[go: up one dir, main page]

WO2025207924A1 - Procédés de désamination sélective utilisant des protéines de liaison à cpg - Google Patents

Procédés de désamination sélective utilisant des protéines de liaison à cpg

Info

Publication number
WO2025207924A1
WO2025207924A1 PCT/US2025/021812 US2025021812W WO2025207924A1 WO 2025207924 A1 WO2025207924 A1 WO 2025207924A1 US 2025021812 W US2025021812 W US 2025021812W WO 2025207924 A1 WO2025207924 A1 WO 2025207924A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
sample
dntp
sequencing
deaminase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/021812
Other languages
English (en)
Inventor
Andrew Kennedy
Annie M. DEMAREE
James GHADIALI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guardant Health Inc
Original Assignee
Guardant Health Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guardant Health Inc filed Critical Guardant Health Inc
Publication of WO2025207924A1 publication Critical patent/WO2025207924A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • the disclosure relates to methods for enrichment of methylated DNA using an mCpG- binding protein and selective deamination of DNA that is not bound to an mCpG-binding protein. Such methods can be useful for accurately detecting the methylation status and variants present in DNA, which, in turn, can be used to infer information about the cells and subject from which the DNA sample is derived.
  • the DNA molecule is from a subject having or suspected of having a disease or disorder, such as cancer.
  • epigenetically modified (e.g., methylated) DNA in certain DNA samples may change upon carcinogenesis.
  • sufficiently sensitive epigenetic (e.g., DNA methylation) profiling can be used to detect aberrant methylation in DNA of a sample.
  • the present disclosure aims to meet the need for improved analysis of methylated DNA, such as in a cfDNA sample, provide other benefits, or at least provide the public with a useful choice.
  • the present disclosure provides methods for enriching methylated DNA in a sample through selective deamination of DNA using a methyl CpG-binding protein (mCpG-binding protein) to selectively block deaminase activity on methylated cytosines.
  • the methylated cytosines are in CpG dinucleotides.
  • the disclosed methods can improve enrichment and analysis of methylated DNA in a sample by contacting DNA in the sample with a mCpG-binding protein and selectively deaminating DNA in the sample with a deaminase.
  • Embodiment 1.1 is a method of selectively deaminating DNA in a sample, the method comprising: (a) contacting the DNA in the sample with an mCpG-binding protein, thereby providing mCpG-bound DNA; and (b) contacting the mCpG-bound DNA with a deaminase, thereby providing a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs.
  • Embodiment 1.2 is the method of the immediately preceding embodiment, wherein the mCpG-binding protein preferentially binds to methylated CpG dinucleotides relative to unmethylated CpG dinucleotides.
  • Embodiment 1.3 is the method of embodiment 1.1 or 1.2, wherein the CpG-binding protein comprises mCpG-binding domain 4 (MBD4), mCpG-binding domain 2 (MBD2), mCpG- binding domain 1 (MBD1), or methyl CpG binding protein 2 (MeCP2).
  • MBD4 mCpG-binding domain 4
  • MBD2 mCpG-binding domain 2
  • MBD1 mCpG- binding domain 1
  • MeCP2 methyl CpG binding protein 2
  • Embodiment 5 is the method of the immediately preceding embodiment, wherein the capture moiety comprises biotin, avidin, streptavidin, neutravidin, an oligonucleotide, digoxygenin, a histidine tag, an affinity tag, an immunoglobulin constant domain, a hapten, or a magnetic particle.
  • the capture moiety comprises biotin, avidin, streptavidin, neutravidin, an oligonucleotide, digoxygenin, a histidine tag, an affinity tag, an immunoglobulin constant domain, a hapten, or a magnetic particle.
  • Embodiment 6 is the method of any one of the preceding, wherein the mCpG-binding protein is immobilized on a solid support.
  • Embodiment 8 is the method of the immediately preceding embodiment, wherein the separating is performed before step (b).
  • Embodiment 14 is the method of any one of the preceding embodiments, further comprising eluting the DNA in the converted sample, thereby providing eluted DNA.
  • Embodiment 18 is the method of the immediately preceding embodiment, wherein the first partitioned subsample is differentially tagged relative to the second partitioned subsample.
  • Embodiment 19 is the method of any one of embodiments 17-18, wherein the partitioning uses the mCpG-binding protein to separate mCpG-bound DNA from DNA not bound to the mCpG-binding protein.
  • Embodiment 24 is the method of the immediately preceding embodiment, wherein the methyl insensitive deaminase is APOBEC3A.
  • Embodiment 26 is the method of the immediately preceding embodiment, wherein the methyl sensitive deaminase is MsddA.
  • Embodiment 30 is the method of embodiment 27 or 28, 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 31 is the method of any one of embodiments 27-30, 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).
  • 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
  • Embodiment 32 is the method of any one of the preceding embodiments, further comprising performing an A-tailing reaction, optionally after a step of subjecting the DNA in the sample to end repair.
  • Embodiment 33 is the method of the immediately preceding embodiment, 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.
  • Embodiment 34 is the method of embodiment 32 or 33, 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 37 is the method of the immediately preceding embodiment, wherein the methylation-preserving amplification is a linear, methylation-preserving amplification.
  • Embodiment 38 is the method of any one of embodiments 36-37, wherein the methylation-preserving amplification comprises contacting the DNA with a methyltransferase.
  • Embodiment 39 is the method of any one of embodiments 36-38, 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 40 is the method of any one of embodiments 36-38, wherein the methylation-preserving amplification comprises thermocycled amplification.
  • Embodiment 41 is the method of any one of embodiments 36-38, wherein the methylation-preserving amplification comprises isothermal amplification.
  • Embodiment 44 is 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 in the converted sample.
  • Embodiment 45 is 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 46 is the method of any one of embodiments 43-45, wherein the sequencing comprises next-generation sequencing (NGS).
  • NGS next-generation sequencing
  • Embodiment 47 is the method of the immediately preceding embodiment, wherein the NGS comprises pyrosequencing, sequencing-by-synthesis, semiconductor sequencing, sequencing-by-ligation, or sequencing-by-hybridization.
  • Embodiment 50 is the method of any one of embodiments 43-45, wherein the sequencing comprises nanopore-based sequencing.
  • Embodiment 51 is the method of any one of embodiments 43-45, wherein the sequencing comprises 5-letter or 6-letter sequencing.
  • Embodiment 53 is the method of embodiment 43-45 or 48, wherein the sequencing comprises single-molecule real time (SMRT) sequencing and the method comprises subjecting the DNA in the sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base including a dNTP comprising a 4mC, a dNTP comprising 5mC, a dNTP comprising 5hmC, a dNTP comprising 6mA, and/or a dNTP comprising 8oxoG, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.
  • SMRT single-molecule real time
  • Embodiment 54 is the method of any one of embodiments 43-53, further comprising analyzing at least some of the sequence data corresponding to regions that are not identified as being synthesized during the end repair to detect the presence or absence of base modifications or mutations present in the DNA sample.
  • Embodiment 55 is the method of any one of embodiments 43-54, wherein the method further comprises detecting the methylation status of cytosines in the DNA in the sample, and further comprises analyzing the sequence data, wherein the analyzing the sequence data filtering out the one or more repaired regions of the end-repaired DNA molecules such that the one or more repaired regions are not used to determine the methylation status of cytosines in the DNA sample.
  • Embodiment 56 is the method of any one of embodiments 43-54, wherein the method is for detecting the single nucleotide variants (SNVs) in the DNA sample, and further comprises analyzing the sequence data, wherein the analyzing the sequence data comprises classifying all base calls within the one or more end repaired regions as not having double stranded support.
  • Embodiment 57 is the method of any one of embodiments 43-56, wherein the DNA sample comprises cell-free DNA (cfDNA).
  • Embodiment 58 is the method of the immediately preceding embodiment, further comprising analyzing the sequence data to determine a level of measured artifacts in the cfDNA.
  • Embodiment 59 is the method of any one of embodiments 27-51, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is other than 5mC or 5hmC, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.
  • Embodiment 60 is the method of any one of embodiments 27-51, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is a methylated cytosine, optionally wherein the methylated base is 5mC or 5hmC, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.
  • Embodiment 61 is the method of any one of embodiments 27-51, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is a methylated cytosine, optionally wherein the methylated base is 5mC or 5hmC, wherein the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations, and the 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.
  • Embodiment 62 is the method of any one of the preceding embodiments, wherein one or more adapters are ligated to the end-repaired DNA molecules or one or more adapters are ligated to the DNA in the sample.
  • Embodiment 64 is the method of any one of embodiments 16-63, wherein the one or more adapters comprise molecular barcodes.
  • Embodiment 65 is the method of any one of embodiments 16-64, wherein at least one cytosine in the one or more adapters is a modification resistant cytosine, optionally wherein each cytosine in the one or more adapters is a modification resistant cytosine.
  • Embodiment 66 is the method of the immediately preceding embodiment, wherein the modification resistant cytosine is a deaminase resistant cytosine.
  • Embodiment 67 is the method of the immediately preceding embodiment, wherein the deaminase resistant cytosine is 5-propynylC (5pyC), 5-pyrrolo-dC (5pyrC), 5- hydroxymethylcytosine (5hmC), glucosylated5-hydroxymethylcytosine (5ghmC), cytosine 5- methylenesulfonate (CMS), or N4-modified cytosine.
  • the deaminase resistant cytosine is 5-propynylC (5pyC), 5-pyrrolo-dC (5pyrC), 5- hydroxymethylcytosine (5hmC), glucosylated5-hydroxymethylcytosine (5ghmC), cytosine 5- methylenesulfonate (CMS), or N4-modified cytosine.
  • the deaminase resistant cytosine is 5-propynylC (5pyC), 5-pyrrolo-dC (5pyrC), 5- hydroxymethylcytosine (5
  • Embodiment 68 is the method of any one of embodiments 16-67, wherein the one or more adapters are Y-shaped adapters.
  • Embodiment 69 is the method of any one of the preceding embodiments, further comprising contacting the DNA in the sample with a methylation sensitive restriction enzyme (MSRE).
  • MSRE methylation sensitive restriction enzyme
  • Embodiment 70 is the method of the immediately preceding embodiment, wherein the contacting the DNA in the sample with the MSRE occurs after ligating one or more adapters to the end-repaired DNA molecules and/or before contacting the mCpG-bound DNA with the deaminase.
  • Embodiment 71 is the method of the immediately preceding embodiment, wherein the one or more adapters is resistant to digestion by the MSRE.
  • Embodiment 72 is the method of the immediately preceding embodiment, wherein the one or more adapters that is resistant to digestion by the MSRE: i. comprises one or more methylated nucleotides, optionally wherein the methylated nucleotides comprise 5- methylcytosine and/or 5-hydroxymethylcytosine; ii. comprises one or more nucleotide analogs resistant to methylation sensitive restriction enzymes; or iii. does not comprise a nucleotide sequence recognized by the MSRE.
  • Embodiment 73 is the method of any one of the preceding embodiments, wherein the DNA in the sample comprises barcodes.
  • Embodiment 75 is the method of the immediately preceding embodiment, wherein the enriching the DNA occurs prior to a step of amplifying DNA in the converted sample, prior to a step of sequencing the DNA, after contacting the DNA in the sample with the deaminase, and/or after partitioning the DNA in the sample into a plurality of subsamples.
  • Embodiment 76 is the method of embodiment 74 or 75, wherein the plurality of target regions comprises epigenetic target regions.
  • Embodiment 77 is the method of embodiment 76, wherein the epigenetic target regions comprise hypermethylation variable target regions.
  • Embodiment 78 is the method of embodiment 76 or 77, wherein the epigenetic target regions comprise hypomethylation variable target regions.
  • Embodiment 79 is the method of any one of embodiments 74-78, wherein the plurality of target regions comprise sequence-variable target regions.
  • Embodiment 80 is the method of any one of the preceding embodiments, wherein the sample comprises cell-free DNA (cfDNA) or DNA from formalin fixed paraffin embedded samples.
  • cfDNA cell-free DNA
  • DNA from formalin fixed paraffin embedded samples.
  • Embodiment 81 is the method of the immediately preceding embodiment, wherein the sample comprises cell -free DNA.
  • Embodiment 82 is the method of any one of the preceding embodiments, wherein the sample is a blood sample and/or a tissue sample.
  • Embodiment 83 is the method of the immediately preceding embodiment, wherein the blood sample is a whole blood sample, a plasma sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample.
  • Embodiment 84 is the method of any one of the preceding embodiments, wherein the sample is from a subject.
  • Embodiment 85 is the method of any one of the preceding embodiments, wherein the sample is from a subject and the method further comprises determining the presence or absence of cancer in the subject based at least in part on the sequencing data.
  • Embodiment 86 is the method of any one of embodiments 84-85, wherein the subject is an animal.
  • Embodiment 87 is the method of the immediately preceding embodiment, wherein the subject is a human.
  • Embodiment 88 is the method of any one of embodiments 84-87, wherein the subject has or is at risk of having a cancer.
  • Embodiment 89 is the method of any one of embodiments 84-88, further comprising determining the presence or status of a cancer in the subject.
  • the results of the methods disclosed herein are used as an input to generate a report.
  • the report may be in a paper or electronic format.
  • the true methylation status of cytosines or variants, as obtained by the methods disclosed herein, or information derived therefrom, can be displayed directly in such a report.
  • diagnostic information or therapeutic recommendations which are at least in part based on the methods disclosed herein can be included in the report.
