DNA.

76. The method of claim 75, wherein the plurality of target regions comprises epigenetic target regions.Attv. Docket No. GH0213WO / 01228-0067-00PCT77. The method of claim 76, wherein the epigenetic target regions comprise hypermethylation variable target regions.

78. The method of claim 76 or 77, wherein the epigenetic target regions comprise hypomethylation variable target regions.

79. The method of any one of claims 75-79, wherein the plurality of target regions comprises sequence-variable target regions.

80. The method of the immediately preceding claim, wherein the DNA comprises cell-free DNA.

81. The method of any one of claims 1-80, wherein the DNA comprises DNA from formalin fixed paraffin embedded samples.

82. The method of any one of claims 1-81, wherein the subject is an animal.

83. The method of the immediately preceding claim, wherein the subject is a human.

84. The method of any one of claims 1-81 wherein the subject has or is at risk of having a cancer.

85. The method of the immediately preceding claim, wherein the cancer has low aberrant hypermethylation.

86. The method of the immediately preceding claim, wherein the cancer is early stage, stage 0 cancer, stage 1 cancer, non-metastatic, or carcinoma in situ.

87. The method of any one of claims 1-86, further comprising determining the presence or status of a cancer in the subject.

88. The method of the immediately preceding claim, wherein the cancer is identified based on the somatic mutation.

89. The method of any one of claims 1-88, further comprising characterizing a cancer or tumor of the subject.

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