  • FIG. 1 is a schematic showing an exemplary selective deamination and methylation enrichment method wherein deaminase activity is blocked at methylated DNA sites using a methyl-binding domain (MBD) protein. End-repair and A-tailing reactions are performed on a DNA sample, followed by ligation of next-generation sequencing (NGS) adapters to the DNA, binding of an MBD protein to at least a portion of the methylated DNA to form a protein- DNA complex, i.e., mCpG-bound DNA, deamination of unbound CpGs in the mCpG-bound DNA using a deaminase, and uracil-tolerant amplification of the DNA.
  • NGS next-generation sequencing
  • FIG. 2 is a schematic showing an exemplary selective deamination methylation enrichment method wherein deaminase activity is blocked at methylated DNA sites using an immobilizable methyl -binding domain (MBD) protein (e.g., MBD2).
  • MBD immobilizable methyl -binding domain
  • End-repair and A-tailing reactions are performed on a DNA sample, followed by ligation of NGS adapters to the DNA, binding of a biotin-immobilized MBD protein to at least a portion of the methylated DNA to form a protein-DNA complex, i.e., mCpG-bound DNA, deamination of unbound CpGs in the mCpG-bound DNA using a deaminase, elution of the immobilized, mCpG-bound, methylated DNA, and uracil-tolerant amplification of the eluted DNA.
  • a protein-DNA complex i.e., mCpG-bound DNA
  • deamination of unbound CpGs in the mCpG-bound DNA using a deaminase
  • elution of the immobilized, mCpG-bound, methylated DNA and uracil-tolerant amplification of the eluted DNA.
  • sequences in the figure are: mCGmCGmCGmCGmCG (SEQ ID NO: 90), mCGmCGmCGCGCG (SEQ ID NO: 91), CGCGmCGmCG (SEQ ID NO: 92), mCGmCGCGCGCG (SEQ ID NO: 96), CGCGCGmCGmCG (SEQ ID NO: 97), mCGCGCGCG (SEQ ID NO: 98), CGCGCGCGmCG (SEQ ID NO: 99), CGCGCGCG (SEQ ID NO: 93), and mCGmCGmCGTGTG (SEQ ID NO: 118), wherein “mC” denotes a methylated cytosine.
  • FIG. 3 is a schematic diagram of an example of a system suitable for use with some embodiments of the disclosure.
  • 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.
  • reaction cleanup refers to the removal of contaminants such as salts, enzymes, unincorporated dNTPs, primers, ethidium bromide, and other impurities that can interfere with downstream analysis.
  • a reaction cleanup when a reaction cleanup is performed between end repair and an A-tailing reaction, it removes unincorporated dNTPs such that the A-tailing reaction can be performed solely in the presence of dATP (z.e. not dCTP, dGTP and dCTP, as used in the end tailing reaction).
  • Reaction cleanups can be performed using commercially available kits such as MinElute Reaction Cleanup Kit (Qiagen)
  • “Repaired regions”, also referred to as “synthesized regions” or “regions of the end-repaired DNA that were synthesized during the end repair” refer to regions of the DNA that were not present in the DNA prior to the end repair and A-tailing reactions. They are regions which have been synthesized by the polymerases used in the end repair and/or A tailing reactions, if present. In instances where the A-tailing is performed in the same tube as the end repair reaction, all four types of dNTPs will be present, and thus the polymerases used for A- tailing may generate synthesized regions, e.g. through nick translation.
  • base pairing specificity refers to the standard DNA base (A, C, G, or T) for which a given base most preferentially pairs.
  • unmodified cytosine and 5 -methyl cytosine 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.
  • “Capable of identifying the base modification in the at least one type of dNTP” refers to the ability of a modification-sensitive sequencing method to detect the presence or absence of the base modification in the at least one type of dNTP comprising a modified base used in the end repair.
  • This detection of the base modification may be direct, such as in nanopore sequencing or single molecule real time sequencing, wherein the sequencing data itself indicates the presence or absence of a base modification.
  • the detection of the base modification may be indirect, for example wherein the method involves a conversion procedure which alters the base pairing specificity dependent on the base modification status. It is these changes in base pairing specificity which can be detected by the sequencing method, e.g. through the comparison of the sequencing data to a reference sequence.
  • a modificationsensitive sequencing method is capable of identifying the base modification in the at least one type of dNTP regardless of whether it can distinguish one base modification from all other base modifications.
  • one form of modification-sensitive sequencing is sequencing after bisulfite conversion. This method is capable of distinguishing 5hmC and 5mC from unmethylated cytosine, but cannot distinguish 5hmC from 5mC.
  • Bases of the “same identity” refer to the same base, regardless of modification status of that base.
  • cytosine is considered to be the “same identity” as 5- methylcytosine (5mC) and/or 5 -hydroxymethyl -cytosine (5hmC), despite them having different modification statuses.
  • Cell-free DNA includes DNA molecules that naturally occur in a subject in extracellular form (e.g., in blood, serum, plasma, or other bodily fluids such as lymph, cerebrospinal fluid, urine, or sputum). While the cfDNA originally existed in a cell or cells in a large complex biological organism, e g., a mammal, it has undergone release from the cell(s) into a fluid found in the organism, and may be obtained from a sample of the fluid without the need to perform an in vitro cell lysis step.
  • cellular nucleic acids means nucleic acids that are disposed within one or more cells from which the nucleic acids have originated, at least at the point a sample is taken or collected from a subject, even if those nucleic acids are subsequently removed (e.g., via cell lysis) as part of a given analytical process.
  • DNA is “derived from cancerous cells” if it originated from a tumor cell.
  • Cell free DNA derived from cancerous cells includes ctDNA or circulating tumor DNA.
  • Tumor cells are neoplastic cells that originated from a tumor, regardless of whether they remain in the tumor or become separated from the tumor (as in the cases, e.g., of metastatic cancer cells and circulating tumor cells).
  • methylation refers to addition of a methyl group to a nucleotide base in a nucleic acid molecule.
  • 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)).
  • DNA methylation refers to addition of a methyl group to adenine, such as in N 6 - methyladenine (6mA).
  • DNA methylation is 5-methylation (modification of the carbon in the 5th position of the cytosine ring).
  • 5-methylation refers to addition of a methyl group to the 5C position of the cytosine to create 5-methylcytosine (5mC).
  • 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).
  • DNA methylation is 3C methylation (modification of the carbon in the 3 rd position of the cytosine ring).
  • 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.
  • the “modified nucleoside profile of DNA” means the position and identity of the nucleoside and the modification status of the nucleoside, such as methylations, within a DNA sequence.
  • different modification sensitive sequencing methods can be used to detect such modifications. This includes methods which involve conversion followed by sequencing detect one or more different types of modified or unmodified nucleoside.
  • the TAPS method detects, but does not distinguish between, 5-methylcytosine (5mC) and 5-hydroxymethyl-cytosine (5hmC).
  • a method for analyzing the modified nucleoside profile of DNA in a sample typically means identifying particular modifications or groups of modification, such as 5mC and/or 5hmC.
  • Modified nucleosides are identified according to the specific method/conversion procedure being used as described above. This generally involves comparing sequence data obtained from DNA that has been subjected to a conversion procedure to a reference sequence. Typically, the method involves (i) comparing the sequence data with (A) one or more pre-determined reference sequence; or (B) sequence data obtained by sequencing a sub-sample of the DNA that was not subjected to the conversion procedure, for example a subsample that was separated before subjecting a separate subsample to the conversion procedure, for example as described herein; and (ii) identifying point differences between the converted DNA sequences and the reference sequence(s) (A) or non-converted DNA sequences (B) as nucleosides (in the initial sample) having a modification status that permits a change in base pairing specificity on exposure to the conversion procedure.
  • 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.
  • nucleobase without substantially altering base-pairing specificity of a given nucleobase means that a majority of molecules comprising that nucleobase that can be sequenced do not have alterations of the base pairing specificity of the second nucleobase relative to its base pairing specificity as it was in the originally isolated sample. In some embodiments, 75%, 90%, 95%, or 99% of molecules comprising that nucleobase that can be sequenced do not have alterations of the base pairing specificity of the second nucleobase relative to its base pairing specificity as it was in the originally isolated sample.
  • base pairing specificity refers to the standard DNA base (A, C, G, or T) for which a given base most preferentially pairs.
  • 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.
  • modified cytosine refers to a cytosine in which at least one position of the cytosine has been substituted with a chemical moiety, such as a methyl or hydroxymethyl, that is different from the substituent at that position in unmodified cytosine.
  • modified cytosine does not include unmodified cytosine.
  • a “combination” comprising a plurality of members refers to either of a single composition comprising the members or a set of compositions in proximity, e.g., in separate containers or compartments within a larger container, such as a multiwell plate, tube rack, refrigerator, freezer, incubator, water bath, ice bucket, machine, or other form of storage.
  • the “capture yield” of a collection of probes for a given target set refers to the amount (e g., amount relative to another target set or an absolute amount) of nucleic acid corresponding to the target set that the collection of probes captures under typical conditions.
  • Exemplary typical capture conditions are an incubation of the sample nucleic acid and probes at 65°C for 10-18 hours in a small reaction volume (about 20 pL) containing stringent hybridization buffer.
  • the capture yield may be expressed in absolute terms or, for a plurality of collections of probes, relative terms.
  • first and second target regions are 50 kb and 500 kb, respectively (giving a normalization factor of 0.1)
  • the DNA corresponding to the first target region set is captured with a higher yield than DNA corresponding to the second target region set when the mass per volume concentration of the captured DNA corresponding to the first target region set is more than 0.1 times the mass per volume concentration of the captured DNA corresponding to the second target region set.
  • the captured DNA corresponding to the first target region set has a mass per volume concentration of 0.2 times the mass per volume concentration of the captured DNA corresponding to the second target region set, then the DNA corresponding to the first target region set was captured with a two-fold greater capture yield than the DNA corresponding to the second target region set.
  • Capturing one or more target nucleic acids refers to preferentially isolating or separating the one or more target nucleic acids from non-target nucleic acids.
  • a “captured set” of nucleic acids refers to nucleic acids that have undergone capture.
  • a “target-region set” or “set of target regions” refers to a plurality of genomic loci targeted for capture and/or targeted by a set of probes (e.g., through sequence complementarity).
  • “Corresponding to a target region set” means that a nucleic acid, such as cfDNA, originated from a locus in the target region set or specifically binds one or more probes for the target-region set.
  • a “differentially methylated region” 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.
  • a DMR has a detectably higher degree of methylation (e.g., a hypermethylated region) in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type or from the same cell or tissue type from a healthy subject.
  • a DMR has a detectably lower degree of methylation (e.g., a hypomethylated region) in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type or from the same cell or tissue type from a healthy subject.
  • binds in the context of an probe or other oligonucleotide and a target sequence means that under appropriate hybridization conditions, the oligonucleotide or probe hybridizes to its target sequence, or replicates thereof, to form a stable probe:target hybrid, while at the same time formation of stable probemon-target hybrids is minimized.
  • a probe hybridizes to a target sequence or replicate thereof to a sufficiently greater extent than to a nontarget sequence, to enable capture or detection of the target sequence.
  • Appropriate hybridization conditions are well-known in the art, may be predicted based on sequence composition, or can be determined by using routine testing methods (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at ⁇ 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly ⁇ 9.50-9.51, 11.12- 11.13, 11.45-11.47 and 11.55-11.57, incorporated by reference herein).
  • Sequence-variable target region set refers to a set of target regions that may exhibit changes in sequence such as nucleotide substitutions (i.e., single nucleotide variations), insertions, deletions, or gene fusions or transpositions in neoplastic cells (e g., tumor cells and cancer cells).
  • Epigenetic target region set refers to a set of target regions that may show sequence-independent changes in neoplastic cells (e.g., tumor cells and cancer cells) or that may show sequence-independent changes in cfDNA from subjects having cancer relative to cfDNA from healthy subjects.
  • sequence-independent changes include, but not limited to, changes in methylation (increases or decreases), nucleosome distribution, CTCF binding, transcription start sites, and regulatory protein binding regions.
  • loci susceptible to neoplasia-, tumor-, or cancer-associated focal amplifications and/or gene fusions may also be included in an epigenetic target region set because detection of a change in copy number by sequencing or a fused sequence that maps to more than one locus in a reference genome tends to be more similar to detection of exemplary epigenetic changes discussed above than detection of nucleotide substitutions, insertions, or deletions, e.g., in that the focal amplifications and/or gene fusions can be detected at a relatively shallow depth of sequencing because their detection does not depend on the accuracy of base calls at one or a few individual positions.
  • hypermethylation refers to an increased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules within a population (e.g., sample) of nucleic acid molecules.
  • 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.
  • hypomethylation refers to a decreased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules within a population (e.g., sample) of nucleic acid molecules.
  • hypomethylated DNA includes unmethylated DNA molecules.
  • 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.
  • methylation status can refer to the presence or absence of methyl group on a DNA base (e.g. cytosine) at a particular genomic position in a nucleic acid molecule. It can also refer to the degree of methylation in a nucleic acid sequence (e.g., highly methylated, low methylated, intermediately methylated or unmethylated nucleic acid molecules). The methylation status can also refer to the number of nucleotides methylated in a particular nucleic acid molecule.
  • 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).
  • SNVs single nucleotide variants
  • Indels insertions or deletions
  • a mutation can be a germline or somatic mutation.
  • 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.
  • neoplasm and “tumor” are used interchangeably. They refer to abnormal growth of cells in a subject.
  • a neoplasm or tumor can be benign, potentially malignant, or malignant.
  • a malignant tumor is referred to as a cancer or a cancerous tumor.
  • next-generation sequencing 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.
  • next-generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization.
  • next-generation sequencing includes the use of instruments capable of sequencing single molecules. Examples of commercially available instruments for performing next-generation sequencing include, but are not limited to, NextSeq, HiSeq, NovaSeq, MiSeq, Ion PGM and Ion GeneStudio S5.
  • nucleic acid tag refers to a short nucleic acid (e.g., less than about 500 nucleotides, about 100 nucleotides, about 50 nucleotides, or about 10 nucleotides in length), used to distinguish nucleic acids from different samples (e.g., representing a sample index), distinguish nucleic acids from different partitions (e g., representing a partition tag) or different nucleic acid molecules in the same sample (e.g., representing a molecular barcode), of different types, or which have undergone different processing.
  • the nucleic acid tag comprises a predetermined, fixed, non-random, random or semi-random oligonucleotide sequence.
  • 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.
  • 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).
  • 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.
  • 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.
  • library adaptors having distinct molecular barcodes encompass library adaptors for uniquely or non-uniquely tagging molecules, in that regardless of whether the adaptors are for unique or non-unique tagging, distinct barcodes will be present in the population of adaptors.
  • DNA that is “not immobilized” or that is “free in solution” refers to DNA that is not bound covalently or non-covalently to a solid support, such as a bead. Such DNA may be free in solution during any step (such as all steps) of the disclosed methods.
  • partitioning refers to physically separating or fractionating a mixture of nucleic acid molecules in a sample based on a characteristic of the nucleic acid molecules.
  • the partitioning can be physical partitioning of molecules. Partitioning can involve separating the nucleic acid molecules into groups or sets based on the level of epigenetic feature (for e.g., methylation). For example, the nucleic acid molecules can be partitioned based on the level of methylation of the nucleic acid molecules.
  • 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.
  • a partitioned set can comprise nucleic acid molecules belonging to a particular level or degree of epigenetic feature (for e.g., methylation).
  • 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 hypom ethylated partitioned set), and a third set for intermediate methylated nucleic acid molecules (third subsample, intermediate partitioned set, intermediately methylated partitioned set, residual partitioned set, or residual partition).
  • 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).
  • a “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.
  • a modified nucleobase is a methylated nucleobase.
  • a modified cytosine is a methyl cytosine, e.g., a 5-methyl cytosine.
  • 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).
  • the DNA may be singlestranded or double-stranded. Suitable agents include agents that recognize modified nucleotides in double-stranded DNA, single-stranded DNA, and both double-stranded and single-stranded DNA
  • polynucleotide refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages.
  • a polynucleotide comprises at least three nucleosides. Oligonucleotides often range in size from a few monomeric units, e.g., 3-4, to hundreds of monomeric units.
  • a polynucleotide is represented by a sequence of letters, such as “ATGCCTG”, the nucleotides are in 5’ -> 3’ order from left to right, and in the case of DNA, “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes deoxythymidine, unless otherwise noted.
  • the letters A, C, G, and T may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases.
  • processing refers to a set of steps used to generate a library of nucleic acids that is suitable for sequencing.
  • the set of steps can include, but are not limited to, partitioning, end repairing, addition of sequencing adapters, tagging, and/or PCR amplification of nucleic acids.
  • quantitative measure refers to an absolute or relative measure.
  • a quantitative measure can be, without limitation, a number, a statistical measurement (e.g., frequency, mean, median, standard deviation, or quantile), or a degree or a relative quantity (e.g., high, medium, and low).
  • a quantitative measure can be a ratio of two quantitative measures.
  • a quantitative measure can be a linear combination of quantitative measures.
  • a quantitative measure may be a normalized measure.
  • reference sequence refers to a known sequence used for purposes of comparison with experimentally determined sequences.
  • a known sequence can be an entire genome, a chromosome, or any segment thereof.
  • a reference sequence can align with a single contiguous sequence of a genome or chromosome or chromosome arm or can include non-contiguous segments that align with different regions of a genome or chromosome. Examples of reference sequences include, for example, human genomes, such as, hgl9 and hg38.
  • sample means anything capable of being analyzed by the methods and/or systems disclosed 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.
  • 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, solidphase 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, singlemolecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiDTM sequencing, MS-PET sequencing, and a combination thereof.
  • sequencing can be performed by a gene
  • sequence information in the context of a nucleic acid polymer means the order and identity of monomer units (e.g., nucleotides, etc.) in that polymer.
  • sequence-variable target region set refers to a set of target regions that may exhibit changes in sequence such as nucleotide substitutions, insertions, deletions, or gene fusions or transpositions in neoplastic cells (e.g., tumor cells and cancer cells).
  • sequence-variable target region set refers to a set of target regions that may exhibit changes in sequence such as nucleotide substitutions, insertions, deletions, or gene fusions or transpositions in neoplastic cells (e.g., tumor cells and cancer cells).
  • sequence-variable target region set refers to a set of target regions that may exhibit changes in sequence such as nucleotide substitutions, insertions, deletions, or gene fusions or transpositions in neoplastic cells (e.g., tumor cells and cancer cells).
  • sequence-variable target region set refers to a set of target regions that may exhibit changes in sequence such as nucleotide substitutions, insertions, deletions, or gene fusions or transposition
  • subject refers to an animal, such as a mammalian species (e.g., human) or avian (e.g., bird) species, or other organism, such as a plant. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companion animals (e.g., pets or support animals).
  • farm animals e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like
  • 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”.
  • 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.
  • the subject can be an individual who is diagnosed of having an autoimmune disease.
  • the subject can be a female individual who is pregnant or who is planning on getting pregnant, who may have been diagnosed of or suspected of having a disease, e.g., a cancer, an auto-immune disease.
  • target-region set or “set of target regions” or “target regions” or “target regions of interest” or “regions of interest” or “genomic regions of interest” refers to a plurality of genomic loci or a plurality of genomic regions targeted for capture and/or targeted by a set of probes (e.g., through sequence complementarity).
  • tumor fraction refers to the proportion of cfDNA molecules that originated from tumor cells for a given sample, or sample-region pair.
  • an “asymmetric adapter” is a double stranded adapter in which the two strands are not completely complementary or are otherwise distinguishable such that synthesis of a complementary sequence of one strand of the adapter results in a sequence that is distinguishable from the sequence of the other strand of the adapter. Examples of asymmetric adapters are Y-shaped adapters and bubble adapters.
  • a “Y-shaped adapter” refers to an adapter comprising two DNA strands comprising complementary and non-complementary parts, wherein the non- complementary parts form single-stranded arms.
  • the adapter can be attached to a sample or insert DNA molecule, e.g., by ligation, such that the complementary (double-stranded) part of the adapter is proximal to the sample or insert DNA molecule.
  • the double stranded portion of the Y-shaped adapter may have a blunt end or an overhang, e.g., of one to three nucleotides.
  • the single stranded arms may or may not be of identical length.
  • a “bubble adapter” refers to an adapter comprising two DNA strands comprising a non-complementary part flanked by complementary parts, such that the adapter has a single stranded region located between double-stranded regions.
  • the adapter can be attached to a sample or insert DNA molecule, e.g., by ligation, such that one of the complementary (double-stranded) parts of the adapter is proximal to the sample or insert DNA molecule.
  • the double stranded portion of the Y-shaped adapter that would be attached to the insert or sample molecule may have a blunt end or an overhang, e.g., of one to three nucleotides.
  • the single stranded portions of the two strands may or may not be of identical length.
  • peripheral blood mononuclear cells 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.
  • amplify refers to a process by which extra or multiple copies of a particular polynucleotide are formed. Amplification methods can include any suitable methods known in the art. As used herein, a nucleic acid molecule amplified using “methylation-preserving amplification” substantially maintains its methylation status post-amplification.
  • the polypeptide is the human polypeptide unless indicated otherwise.
  • the polypeptide comprising the XninnXi 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.
  • a “T1372S mutation” in TET2 refers to a substitution in a TET2 enzyme of the threonine present at position 1372 of the full-length wildtype 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.
  • 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.
  • Cancer formation and progression may arise from both genetic modification and epigenetic features of deoxyribonucleic acid (DNA).
  • DNA deoxyribonucleic acid
  • the present disclosure provides methods and systems for analyzing DNA, such as cell-free DNA (cfDNA).
  • cfDNA cell-free DNA
  • the present disclosure provides methods for analyzing epigenetic and/or sequence-variable target regions.
  • cells in or around a cancer or neoplasm may shed more DNA than cells of the same tissue type in a healthy subject.
  • the distribution of tissue of origin of certain DNA samples, such as cfDNA may change upon carcinogenesis.
  • an increase in the level of hypermethylation variable target regions that show lower methylation in healthy cfDNA than in at least one other tissue type can be an indicator of the presence (or recurrence, depending on the history of the subject) of cancer.
  • an increase in the level of hypomethylation variable target regions in the sample can be an indicator of the presence (or recurrence, depending on the history of the subject) of cancer.
  • DNA methylation comprises addition of a methyl group to a cytosine residue 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).
  • DNA methylation comprises addition of a methyl group to an adenine residue, such as in N6- methyladenine.
  • DNA methylation is 5-methylation (modification of the carbon in the 5th position of the cytosine ring).
  • 5-methylation comprises addition of a methyl group to the 5C position of the cytosine residue to create 5-methylcytosine (m5c or 5-mC or 5mC).
  • methylation comprises a derivative of m5c.
  • Derivatives of m5c include, but are not limited to, 5-hydroxymethylcytosine (5-hmC or 5hmC), 5-formylcytosine (5-fC), and 5-caryboxylcytosine (5-caC).
  • DNA methylation is 3C methylation (modification of the carbon in the 3 rd position of the cytosine ring).
  • 3C methylation comprises addition of a methyl group to the 3C position of the cytosine residue 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.
  • the present disclosure provides methods and systems for selectively deaminating DNA, such as cell-free DNA (cfDNA), in a sample.
  • cfDNA cell-free DNA
  • mCpG-bound DNA can be eluted from unbound DNA before the downstream analyses, such that resources used downstream, e.g., during sequencing and analysis, can be more efficiently focused on methylated DNA.
  • the methylated DNA comprises methylated cytosines of CpG dinucleotides.
  • Some embodiments of the disclosed methods of selectively deaminating DNA in a sample comprise: (a) contacting the DNA in the sample with a methyl-CpG (mCpG) binding protein, thereby providing mCpG-bound DNA; and (b) contacting the mCpG-bound DNA with a deaminase.
  • the contacting the mCpG-bound DNA with a deaminase provides a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs.
  • the mCpG-binding domain protein comprises mCpG- binding domain 4 (MBD4), mCpG-binding domain 2 (MBD2), mCpG-binding domain 1 (MBD1), or methyl CpG binding protein 2 (MeCP2).
  • the mCpG-binding protein domain preferentially binds to methylated CpG dinucleotides relative to unmethylated CpG dinucleotides.
  • the mCpG-binding protein has a greater affinity for a methylated CpG dinucleotide relative to an unmethylated CpG dinucleotide.
  • the mCpG- binding protein has an affinity (e.g., Ka) for a methylated CpG dinucleotide that is stronger than the affinity for an unmethylated CpG dinucleotide by a factor of about 10, about 5, about 2, or about 1.5.
  • the mCpG-binding protein comprises mCpG-binding domain 4 (MBD4). In some embodiments, the mCpG-binding protein comprises mCpG-binding domain 2 (MBD2). In some embodiments, the mCpG-binding protein comprises methyl CpG binding protein 2 (MeCP2). In some embodiments, the mCpG-binding protein comprises mCpG-binding domain 1 (MBD1). In some embodiments, the MBD1 contains three internal CXXC zinc finger domains: CXXC-1, CXXC-2, and CXXC-3.
  • MBD1 contains two internal CXXC zinc finger domains: CXXC-1 and CXXC-2 In some embodiments, MBD1 contains internal CXXC zinc finger domains CXXC-1 and CXXC-2 and does not contain CXXC-3.
  • the method of selectively deaminating DNA in a sample further comprises amplifying DNA in the converted sample using a DNA polymerase.
  • the DNA polymerase is a uracil-tolerant polymerase.
  • the uracil -tolerant polymerase may be Q5U® Hot Start High-Fidelity DNA Polymerase, One'/ag" DNA Polymerase, Tag DNA Polymerase, LongAmp® Taq DNA Polymerase, Hemo Klen Taq, Epimark® Hot Start Taq DNA Polymerase, Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, Bst 2.0 DNA Polymerase, Bst 3.0 DNA Polymerase, Bsu DNA Polymerase, Large Fragment, phi29 DNA Polymerase, phi29-XT DNA Polymerase, TherminatorTM DNA Polymerase, DNA Polymerase I (E. coll), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”), Klenow Fragment (3 ' — >5' exo-), or any combination thereof.
  • the mCpG-binding protein comprises a capture moiety.
  • the capture moiety comprises biotin, avidin, streptavidin, neutravidin, an oligonucleotide, digoxygenin, a histidine tag, an affinity tag, an immunoglobulin constant domain, a hapten, or a magnetic particle.
  • the mCpG-binding protein that recognizes a modified nucleobase is immobilized on a solid support.
  • the method of selectively deaminating DNA in a sample further comprises separating the mCpG-bound DNA from unbound DNA using the mCpG- binding protein. In some embodiments, the separating provides methylation-separated DNA. In some embodiments, the separating is performed before contacting the mCpG-bound DNA with a deaminase.
  • the method further comprises separating the converted DNA from mCpG-bound DNA using the mCpG-binding protein. In some embodiments, the separating provides methylation-separated DNA. In some embodiments, the separating is performed after contacting the mCpG-bound DNA with a deaminase.
  • the separating comprises partitioning DNA in the sample into a plurality of partitioned subsamples.
  • the plurality of partitioned subsamples comprises a first partitioned subsample and a second partitioned subsample.
  • the first partitioned subsample comprises methylated DNA in a greater proportion than the second partitioned subsample.
  • the method comprises differentially tagging the first partitioned subsample and the second partitioned subsample.
  • the first partitioned subsample is differentially tagged relative to the second partitioned subsample.
  • the second partitioned subsample comprises hypomethylated DNA.
  • the method further comprises eluting the DNA in the converted sample. In some embodiments, the eluting provides eluted DNA. In some embodiments, the eluted DNA is single-stranded DNA. In some embodiments, the singlestranded DNA is hypomethylated DNA. In some embodiments, the method further comprises ligating one or more adapters to the single-stranded DNA.
  • the method further comprises partitioning the eluted DNA into a plurality of partitioned subsamples.
  • the plurality of partitioned subsamples comprises a first partitioned subsample and a second partitioned subsample.
  • the first partitioned subsample comprises mCpG-bound DNA in a greater proportion than the second partitioned subsample.
  • the first partitioned subsample is differentially tagged relative to the second partitioned subsample.
  • the partitioning uses the mCpG-binding protein to separate mCpG-bound DNA from DNA not bound to the mCpG-binding protein. In some embodiments, the partitioning comprises precipitating the mCpG-bound DNA. In some embodiments, the precipitation of the mCpG-bound DNA separates it from the unbound DNA.
  • the deaminase is thermally inactivated after contacting the mCpG-bound DNA with the deaminase.
  • the thermal inactivation comprises heating or cooling of the deaminase to a temperature at which the deaminase has reduced or inhibited activity relative to a deaminase that has not been subjected to heating or cooling.
  • the thermal inactivation completely inhibits the activity of the deaminase or reduces the activity of the deaminase by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 90%, about 95%, about 98%, about 99%, or 100% relative to a deaminase that has not been subjected to heating or cooling.
  • the deaminase is a double-stranded DNA (dsDNA) deaminase. In some embodiments, the deaminase is a single-stranded DNA (ssDNA) deaminase. In some embodiments, the deaminase is a methyl-insensitive deaminase, including e.g., APOBEC3A (A3A).
  • the deaminase is a methyl-sensitive deaminase, including e.g., modification-sensitive DNA deaminase A (MsddA) or a modification-sensitive DNA deaminase A (MsddA)-like deaminase.
  • MsddA modification-sensitive DNA deaminase A
  • MsddA modification-sensitive DNA deaminase A
  • the population includes nucleic acids having varying levels of sequence variation and/or epigenetic variation, such as post-translation modifications (PTMs) of chromatin and/or nucleobase modifications, e.g., modifications of cytosine, particularly at the 5-position of the nucleobase, e.g., 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine.
  • PTMs post-translation modifications
  • 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 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.
  • 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.
  • Exemplary amounts of cell-free nucleic acids (e.g. cfDNA) 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.
  • the amount can be up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acid molecules.
  • the amount can be at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of cell-free nucleic acid molecules.
  • the amount can be up to 1 femtogram (fg), 10 fg, 100 fg, 1 picogram (pg), 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng, or 200 ng of cell-free nucleic acid molecules.
  • the method can comprise obtaining 1 femtogram (fg) to 200 ng cell-free nucleic acid molecules from samples.
  • Cell-free nucleic acids have an exemplary size distribution of about 100-500 nucleotides, with molecules of 110 to about 230 nucleotides representing about 90% of molecules, with a mode of about 168 nucleotides and a second minor peak in a range between 240 to 440 nucleotides.
  • Cell-free nucleic acids can be isolated from bodily fluids through a fractionation or partitioning step in which cell-free nucleic acids, as found in solution, are separated from intact cells and other non-soluble components of the bodily fluid. Partitioning may include techniques such as centrifugation or filtration. Alternatively, cells in bodily fluids can be lysed and cell-free and cellular nucleic acids processed together. Generally, after addition of buffers and wash steps, nucleic acids can be precipitated with an alcohol. Further clean up steps may be used such as silica-based columns to remove contaminants or salts. Non-specific bulk carrier nucleic acids, DNA or protein for sequencing, hybridization, and/or ligation, may be added throughout the reaction to optimize certain aspects of the procedure such as yield.
  • samples can include various forms of nucleic acid including double stranded DNA, single stranded DNA and single stranded RNA.
  • single stranded DNA and RNA can be converted to double stranded forms so they are included in subsequent processing and analysis steps.
  • the methods disclosed herein are also particularly suited for the analysis of DNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. While the formalin fixation process adequately preserves the ultrastructure of the tissues, it results in various types of damage to the DNA within the tissues, such as nicks in the DNA. As explained elsewhere herein, these nicks can lead to synthesis of regions of the DNA molecule in the end repair process. The methods disclosed herein allow for these regions to be identified and the sequence data to be interpreted accordingly.
  • FFPE formalin-fixed paraffin-embedded
  • Reference or control molecules can be added to or spiked into a sample as a control or normalization standard. For example, a certain amount of modified DNA from a species other than the species of the subject from which the sample was obtained or synthetic nucleic acids comprising certain modifications may be added to the sample. In some embodiments, the reference or control molecules are distinguishable from the molecules originally present in the sample. In some embodiments, the detected DNA sequences are normalized to the reference or control molecules.
  • the disclosed methods comprise subjecting the DNA in the sample to end repair to generate end-repaired DNA molecules.
  • the end repair is performed before contacting the DNA in the sample with an mCpG-binding protein.
  • the end repair is performed before contacting the DNA in the sample with a deaminase.
  • the end repair is performed using deoxy nucleotide triphosphates (dNTPs).
  • dNTPs deoxy nucleotide triphosphates
  • 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.
  • 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 nonblunt 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.
  • Fragmented and/or damaged DNA e.g. cfDNA or DNA from FFPE samples
  • 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.
  • 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.
  • 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.
  • end repair is conducted in the presence of dATP, dCTP, dGTP and dTTP.
  • A-tailing refers to the addition of a single deoxyadenosine residue to the end of a blunt-ended double-stranded DNA fragment to form a 3' deoxyadenosine single-base overhang.
  • 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.
  • 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 compatible with 3' T-overhangs, which can be used on adapters.
  • Methods comprising end repair, A-tailing and ligation to adapters with 3' T-overhangs can result in higher efficiency ligation, compared to blunt ended ligation, as blunt ligation can lead to self-ligation of the adapters and/or DNA molecules.
  • the methods disclosed herein comprise end repair of the DNA molecules followed by blunt end ligation of adapters.
  • the methods disclosed herein comprise end repair of the DNA molecules followed by A-tailing and sticky-end ligation of T-tailed adapters.
  • the methods disclosed herein comprise an A-tailing step, it may be performed separately from the end repair with an intervening reaction clean-up step or it may be performed in the same reaction as the end repair (e.g. using NEBNext® UltraTM II End Repair/dA-Tailing Module (E7546)).
  • the reaction clean-up step removes unincorporated dNTPs.
  • the sticky-end ligation may be performed with a mixture of T-tailed adapters and C-tailed adapters.
  • 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.
  • 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.
  • 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.
  • dCTP non-methylated deoxy cytidine triphosphate
  • both the DNA polymerases used in end repair and A tailing can lead to the generation of synthesized regions.
  • the gaps can be fdled in with DNA polymerases used in the end repair reaction, regardless of whether they possess 5’ to 3’ exonuclease activity or strand displacement activity. After this gap filling, a nick will still exist between the synthesized region and the region of the original DNA molecule 3’ of the gap.
  • the A-tailing enzymes may then introduce further synthesized regions through nick translation, as described for the nicked DNA. This synthesized region may extend to the 3 ’end of the DNA molecule.
  • the end-repair and the A-tailing reactions are performed in a single tube.
  • the A tailing reaction can be performed at a higher temperature than the end repair.
  • end repair is performed at ambient temperature (e.g. 15-35°C) and A tailing is performed at a temperature over 60°C, including e.g., about 60°C-75°C.
  • the A tailing reaction can be performed using a thermostable polymerase (e.g. Taq DNA polymerase, TH 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.
  • 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 fdtered 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.
  • 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).
  • 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 cleanup means that only dATP (not dCTP, dTTP or dGTP) is present in the A tailing reaction.
  • 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.
  • the end-repair is performed with a polymerase which lacks 5’to 3’ exonuclease activity and/or strand displacement activity.
  • 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 /// ⁇ /, phi29 DNA Polymerase, T7 DNA Polymerase, DNA Polymerase I (E. coli), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”) or T4 DNA Polymerase.
  • the polymerase used in the end repair is T4 DNA Polymerase or Klenow fragment.
  • the end repair is performed with a DNA polymerase which has 5 ’-3’ exonuclease activity and/or is a strand displacing DNA polymerase.
  • 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.
  • the A tailing reaction is performed using Klenow Fragment lacking 3'-5' exonuclease activity.
  • 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.
  • a dNTP comprising a modified base can be used in a combined end repair and A-tailing reaction.
  • 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).
  • 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).
  • a dNTP comprising a modified base when 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.
  • 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.
  • dNTP double-modified DNA
  • 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.
  • 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.
  • an mCpG-binding protein binds mCpG/mCpG dsDNA dyads.
  • the end repair reaction is conducted using an unmethylated deoxycytidine triphosphate (dCTP)
  • synthesized regions in the end-repaired molecules are called as unmethylated cytosines (on both strands) at these positions.
  • dmCTP methylated deoxy cytidine triphosphate
  • synthesized regions (on both strands) in the end-repaired molecules are called as methylated cytosines at these positions if the opposing, template strand comprises an mCpG.
  • the methods comprise ligating adapters to DNA.
  • DNA molecules can be subjected to blunt-end ligation with blunt-ended adapters.
  • DNA molecules can be subjected to sticky-end ligation with sticky-ended adapters.
  • 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).
  • the ligation step can take place before or after the conversion step.
  • conversion step or “conversion procedure” refers to any step or procedure that changes the base pairing specificity of one or more nucleotides.
  • the conversion step comprises contacting DNA (e.g., mCpG-bound DNA) with a deaminase.
  • the conversion step of contacting DNA (e.g., mCpG-bound DNA) with a deaminase provides a converted sample in which unmethylated CpGs in the DNA are converted to UpGs.
  • 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 contacting the DNA with an mCpG-binding protein. In some embodiments, the ligation step occurs before contacting the DNA with a deaminase and before contacting the DNA with an mCpG-binding protein. In some embodiments, the ligation step occurs after separating mCpG-bound DNA from unbound DNA. 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.
  • 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.
  • 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.
  • two adapters can be ligated to a single sample DNA molecule, with one adapter ligated to each end of the sample nucleic acid molecule.
  • the ligase used in ligation reactions can act on both single strand DNA nicks and double stranded DNA ends.
  • 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.
  • NGS next generation sequencing
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the modified nucleosides comprise modification resistant cytosines.
  • each cytosine in each adapter is a modification resistant cytosine.
  • the modification resistant cytosine is a deamination resistant cytosine.
  • the deamination resistant cytosine comprises 5-propynylC (5pyC), 5-pyrrolo-dC (5pyrC), 5-hydroxymethylcytosine (5hmC), glucosylated5- hydroxymethylcytosine (5ghmC), cytosine 5-methylenesulfonate (CMS), or N4-modified cytosine.
  • the adapters are resistant to digestion by an MSRE.
  • the MSRE digestion-resistant adapters comprise one or more methylated nucleotides, comprise one or more nucleotide analogs resistant to methylation sensitive restriction enzymes, or do not comprise a nucleotide sequence recognized by the MSRE.
  • the one or more methylated nucleotides in the MSRE digestion-resistant adapters comprise 5 -methylcytosine and/or 5-hydroxymethylcytosine.
  • either or both of the adapters may comprise one or more known modified nucleosides.
  • 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.
  • adapters may be added to the DNA or a subsample thereof.
  • Adapters can be ligated to DNA at any point in the methods herein.
  • adapters are ligated to the DNA in a sample, to DNA after contacting the DNA with an mCpG- binding protein, or to DNA in a converted sample.
  • adapters are ligated to the DNA in the sample prior to contacting the mCpG-bound DNA with a deaminase.
  • adapters are ligated to the DNA in the sample prior to contacting the DNA in the sample with an mCpG-binding protein.
  • adapters are ligated to DNA eluted from a converted sample.
  • the eluted DNA is single-stranded DNA.
  • the single-stranded DNA is hypomethylated DNA.
  • DNA in the sample may be contacted with a methylation sensitive restriction enzyme (MSRE).
  • MSRE methylation sensitive restriction enzyme
  • DNA in the sample is contacted with an MSRE after ligating adapters to end-repaired DNA molecules and/or before contacting mCpG-bound DNA with a deaminase.
  • adapters are ligated to the DNA of a sample or subsample thereof prior to annealing primers to the DNA for capture probe generation.
  • the adapter-ligated DNA is amplified prior to annealing primers to the DNA for capture probe generation.
  • adapters are ligated to the DNA of a sample or subsample thereof before the DNA is contacted with the capture probes.
  • 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.
  • 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.
  • the adapters ligated to DNA captured by the 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.
  • adapter-ligated DNA can be separated from DNA that does not comprise adapters.
  • the disclosed methods comprise analyzing DNA in a sample.
  • 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.
  • adapters are added by other approaches, such as ligation.
  • first adapters are added to the 3’ ends of the nucleic acids by ligation, which may include ligation to single-stranded DNA.
  • the first adapter comprises an affinity tag, such as biotin, and nucleic acid ligated to the first adapter is bound to a solid support (e.g., bead), which may comprise a binding partner for the affinity tag such as streptavidin.
  • a solid support e.g., bead
  • a binding partner for the affinity tag such as streptavidin.
  • 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.
  • template DNA is first heat denatured and then immediately cold shocked to render the template DNA molecules singlestranded.
  • the DNA can be maintained as single-stranded throughout the ligation reaction by the inclusion of a thermostable single-stranded binding protein (SSB).
  • SSB thermostable single-stranded binding protein
  • 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 singlestranded 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.
  • the forward adapter e g., (P5) Illumina adapter
  • the reverse adapter e.g., (P7) Illumina adapter
  • the native polarity of input DNA molecules can be retained.
  • T4 Polynucleotide Kinase 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.
  • the random nucleotides of the splint adapter can be annealed to the single-stranded template molecule.
  • 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.
  • 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.
  • the nucleic acids are linked at both ends to Y-shaped adapters including primer binding sites and tags.
  • the molecules are amplified.
  • the DNA molecules of the sample may be tagged with sample indexes and/or molecular barcodes (referred to generally as “tags”).
  • the DNA molecules of the sample comprise barcodes.
  • Tags can be molecules, such as nucleic acids, containing information that indicates a feature of the molecule with which the tag is associated.
  • 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)).
  • Tagging strategies can be divided into unique tagging and non-unique tagging strategies.
  • 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”.
  • 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 “nonunique 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.
  • a tag can comprise one or a combination of barcodes.
  • 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.
  • 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.
  • 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 e.g., following pooling of a plurality of samples for parallel sequencing.
  • Tags comprising barcodes can be incorporated into or otherwise joined to adapters.
  • Tags can be incorporated by ligation, overlap extension PCR among other methods.
  • Tags can be used to label the individual polynucleotide population partitions so as to correlate the tag (or tags) with a specific partition.
  • tags can be used in embodiments of the disclosure that do not employ a partitioning step.
  • a single tag can be used to label a specific partition.
  • multiple different tags can be used 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.
  • the tags may have additional functions, for example the tags can be used to index sample sources or used as unique molecular identifiers (which can be used to improve the quality of sequencing data by differentiating sequencing errors from mutations, for example as in Kinde et al., Proc Nat’l Acad Sci USA 108: 9530-9535 (2011), Kou et al., PLoS ONE, 11 : eO 146638 (2016)) or used as non-unique molecule identifiers, for example as described in US Pat. No. 9,598,731.
  • the tags may have additional functions, for example the tags can be used to index sample sources or used as nonunique molecular identifiers (which can be used to improve the quality of sequencing data by differentiating sequencing errors from mutations).
  • Molecular barcodes and/or sample indexes may be introduced simultaneously, or in any sequential order.
  • 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.
  • 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.
  • both the molecular barcodes and the sample indexes are introduced prior to performing probe-based capturing steps, if present.
  • the sample indexes are introduced after sequence capturing steps are performed, if present.
  • sample indexes are incorporated through overlap extension polymerase chain reaction (PCR).
  • the tags may be located at one end or at both ends of the sample DNA molecule.
  • tags are predetermined or random or semirandom sequence oligonucleotides.
  • 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.
  • 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 typically allows for the assignment of a unique identity to a particular molecule.
  • 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, sub-sequences 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.
  • 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.
  • end region comprises the last 1, last 2, the last 5, the last 10, the last 15, the last 20, the last 25, the last 30 or at least the last 30 base positions at the 3' end of the sequencing read that align to the reference sequence.
  • the length, or number of base pairs, of an individual sequence read are also optionally used to assign a unique identity to a given molecule.
  • This number is a function of the number of molecules falling into the calls.
  • 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).
  • 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).
  • the assignment of unique or non-unique molecular barcodes in reactions is performed using methods and systems described in, for example, U.S. Patent Application Nos. 20010053519, 20030152490, and 20110160078, and U.S. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a format uses 20-50 different tags (e.g., barcodes) ligated to both ends of target nucleic acids.
  • 35 different tags e.g., barcodes
  • 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.
  • unique tags may be predetermined or random or semi-random sequence oligonucleotides.
  • a plurality of barcodes may be used such that barcodes are not necessarily unique to one another in the plurality.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method may comprise detecting (e g., sequencing) molecules amplified from and/or captured by the one or more internal control DNAs.
  • the method can comprise comparing an amount of internal control DNAs (e.g., number of molecules or reads detected that correspond to an internal control DNA sequence) to a predetermined threshold, and either rejecting sequencing results if the predetermined threshold is not met or accepting sequencing results if the predetermined threshold is met.
  • the predetermined threshold may be established, e.g., based on historical data or by testing the method on samples of DNA from test subjects, such as healthy volunteers. For example, amplification and detection of the one or more internal control DNAs provides confirmation that the amplification process proceeded properly, thus reducing the likelihood of a false negative.
  • the deaminase is a methyl-sensitive deaminase or a methyl-insensitive deaminase. In some embodiments, the deaminase is a dsDNA deaminase and/or a ssDNA deaminase.
  • This step of contacting the mCpG-bound DNA with a deaminase can be referred to as, or be included in, a conversion procedure, such as any of the conversion procedures described elsewhere herein. For an exemplary description of conversion using a deaminase, see, e.g., Schutsky et al., Nature Biotechnology 2018; 36: 1083-1090.
  • DNA in the converted sample is then amplified, separated, partitioned, eluted, and/or enriched.
  • the converted DNA is sequenced, and a level of methylation at one or more differentially methylated regions of the DNA is quantified.
  • Such embodiments may also comprise a step of end-repair prior to the sequencing and/or prior to contacting the DNA in the sample with an mCpG-binding protein.
  • the deaminase (e.g., the methyl -insensitive deaminase or the methyl-sensitive deaminase) comprises a mutant deaminase or an alternatively truncated deaminase.
  • the mutant deaminase or an alternatively truncated comprises any one or more a mutant StsDaOl comprising the C-terminus of MGYPDa829, in which the mutant StsDaOl comprises the amino acid sequence of
  • MLPCPSELEAGLEKAKNAS SFERPGGMSGHAKLSDGTSHDLS SGGDGRNLRSDWEAPP GTTDENFHHLENQTAALMRQSGSHEAYLYLHKAEGAAYGACKYCVSAMREMLPQGS KLMVIWRNEEGAIRNRVFIGTSNDPKMSSRYKGN (SEQ ID NO: 78); an alternatively truncated MGYPDa829 with 11 amino acids at the C terminus deleted, in which the alternatively truncated MGYPDa829 comprises the amino acid sequence of
  • HcDaOl MNLPEYDGKTTHGVLVLDNGTQVQLVSGNGDPRYTNYRNNGHVEQKAAIYMRENNIS NATVYHNNTNGTCGYCNTMTATFLPEGATLTVVPPKNAVANNSRAIAYVKTYTGTSN (SEQ ID NO: 79); an alternatively truncated HcDaOl with 21 amino acids at the N terminus deleted, in which the alternatively truncated HcDaOl comprises the amino acid sequence of MITDSNILSKLGTVAAENVPLARKSLSKASELSSMLGYGSKAIASDGTTNTLSGWAEITQ GAEYFNRAATPQDVIAKSNEIGHTLKTDIFDNGIPGQYNASHAEKQLSLLSNKALGVSRP MCSDCVEYFKKLAKFSNTEKVVSDSNITRIFMPDGITIIEIPH (SEQ ID NO: 80); an alternatively truncated HmDaO2 with 16 amino acids at the N terminus deleted, in which the
  • MPRLGPRGVDPAHHNANIMVRDASGRLRHHERIVSGNMTPEEQALGFPRNTLASHTEA RAIRNIPLHRGETMIITGQRPPCPTCKGIMNQAARESGATIIYRWRENGVTRTWTAGN (SEQ ID NO: 81); an alternatively truncated MGYPDal7 with 18 amino acids at the N terminus deleted, in which the alternatively truncated MGYPDal7 comprises the amino acid sequence of MNYDVNSAEQYLRAVLDYENLSPEAWKSIDQFRESHGLNTLGNGLPKKGDGTV AFINA NGEKIFGINSSLLSEDKKMLGKKYYESMKEAGYFDNVTSYGNGSGQVFTHAEGNSLMN VYDLYGKSIGKDITIFCDRTTCGICKNNLGYFKDYFGIDSLTVLNKNGDIVIINKGTYIKIK S (SEQ ID NO: 82); an alternatively truncated MGYPDal7 with 16 amino acid-extension at the
  • RhDaOl comprises the amino acid sequence of MVTAGVGEAGKKEPWEAIDRFRKSNGLEPLGDRIPVRGDGLETVALMEVSGNKVFGVN SSLLSDELKNLGRDFFKVIKEKGLLGNAKHYGSGEAQVLTHAEAHALMKARKEAGGHL GDSVVLYVDRLTCPNCQKYLPEVRAAMGIKTLKVITKGGIELIL (SEQ ID NO: 89).
  • the contacting the mCpG-bound DNA with a deaminase provides a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs.
  • all unmethylated CpGs in the sample can be converted to UpGs using the deaminase.
  • at least a portion of unmethylated CpGs are not converted to UpGs using the deaminase.
  • at least a portion of methylated CpGs can be converted to UpGs using the deaminase.
  • Table 1 summarizes exemplary methods of deamination with the type of modified bases detectable with these methods. These are described in more detail below.
  • 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)).
  • an unmodified nucleoside e.g. cytosine
  • the corresponding modified nucleoside e.g. methylated cytosine, such as 5hmC and/or 5mC
  • any modified nucleoside e.g. modified cytosine, adenosine, guanosine and thymidine (or uracil)
  • 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.
  • 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.
  • any enzymatic conversion procedure could be used allowing 5-methylcytosine (m5c or 5-mC or 5mC) to be distinguished from unmodified cytosine.
  • the conversion procedure converts unmodified (e.g., unmethylated) cytosines to uracils using a deaminase.
  • 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.
  • dNTPs wherein at least one type of dNTP comprises a 5hmC
  • 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.
  • 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 .
  • TET2 and T4-0GT or 5-hydroxymethylcytosine carbamoyltransferase 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.
  • the conversion procedure comprises enzymatic conversion of a nucleobase using a non-specific, modification-sensitive double-stranded DNA deaminase, e.g., as in SEM-seq.
  • a non-specific, modification-sensitive double-stranded DNA deaminase e.g., as in SEM-seq.
  • Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA.
  • bioRxiv; DOI: 10.1101/2023.06.29.547047 available at https://www.biorxiv.org/content/10.1101/2023.06.29.547047vl.
  • 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 T4-0GT or 5- hydroxymethylcytosine carbamoyltransferase protection and denaturing steps that are of use, e.g., in APOBEC3A-based protocols. Additionally, MsddA does not deaminate 5-formylated cytosines (5fC) or 5-carboxylated cytosines (5-caC).
  • MsddA nonspecific, modification-sensitive double- stranded DNA deaminase
  • SEM-seq nondestructive single-enzyme 5-methylctyosine sequencing
  • unmodified cytosines in the DNA are deaminated to uracil and is read as “T” during sequencing.
  • Modified cytosines e.g., 5mC
  • 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.
  • the conversion procedure comprises enzymatic conversion of unmodified cytosine using MsddA or a modification-sensitive DNA deaminase A (MsddA)-like deaminase.
  • MsddA modification-sensitive DNA deaminase A
  • MsddA-like deaminases have reduced activity on each of 5mC, 5hmC, and 5gmC relative to unmodified cytosine in dsDNA, e.g., a reduction of about 75%, 80%, or more on each of 5mC, 5hmC, and 5gmC relative to unmodified cytosine (e.g., using assay conditions as described in Vaisvila et al., such as analysis of deamination of C in E.
  • Deamination can be performed by contacting substrate DNA with deaminase and analyzed using NGS as follows: 50 ng of unmodified E.
  • coli C2566 genomic DNA can be combined with the control DNAs (about 1 ng of Lambda, XP12, and T4147, and 0.1 ng of the 5hmC Adenovirus PCR fragment), sheared to about 300 bp and ligated to pyrrolo-dC adapters with 1 uL of in vitro synthesized deaminase (e.g., synthesized using the PURExpress In Vitro Protein Synthesis kit (NEB, Ipswich, MA) following manufacturer’s recommendations with 100-400 ng of PCR fragment template DNA containing codon-optimized deaminase coding sequence and T7 promoter and terminator).
  • control DNAs about 1 ng of Lambda, XP12, and T4147, and 0.1 ng of the 5hmC Adenovirus PCR fragment
  • the control DNAs about 1 ng of Lambda, XP12, and T4147, and 0.1 ng of the 5hmC Adeno
  • Exemplary deamination reaction conditions are 50 mM Bis-Tris pH 6.0, 0.1% Triton X-100 for 1 hour at 37 degrees C.
  • 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.
  • 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,
  • the amino acid sequence of the methylsensitive deaminase comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, which is the amino acid sequence of MsddA (Accession:
  • 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 SEQ ID NO: 7, which is the amino acid sequence of XcDaOl (Accession: B0RYS5; database: UniProt):
  • 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 SEQ ID NO: 8, which is the amino acid sequence of KsDaOl (Accession: E4NEH0; database: UniProt):
  • 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 SEQ ID NO: 9, which is the amino acid sequence of PwDaOl (Accession: UPI0001B0E918; database: UniParc): MREEILSRIARVGAMHAGNRPKPPPDLPEPGQGKPPTSPGKAIKHSSFWGALAGAAVGA LIAAAAFTVAAAAVAGAVALTGITGGAGLALVVGVVKLAVGFGAVFALGDLIGGVTNR VSAMVDSASPSSGAVKDGSKTVFVEGNPVSRAEIDAVLCDKHSGPQLIAQGSATVFVEG YYAARVGDKTVCGATIKEGASTVFFGSGQESPLSVADEFSGWEKALILAVEFLMPPSRG LFRGLGKLFTKGPMAVLRGMKAGAAHALEGLRSAVQCAKNGFKESKG
  • 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 SEQ ID NO: 11, which is the amino acid sequence of SrDaOl (Accession: D6Z8J1; database: UniProt):
  • 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 SEQ ID NO: 12, which is the amino acid sequence of NgDaOl (Accession: G3Z1X9; database: UniProt):
  • 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 SEQ ID NO: 13, which is the amino acid sequence of NsDaOl (Accession: D3A5B8; database: UniProt):
  • 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 SEQ ID NO: 20, which is the amino acid sequence of MGYPDa25 (Accession: MGYP00 1677015708; database: MGnify):
  • 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 SEQ ID NO: 21, which is the amino acid sequence of MGYPDa26 (Accession: MGYP003599500301; database: MGnify):
  • 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 SEQ ID NO: 22, which is the amino acid sequence of AshDaOl (Accession: A0A7H8HBT9; database: UniProt):
  • 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 SEQ ID NO: 23, which is the amino acid sequence of MGYPDa21 (Accession: MGYP001278432191; database: MGnify):
  • 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 SEQ ID NO: 24, which is the amino acid sequence of PpDa03 (Accession: A0A3S0YAN6; database: UniProt):
  • 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 SEQ ID NO: 27, which is the amino acid sequence of PpDaO4 (Accession: A0A5M9IK01; database: UniProt):
  • 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 SEQ ID NO: 28, which is the amino acid sequence of CsDaOl (Accession: A0A3E1NUV0; database: UniProt):
  • 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 SEQ ID NO: 29, which is the amino acid sequence of MGYPDa22 (Accession:
  • 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 SEQ ID NO: 31, which is the amino acid sequence of MGYPDa24 (Accession: MGYP000620945751; database: MGnify):
  • 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 SEQ ID NO: 32, which is the amino acid sequence of AaDaO2 (Accession: A0A2S8AG27; database: UniProt):
  • 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 SEQ ID NO: 33, which is the amino acid sequence of MmgDaOl (Accession: JANFCG010307071; database: GeneBank):
  • 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 SEQ ID NO: 34, which is the amino acid sequence of PbDaOl (Accession: MCL1918637.1; database: GeneBank):
  • 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 SEQ ID NO: 36, which is the amino acid sequence of LsfDaOl (Accession: UHQ2 1442.1; database: GeneBank):
  • 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 SEQ ID NO: 37, which is the amino acid sequence of SmgDaOl (Accession: JAHZIM010172802.1; database: GeneBank):
  • 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 SEQ ID NO: 38, which is the amino acid sequence of DaDaOl (Accession: tr
  • 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 SEQ ID NO: 40, which is the amino acid sequence of EcDaO2 (Accession: ABG02915.1; database: GeneBank):
  • 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 SEQ ID NO: 41, which is the amino acid sequence of NgDaO2 (Accession: WP_003703542.1 ; database: GeneBank):
  • 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 SEQ ID NO: 42, which is the amino acid sequence of PaDaOl (Accession: WP_006660219.1; database: GeneBank):
  • 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 SEQ ID NO: 43, which is the amino acid sequence of AsDaOl (Accession: WP_005802165.1; database: GeneBank):
  • 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 SEQ ID NO: 44, which is the amino acid sequence of HgmDaOl (Accession:
  • 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 SEQ ID NO: 45, which is the amino acid sequence of MsDaO2 (Accession: tr
  • 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 SEQ ID NO: 46, which is the amino acid sequence of XinDaOl (Accession: tr
  • 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 SEQ ID NO: 47, which is the amino acid sequence of XjaDaOl (Accession: tr
  • 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 SEQ ID NO: 48, which is the amino acid sequence of RhDaOl (Accession: tr
  • 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 SEQ ID NO: 49, which is the amino acid sequence of MGYPDaO4 (Accession: MGYP001129217467; database: MGnify):
  • 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 SEQ ID NO: 50, which is the amino acid sequence of MGYPDa05 (Accession: MGYP00 1094202578; database: MGnify):
  • 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 SEQ ID NO: 51, which is the amino acid sequence of BaDaOl (Accession: tr
  • 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 SEQ ID NO: 52, which is the amino acid sequence of WWTPDaO4 (Accession: tig00000754- 10-8122160_273; database: GeneBank):
  • 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 SEQ ID NO: 53, which is the amino acid sequence of PbDaO2 (Accession: A0A356KTR0; database: UniProt):
  • 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 SEQ ID NO: 55, which is the amino acid sequence of MGYPDal 5 (Accession: MGYP00 1492404292; database: MGnify):
  • 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 SEQ ID NO: 56, which is the amino acid sequence of MGYPDal6 (Accession: MGYP00 1076701719; database: MGnify):
  • 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 SEQ ID NO: 57, which is the amino acid sequence of MGYPDal7 (Accession: MGYP001100966096; database: MGnify):
  • 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 SEQ ID NO: 60, which is the amino acid sequence of MGYPDal8 (Accession: MGYP000969250293; database: MGnify):
  • 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 SEQ ID NO: 66, which is the amino acid sequence of FbiDaOl (Accession:
  • 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 SEQ ID NO: 67, which is the amino acid sequence of PvmDaOl (Accession:
  • 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 SEQ ID NO: 68, which is the amino acid sequence of SjDaOl (Accession: tr
  • 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 SEQ ID NO: 69, which is the amino acid sequence of HmDaO2 (Accession: 3300044995_Ga0484949_008810_2_589; database: IMG/M hot metagenome bin):
  • 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 SEQ ID NO: 71, which is the amino acid sequence of RaDaOl (Accession: A0A373WC03; database: UniProt):
  • 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 SEQ ID NO: 73, which is the amino acid sequence of AvDaOl (Accession: tr
  • 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 SEQ ID NO: 74, which is the amino acid sequence of CbDaOl (Accession: NLK69555.1; database: GeneBank):
  • 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 SEQ ID NO: 75, which is the amino acid sequence of PfDaOl (Accession: tr
  • 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 SEQ ID NO: 76, which is the amino acid sequence of NoDaO 1 (Accession: WP_223985731.1; database: GeneBank):
  • 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 SEQ ID NO: 77, which is the amino acid sequence of SsdA (Accession:
  • the methyl-sensitive deaminase comprises the
  • 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 SEQ ID NO: 2 and contains the Y130L mutation.
  • the Y130 substitution mutation is Y130W (e.g., SEQ ID NO: 3):
  • the Y132 substitution mutation is Y132P (e g., SEQ ID NO: 4):
  • the methyl -sensitive deaminase has substitution mutations Y130V and Y132H substitution mutation (e.g., SEQ ID NO: 5): MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQ AKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQEN THVRLRIFAARIVDHDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQP WDGLDEHSQALSGRLRAILQNQGN.
  • the methyl -sensitive deaminase has substitution mutations Y130W and Y132H (e.g., SEQ ID NO: 6):
  • 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 SEQ ID NO: 2 and contains the Y130L mutation.
  • 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 SEQ ID NO: 3 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 SEQ ID NO: 4 and contains the Y132P mutation.
  • 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 SEQ ID NO: 5 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 SEQ ID NO: 6 and contains the Y130W and Y132H.
  • the deaminase (e.g., the methyl-insensitive deaminase or the methyl-sensitive deaminase) comprises any one or more of the deaminases or a truncated version thereof, disclosed in WO 2024/073043, which is incorporated by reference herein in its entirety.
  • the conversion procedure further includes enzymatic protection of 5hmCs, such as by glucosylation of the 5hmCs (e.g., using 0GT) or by carbamoylation of the 5hmCs (e.g., using 5-hydroxymethylcytosine carbamoyltransferase), in the DNA prior to the deamination of unprotected modified cytosines.
  • enzymatic protection of 5hmCs such as by glucosylation of the 5hmCs (e.g., using 0GT) or by carbamoylation of the 5hmCs (e.g., using 5-hydroxymethylcytosine carbamoyltransferase), in the DNA prior to the deamination of unprotected modified cytosines.
  • 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.
  • PTT P-glucosyl transferase
  • 5cmC 5- hydroxymethylcytosine carbamoyltransferase
  • a TET protein can be used to convert 5mC and optionally 5hmC (but not unmodified C) into substrates (e.g., 5caC) that cannot be deaminated by a deaminase, and then a deaminase (e.g., APOBEC3A) can be used to deaminate unmodified cytosines, converting them to uracils.
  • a deaminase e.g., APOBEC3A
  • TET enzymes may be used in the disclosed methods as appropriate.
  • 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.
  • the one or more TET enzymes comprise a V1900 TET mutant, such as a VI 900 A, V 1900C, V 1900G, V 19001, or VI 900P TET mutant.
  • the one or more TET enzymes comprise a VI 900 TET2 mutant, such as a V1900A, V1900C, V1900G, VI 9001, or V1900P TET2 mutant.
  • the TET enzyme comprises a mutation that increases formation of 5-caC. Exemplary mutations are set forth above.
  • a TET2 comprising a T1372S mutation is described in US Patent 10,961,525 and may be expressed and used as a fragment comprising TET2 residues 1129-1480 joined to TET2 residues 1844-1936 by a linker. Position 1372 of TET2 corresponds to position 258 of SEQ ID NO: 21 (wild type TET2 catalytic domain) of US Patent 10,961,525. Thus, the sequence of a T1372S TET2 catalytic domain may be obtained by changing the threonine at position 258 of SEQ ID NO: 21 of US Patent 10,961,525 to serine.
  • TET2 comprising a T1372S mutation is also described in Liu et al., Nat Chem Biol.
  • TET2 comprising a T1372S mutation can more efficiently oxidize 5mC to produce 5-carboxylcytosine (5-caC) than other versions of TET2 such as TET2 lacking a T1372S mutation.
  • the thermal inactivation completely inhibits the activity of the deaminase or reduces the activity of the deaminase by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 90%, about 95%, about 98%, about 99%, or 100% relative to a deaminase that has not been subjected to heating or cooling.
  • the separating step can occur before or after contacting the DNA (e.g., mCpG- bound DNA) with a deaminase.
  • the separating step can occur after contacting the DNA (e.g., DNA in the sample) with an mCpG-binding protein.
  • the separating step can occur after contacting the DNA (e.g., mCpG-bound DNA) with a deaminase.
  • the separating step can occur before contacting the DNA (e.g., mCpG-bound DNA) with a deaminase.
  • separating comprises partitioning.
  • a heterogeneous nucleic acid sample is partitioned into two or more partitions (sub-samples).
  • 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.
  • the separating comprises partitioning the DNA in the sample into a plurality of partitioned subsamples.
  • the plurality of partitioned subsamples comprises a first partitioned subsample and a second partitioned subsample.
  • the first partitioned subsample comprises methylated DNA in a greater proportion than the second partitioned subsample.
  • the partitioning step can occur before or after contacting the DNA (e.g., mCpG-bound DNA) with a deaminase.
  • the partitioning step can occur after contacting the DNA (e.g., DNA in the sample) with an mCpG-binding protein.
  • the partitioning step occurs before contacting the DNA (e.g., mCpG-bound DNA) with a deaminase.
  • the partitioning step occurs after contacting the DNA (e.g., mCpG-bound DNA) with a deaminase. In some embodiments, the partitioning step occurs after contacting the DNA with a deaminase and after contacting the mCpG-binding protein to the DNA in the sample. In some embodiments, the partitioning step occurs before or after a conversion step. In some embodiments, the conversion step comprises contacting DNA (e.g., mCpG-bound DNA) with a deaminase.
  • 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.
  • a heterogeneous population of nucleic acids can be partitioned into nucleic acid molecules associated with nucleosomes and nucleic acid molecules devoid of nucleosomes.
  • a heterogeneous population of nucleic acids may be partitioned into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).
  • 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).
  • the DNA of at least one partition is subjected to an end repair and sequencing procedure described herein. In some embodiments at least one partition is not subjected to the end repair and sequencing procedure described herein.
  • 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.
  • partition tagging comprises tagging molecules in each partition with a partition tag.
  • partition tags identify the source partition.
  • different partitions are tagged with different sets of molecular tags, e.g., comprised of a pair of barcodes.
  • 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.
  • the molecules may be pooled for sequencing in a single run.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • NDR nucleosome depleted region
  • Disclosed methods herein comprise analyzing DNA in a sample.
  • the disclosed methods comprise partitioning DNA.
  • different forms of DNA e.g., hypermethylated and hypomethylated DNA
  • This approach can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated.
  • 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.
  • 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.
  • 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.
  • extent of methylation e.g., relative number of methylated nucleobases per molecule
  • the partitioning comprises contacting the DNA with an agent that recognizes a modification associated with (e.g., in) the DNA.
  • the agent that recognizes the modification is an antibody or an mCpG-binding protein.
  • the agent is immobilized on a solid support.
  • the solid support comprises a bead.
  • the partitioning comprises immunoprecipitation, e.g., using the antibody agent, such as an antibody or an mCpG-binding protein, immobilized on solid support.
  • the separating comprises precipitating the mCpG-bound DNA. In some embodiments, the separating comprises precipitating the mCpG-bound DNA to separate it from the unbound DNA. In some embodiments, the partitioning comprises precipitating the mCpG-bound DNA. In some embodiments, the partitioning comprises precipitating the mCpG-bound DNA to separate it from the unbound DNA. In some embodiments, the precipitating the mCpG-bound DNA can be performed using any pair of binding partners. In some embodiments, one of the binding partners may be linked to the mCpG protein, and the other binding partner may be linked to a solid support. In some embodiments, the binding partner comprises biotin and streptavidin.
  • the biotin may be linked to the mCpG-binding protein, and the streptavidin may be linked to a solid support.
  • the mCpG-binding protein is linked to a solid support, optionally using any pair of binding partners.
  • the separating comprises immunoprecipitating the mCpG-bound DNA. In some embodiments, the separating comprises immunoprecipitating the mCpG-bound DNA separately from the unbound DNA. In some embodiments, the partitioning comprises immunoprecipitating the mCpG-bound DNA. In some embodiments, the partitioning comprises immunoprecipitating the mCpG-bound DNA separately from the unbound DNA.
  • the modification is methylation
  • the partitioning comprises partitioning on the basis of methylation level.
  • the agent is a methyl binding reagent.
  • the methyl binding reagent specifically recognizes 5-methylcytosine.
  • the agent is a hydroxymethyl binding reagent.
  • the methyl binding reagent specifically recognizes 5-hydroxymethylcytosine, biotinylated 5-hydroxymethylcytosine, glucosylated 5-hydroxymethylcytosine, or sulfonylated 5-hydroxymethylcytosine.
  • 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.
  • binding reagent specifically binds a methylated protein, an acetylated protein, such as a methylated or acetylated histone.
  • the binding reagent specifically binds an unmethylated or unacetylated protein epitope.
  • the modification is hydroxymethylation
  • the partitioning comprises partitioning on the basis of hydroxymethylation level.
  • the agent is a hydroxymethyl binding reagent, such as an antibody.
  • the hydroxymethyl binding reagent e.g., antibody
  • the hydroxymethyl binding reagent specifically recognizes 5-hydroxymethylcytosine (5-hmC).
  • 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.
  • 5- hmC can be enzymatically glucosylated and then partitioned based on binding to I-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).
  • 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.
  • Partitioning nucleic acid molecules in a sample can increase a rare signal, e.g., by enriching rare nucleic acid molecules that are more prevalent in one partition of the sample. For example, a genetic variation present in hypermethylated DNA but less (or not) present in hypomethylated DNA can be more easily detected by partitioning a sample into hypermethylated and hypomethylated nucleic acid molecules.
  • 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).
  • cfDNA cell free DNA
  • ctDNA circulating tumor DNA
  • cfNA cell free nucleic acids
  • 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.
  • heterogeneous DNA in a sample is partitioned into two or more partitions (e.g., at least 3, 4, 5, 6 or 7 partitions).
  • 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.
  • the differentially tagged partitions are separately sequenced.
  • 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.
  • CNVs copy number variations
  • SNVs single nucleotide variations
  • indels insertions/deletions
  • in silico analysis can include analysis to determine epigenetic variation (one or more of methylation, chromatin structure, etc.). Analysis can include in silico using sequence information, genomic coordinates length, coverage, and/or copy number. For example, coverage of sequence reads can be used to determine nucleosome positioning 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).
  • NDR nucleosome depleted region
  • 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.
  • the agent used in the partitioning is an agent that recognizes a modified nucleobase.
  • the modified nucleobase recognized by the agent is a modified cytosine, such as a methylcytosine (e.g., 5-methylcytosine).
  • 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.
  • 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.
  • 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).
  • 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 single- stranded form.
  • 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.
  • hypomethylated (and optionally intermediately methylated) subsamples may then be contacted with a methylation dependent nuclease that cleaves hemi-methylated DNA.
  • partitioning agents are histone binding proteins which can separate nucleic acids bound to histones from free or unbound nucleic acids.
  • histone binding proteins examples include RBBP4, RbAp48 and SANT domain peptides.
  • partitioning can comprise both binary partitioning and partitioning based on degree/level of modifications.
  • 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).
  • MethylMinder Methylated DNA Enrichment Kit ThermoFisher Scientific.
  • 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.
  • 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).
  • the DNA of interest is one or more differentially methylated regions of the DNA.
  • 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.
  • 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).
  • 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.
  • different forms of DNA e.g., hypermethylated and hypomethylated DNA
  • a methylated DNA binding protein e.g., an MBD such as MBD1, MBD2, MBD4, or MeCP2
  • an antibody specific for 5-methylcytosine as in MeDIP
  • a DNA fragmentation pattern can be determined based on endpoints and/or centerpoints of DNA molecules, such as cfDNA molecules.
  • 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 bom 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.
  • methylation 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.
  • 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.
  • a salt concentration 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.
  • 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).
  • a hypomethylated partition enriched in nucleic acids comprising no methylation
  • a methylated partition enriched in nucleic acids comprising low levels of methylation
  • a hyper methylated partition enriched in nucleic acids comprising high levels of methylation
  • portioning nucleic acid samples based on characteristics such as methylation see WO2018/119452, which is incorporated herein by reference.
  • 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).
  • ChIP chromatin-immuno-precipitation
  • AF4 asymmetrical field flow fractionation
  • the partitioning comprises contacting the DNA with a methylation sensitive restriction enzyme (MSRE) and/or a methylation dependent restriction enzyme (MDRE).
  • MSRE methylation sensitive restriction enzyme
  • MDRE methylation dependent restriction enzyme
  • 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.
  • the partitioning is performed by contacting the nucleic acids with a methyl binding domain (“MBD”) of a methyl binding protein (“MBP”).
  • MBD methyl binding domain
  • MBP methyl binding protein
  • the nucleic acids are contacted with an entire MBP.
  • an MBD binds to 5-methylcytosine (5mC)
  • an MBP comprises an MBD and is referred to interchangeably herein as a methyl binding protein or a methyl binding domain protein.
  • 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.
  • agents that recognize a modified nucleobase contemplated herein include, but are not limited to:
  • MeCP2 is a protein that preferentially binds to 5-methyl-cytosine over unmodified cytosine.
  • Sequences that comprise aberrantly high copy numbers may tend to be hypermethylated.
  • the DNA contacted with capture probes specific for members of an epigenetic target region set comprising a plurality of target regions that are both type-specific differentially methylated regions and copy number variants comprises at least a portion of a hypermethylated partition.
  • the DNA from or comprising at least a portion of the hypermethylated partition may or may not be combined with DNA from or comprising at least a portion of one or more other partitions, such as an intermediate partition or a hypomethylated partition.
  • 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.
  • 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.
  • 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.
  • extent of methylation e.g., relative number of methylated nucleobases per molecule
  • Amplification methods can involve cycles of denaturation, annealing and extension, resulting from thermocycling, such as polymerase chain reaction (PCR), or can be isothermal, such as in linear amplification methods, transcription-mediated amplification, recombinase polymerase amplification (RPA), helices dependent amplification (HDA), loop-mediated isothermal amplification (LAMP) (Notomi et al., Nuc. Acids Res., 28, e63, 2000), rolling-circle amplification (RCA) (Blanco et al., J. Biol. Chem., 264, 8935-8940, 1989), or hyperbranched rolling circle amplification (Lizard et al., Nat.
  • PCR polymerase chain reaction
  • amplification methods include the ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustained sequence based replication.
  • the methylation-preserving amplification comprises linear amplification with thermocycling.
  • methylation-preserving amplification comprises amplification performed in the presence of a methyltransferase.
  • Methylating agents of use in methylation-preserving amplification methods described herein are known to those of ordinary skill in the art, and can include, for example, any suitable methyltransferase.
  • the methylating agent is DNMT1.
  • DNMT1 is the most abundant DNA methyltransferase in mammalian cells and predominantly methylates hemimethylated CpG dinucleotides 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).
  • DNMT1 is used at a concentration of about 50-10000 U/mL, such as about 50-2000, about 50-5000, about 2500- 7500, or about 5000-10000 U/mL. In some embodiments, DNMT1 is used at a concentration of about 100-500, about 500-1000, about 100-1000, about 1000-1500, about 500-1500, about 600- 1400, about 700-1300, about 800-1200, about 900-1100, or about 950-1050 U/mL.
  • 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. [00409] In some embodiments, enriching methylated DNA in a sample comprises amplification, such as embodiments comprising quantitative PCR (qPCR) or digital PCR.
  • qPCR quantitative PCR
  • digital PCR digital PCR
  • the present methods perform dsDNA ligations with T- tailed and C-tailed adapters.
  • the addition of C-tailed adapters can increase ligation efficiency because the A-tailing reaction can also add G-tails to a small portion of the DNA molecules, when the A tailing is performed in the presence of dGTP, such as when the A-tailing is performed in the same reaction as the end repair.
  • the use of T-tailed and C-tailed adapters can result in amplification of at least 50, 60, 70 or 80% of double stranded nucleic acids.
  • the present methods can increase the amount or number of amplified molecules relative to control methods performed with T-tailed adapters alone by at least 10, 15, or 20%.
  • an amplification of the DNA in a sample comprises amplifying rolling-circle amplification (RCA).
  • RCA comprises circularizing a DNA template (e.g., DNA in the converted sample).
  • RCA comprises copying the circularized DNA template using a rolling circle polymerase to generate a plurality of circularized DNA templates.
  • 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).
  • the RCA occurs prior to a step of sequencing the DNA.
  • adapted DNA is amplified before sequencing. This may be an additional amplification step subsequent to an earlier amplification step, such as amplification as described elsewhere herein.
  • amplification of adapted DNA comprises RCA, e.g., as described above.
  • RCA comprises copying the circularized DNA template using a rolling circle polymerase to generate a plurality of circularized DNA templates.
  • the rolling circle polymerase is a phi29 DNA polymerase.
  • Amplification may in some cases be before one or more capture steps.
  • the ligation step occurs after the conversion step. In some embodiments, the ligation occurs before or simultaneously with amplification.
  • sequencing DNA that was amplified using RCA provides sequence reads comprising multiple copies of the sequence of an original sample molecule or converted molecule and the copies are used to determine a consensus sequence of the original sample molecule or converted molecule.
  • DNA molecules in a sample can be subjected to a capture step (also referred to herein as a “enriching” or “enrichment” step), in which molecules having target sequences are captured for subsequent analysis.
  • a capture step also referred to herein as a “enriching” or “enrichment” step
  • methods disclosed herein comprise a step of capturing (i.e., enriching) one or more sets of target regions of DNA, such as cfDNA.
  • the capture step is performed prior to a step of amplifying DNA in the converted sample, prior to a step of sequencing the DNA, after contacting the DNA in the sample with the deaminase, and/or after partitioning the DNA in the sample into a plurality of subsamples.
  • the capture step is performed prior to a step of amplifying DNA in the converted sample, prior to a step of sequencing the DNA, after contacting the DNA in the sample with the deaminase, and after partitioning the DNA in the sample into a plurality of subsamples. In some embodiments, the capture step is performed prior to a step of amplifying DNA in the converted sample, prior to a step of sequencing the DNA, and after contacting the DNA in the sample with the deaminase. In some embodiments, the capture step is performed prior to a step of amplifying DNA in the converted sample, prior to a step of sequencing the DNA, and after partitioning the DNA in the sample into a plurality of subsamples.
  • the capture step is performed prior to a step of amplifying DNA in the converted sample, after contacting the DNA in the sample with the deaminase, and after partitioning the DNA in the sample into a plurality of subsamples. In some embodiments, the capture step is performed prior to a step of sequencing the DNA, after contacting the DNA in the sample with the deaminase, and after partitioning the DNA in the sample into a plurality of subsamples. In some embodiments, the capture step is performed prior to a step of amplifying DNA in the converted sample and prior to a step of sequencing the DNA.
  • the capture step is performed prior to a step of amplifying DNA in the converted sample and after contacting the DNA in the sample with the deaminase. In some embodiments, the capture step is performed prior to a step of amplifying DNA in the converted sample and after partitioning the DNA in the sample into a plurality of subsamples. In some embodiments, the capture step is performed prior to a step of sequencing the DNA and after contacting the DNA in the sample with the deaminase. In some embodiments, the capture step is performed prior to a step of sequencing the DNA and after partitioning the DNA in the sample into a plurality of subsamples.
  • the capture step is performed after contacting the DNA in the sample with the deaminase and after partitioning the DNA in the sample into a plurality of subsamples.
  • Capture may be performed using any suitable approach known in the art.
  • Target capture can involve use of a bait set comprising oligonucleotide baits (a type of probe useful herein) labeled with a capture moiety, such as biotin or the other examples noted below.
  • the probes can have sequences selected to tile across a panel of regions, such as genes.
  • Such bait sets are combined with a sample under conditions that allow hybridization of the target molecules with the baits.
  • captured molecules are isolated using the capture moiety.
  • a biotin capture moiety by bead-based streptavidin.
  • Capture moieties include, without limitation, biotin, avidin, streptavidin, a nucleic acid comprising a particular nucleotide sequence, digoxygenin, a histidine tag, an affinity tag, an immunoglobulin constant domain, a hapten recognized by an antibody, and magnetically attractable particles.
  • the immunoglobulin constant domain may be bound using protein A, protein G, or a secondary antibody.
  • the secondary antibody comprises an anti -mouse secondary antibody.
  • the anti-mouse secondary antibody is a goat anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, or a donkey anti-mouse secondary antibody.
  • an mCpG-binding protein comprises a capture moiety.
  • the extraction moiety can be a member of a binding pair, such as biotin/ streptavidin or hapten/antibody.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • DNA is captured from at least the first subsample or the second subsample, e.g., at least the first subsample and the second subsample.
  • the subsamples are differentially tagged (e.g., as described herein) and then pooled before undergoing capture.
  • 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.
  • 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 set at a greater capture yield than cfDNA corresponding to the epigenetic target region set.
  • the volume of data needed to determine fragmentation patterns (e.g., to test fsor perturbation of transcription start sites or CTCF binding sites) or fragment abundance (e.g., in hypermethylated and hypom ethylated partitions) is generally less than the volume of data needed to determine the presence or absence of cancer-related sequence mutations.
  • Capturing the target region sets at different yields can facilitate sequencing the target regions to different depths of sequencing in the same sequencing run (e.g., using a pooled mixture and/or in the same sequencing cell).
  • the methods further comprise sequencing the captured cfDNA, e.g., to different degrees of sequencing depth for the epigenetic and sequence-variable target region sets, consistent with the discussion herein.
  • complexes of target-specific probes and DNA are separated from DNA not bound to target-specific probes.
  • a washing or aspiration step can be used to separate unbound material.
  • the complexes have chromatographic properties distinct from unbound material (e.g., where the probes comprise a ligand that binds a chromatographic resin), chromatography can be used.
  • the set of target-specific probes may comprise a plurality of sets such as probes for a sequence-variable target region set and probes for an epigenetic target region set.
  • the capturing step is performed with the probes for the sequence-variable target region set and the probes for the epigenetic target region set in the same vessel at the same time, e.g., the probes for the sequence-variable and epigenetic target region sets are in the same composition.
  • the concentration of the probes for the sequence- variable target region set is greater than the concentration of the probes for the epigenetic target region set.
  • the capturing step is performed with the sequence-variable target region probe set in a first vessel and with the epigenetic target region probe set in a second vessel, or the contacting step is performed with the sequence-variable target region probe set at a first time and a first vessel and the epigenetic target region probe set at a second time before or after the first time.
  • This approach allows for preparation of separate first and second compositions comprising captured DNA corresponding to the sequence-variable target region set and captured DNA corresponding to the epigenetic target region set.
  • the compositions can be processed separately as desired (e.g., to fractionate based on methylation as described elsewhere herein) and recombined in appropriate proportions to provide material for further processing and analysis such as sequencing.
  • a captured set of DNA (e.g., cfDNA) is provided.
  • the captured set of DNA may be provided, e g., by performing a capturing step prior to a sequencing step as described herein.
  • the captured set may comprise DNA corresponding to a sequence-variable target region set, an epigenetic target region set, or a combination thereof.
  • a capture step is performed prior to a conversion step or after a conversion step.
  • a first target region set is captured (e.g., from a sample or a first subsample), comprising at least epigenetic target regions.
  • the epigenetic target regions captured from the first subsample may comprise hypermethylation variable target regions.
  • the hypermethylation variable target regions are CpG-containing regions that are unmethylated or have low methylation in cfDNA from healthy subjects (e.g., below- average methylation relative to bulk cfDNA).
  • the hypermethylation variable target regions are regions that show lower methylation in healthy cfDNA than in at least one other tissue type.
  • cancer cells may shed more DNA into the bloodstream than healthy cells of the same tissue type.
  • the distribution of tissue of origin of cfDNA may change upon carcinogenesis.
  • 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.
  • the DNA corresponding to the sequence-variable target region set may be present at a greater concentration than the DNA corresponding to the epigenetic target region set, e.g., a 1.1 to 1.2-fold greater concentration, a 1.2- to 1.4-fold greater concentration, a 1.4- to 1.6-fold greater concentration, a 1.6- to 1.8-fold greater concentration, a 1.8- to 2.0-fold greater concentration, a 2.0- to 2.2-fold greater concentration, a 2.2- to 2.4-fold greater concentration a 2.4- to 2.6-fold greater concentration, a 2.6- to 2.8-fold greater concentration, a 2.8- to 3.0-fold greater concentration, a 3.0- to 3.5-fold greater concentration, a 3.5- to 4.0, a 4.0- to 4.5-fold greater concentration, a 4.5- to 5.0
  • the sequence-variable target region set comprises a plurality of regions known to undergo somatic mutations in cancer.
  • the sequence-variable target region set targets a plurality of different genes or genomic regions (“panel”) selected such that a determined proportion of subjects having a cancer exhibits a genetic variant or tumor marker in one or more different genes or genomic regions in the panel.
  • the panel may be selected to limit a region for sequencing to a fixed number of base pairs.
  • the panel may be selected to sequence a desired amount of DNA, e.g., by adjusting the affinity and/or amount of the probes as described elsewhere herein.
  • the panel may be further selected to achieve a desired sequence read depth.
  • the panel may be selected to achieve a desired sequence read depth or sequence read coverage for an amount of sequenced base pairs.
  • the panel may be selected to achieve a theoretical sensitivity, a theoretical specificity, and/or a theoretical accuracy for detecting one or more genetic variants in a sample.
  • Probes for detecting the panel of regions can include those for detecting genomic regions of interest (hotspot regions). Information about chromatin structure can be taken into account in designing probes, and/or probes can be designed to maximize the likelihood that particular sites (e.g., KRAS codons 12 and 13) can be captured, and may be designed to optimize capture based on analysis of cfDNA coverage and fragment size variation impacted by nucleosome binding patterns and GC sequence composition. Regions used herein can also include non-hotspot regions optimized based on nucleosome positions and GC models.
  • Probes for detecting the panel of regions can include those for detecting genomic regions of interest (hotspot regions). Information about chromatin structure can be taken into account in designing probes, and/or probes can be designed to maximize the likelihood that particular sites (e.g., KRAS codons 12 and 13) can be captured, and may be designed to optimize capture based on analysis of cfDNA coverage and fragment size variation impacted by nucleosome binding patterns and GC sequence composition. Regions used herein can also include non-hotspot regions optimized based on nucleosome positions and GC models.
  • a sequence-variable target region set used in the methods of the present disclosure comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 of the genes of Table 3 of WO 2020/160414.
  • a sequence-variable target region set used in the methods of the present disclosure comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the genes of Table 4 of WO 2020/160414.
  • suitable target region sets are available from the literature. For example, Gale et al., PLoS One 13: e0194630 (2018), which is incorporated herein by reference, describes a panel of 35 cancer-related gene targets that can be used as part or all of a sequence-variable target region set.
  • the sequence-variable target region set comprises target regions from at least 10, 20, 30, or 35 cancer-related genes, such as the cancer-related genes listed above and in WO 2020/160414.
  • a collection of capture probes is used in methods described herein, e.g., comprising capture probes prepared by any method disclosed herein for doing so.
  • the collection of capture probes further comprises target-binding probes specific for a sequence-variable target region set and/or target-binding probes specific for an epigenetic target region set.
  • the capture yield of the target-binding probes specific for the sequence-variable target region set is higher (e.g., at least 2-fold higher) than the capture yield of the target-binding probes specific for the epigenetic target region set.
  • the collection of capture probes is configured to have a capture yield specific for the sequence-variable target region set higher (e.g., at least 2-fold higher) than its capture yield specific for the epigenetic target region set.
  • the capture yield of the target-binding probes specific for the sequence-variable target region set is at least 1.25-, 1.5-, 1.75-, 2-, 2.25-, 2.5-, 2.75-, 3-, 3.5-, 4-, 4.5-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, or 15-fold higher than the capture yield of the target -binding probes specific for the epigenetic target region set.
  • the capture yield of the target-binding probes specific for the sequence-variable target region set is
  • the collection of capture probes is configured to have a capture yield specific for the sequence-variable target region set at least 1.25-, 1.5-, 1.75-, 2-,
  • the collection of capture probes is configured to have a capture yield specific for the sequence-variable target region set is 1.25- to 1.5-, 1.5- to 1.75-, 1.75- to 2-, 2- to 2.25-, 2.25- to 2.5-, 2.5- to 2.75-, 2.75- to 3-, 3- to 3.5-, 3.5- to 4-, 4- to 4.5-, 4.5- to 5-, 5- to 5.5-, 5.5- to 6-, 6- to 7-, 7- to 8-, 8- to 9-, 9- to 10-, 10- to 11-, 11- to 12-, 13- to 14-, or 14- to 15-fold higher than its capture yield specific for the epigenetic target region set.
  • the collection of probes can be configured to provide higher capture yields for the sequence-variable target region set in various ways, including concentration, different lengths and/or chemistries (e.g., that affect affinity), and combinations thereof. Affinity can be modulated by adjusting probe length and/or including nucleotide modifications as discussed below.
  • the capture probes specific for the sequence-variable target region set are present at a higher concentration than the capture probes specific for the epigenetic target region set.
  • concentration of the target-binding probes specific for the sequence-variable target region set is at least 1.25-, 1.5-, 1.75-, 2-, 2.25-, 2.5-, 2.75-, 3-, 3.5-, 4-, 4.5-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, or 15-fold higher than the concentration of the target -binding probes specific for the epigenetic target region set.
  • the concentration of the target-binding probes specific for the sequence-variable target region set is 1.25- to 1.5-, 1.5- to 1.75-, 1.75- to 2-, 2- to 2.25-, 2.25- to 2.5-, 2.5- to 2.75-, 2.75- to 3-, 3- to 3.5-, 3.5- to 4-, 4- to 4.5-, 4.5- to 5-, 5- to 5.5-, 5.5- to 6-, 6- to 7-, 7- to 8-, 8- to 9-, 9- to 10-, 10- to 11-, 11- to 12-, 13- to 14-, or 14- to 15-fold higher than the concentration of the target-binding probes specific for the epigenetic target region set.
  • concentration may refer to the average mass per volume concentration of individual probes in each set.
  • the capture probes specific for the sequence-variable target region set have a higher affinity for their targets than the capture probes specific for the epigenetic target region set.
  • Affinity can be modulated in any way known to those skilled in the art, including by using different probe chemistries.
  • certain nucleotide modifications such as cytosine 5-methylation (in certain sequence contexts), modifications that provide a heteroatom at the 2’ sugar position, and LNA nucleotides, can increase stability of double-stranded nucleic acids, indicating that oligonucleotides with such modifications have relatively higher affinity for their complementary sequences. See, e.g., Severin et al., Nucleic Acids Res.
  • the capture probes specific for the sequence-variable target region set have modifications that increase their affinity for their targets. In some embodiments, alternatively or additionally, the capture probes specific for the epigenetic target region set have modifications that decrease their affinity for their targets.
  • the capture probes comprise a capture moiety.
  • the capture moiety may be any of the capture moieties described herein, e.g., biotin.
  • the capture probes are linked to a solid support, e.g., covalently or non-covalently such as through the interaction of a binding pair of capture moieties.
  • the solid support is a bead, such as a magnetic bead.
  • the capture probes specific for the sequence-variable target region set and/or the capture probes specific for the epigenetic target region set are a capture probe set as discussed above, e.g., probes comprising capture moieties and sequences selected to tile across a panel of regions, such as genes.
  • the capture probes are provided in a single composition.
  • the single composition may be a solution (liquid or frozen). Alternatively, it may be a lyophilizate.
  • the capture probes may be provided as a plurality of compositions, e.g., comprising a first composition comprising probes specific for the epigenetic target region set and a second composition comprising probes specific for the sequence-variable target region set.
  • These probes may be mixed in appropriate proportions to provide a combined probe composition with any of the foregoing fold differences in concentration and/or capture yield.
  • they may be used in separate capture procedures (e.g., with aliquots of a sample or sequentially with the same sample) to provide first and second compositions comprising captured epigenetic target regions and sequence-variable target regions, respectively.
  • the probes specific for hypermethylation variable target regions comprise probes specific for 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.
  • the probes specific for hypermethylation variable target regions comprise probes specific for a plurality of loci listed in Table 2 or Table 3, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 2 or Table 3.
  • probes specific for hypomethylation variable target regions include probes specific for repeated elements and/or intergenic regions.
  • probes specific for repeated elements include probes specific for one, two, three, four, or five of LINE1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and/or satellite DNA.
  • Exemplary probes specific for genomic regions that show cancer-associated hypomethylation include probes specific for nucleotides 8403565-8953708 and/or 151104701- 151 106035 of human chromosome 1.
  • the probes specific for hypomethylation variable target regions include probes specific for regions overlapping or comprising nucleotides 8403565-8953708 and/or 151104701-151106035 of human chromosome 1.
  • the probes for the epigenetic target region set include probes specific for CTCF binding regions.
  • the probes specific for CTCF binding regions comprise probes specific for at least 10, 20, 50, 100, 200, or 500 CTCF binding regions, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 CTCF binding regions, e.g., such as CTCF binding regions described above or in one or more of CTCFBSDB or the Cuddapah et al., Martin et al., or Rhee et al. articles cited above.
  • the probes for the epigenetic target region set comprise at least 100 bp, at least 200 bp at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, or at least 1000 bp upstream and downstream regions of the CTCF binding sites. d. Transcription start sites
  • the probes for the epigenetic target region set include probes specific for transcriptional start sites.
  • the probes specific for transcriptional start sites comprise probes specific for at least 10, 20, 50, 100, 200, or 500 transcriptional start sites, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 transcriptional start sites, e.g., such as transcriptional start sites listed in DBTSS.
  • the probes for the epigenetic target region set comprise probes for sequences at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, or at least 1000 bp upstream and downstream of the transcriptional start sites.
  • 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.
  • regions that may show focal amplifications in cancer can be included in the epigenetic target region set, as discussed above.
  • the probes specific for the epigenetic target region set include probes specific for focal amplifications.
  • the probes specific for focal amplifications include probes specific for one or more of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAFI .
  • the probes specific for focal amplifications include probes specific for one or more of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the foregoing targets. f. Control regions
  • the probes specific for the epigenetic target region set include probes specific for control methylated regions that are expected to be methylated in essentially all samples. In some embodiments, the probes specific for the epigenetic target region set include probes specific for control hypomethylated regions that are expected to be hypomethylated in essentially all samples.
  • the probes for the sequence-variable target region set may comprise probes specific for a plurality of regions known to undergo somatic mutations in cancer.
  • the probes may be specific for any sequence-variable target region set described herein. Exemplary sequence-variable target region sets are discussed in detail herein, e.g., in the sections above concerning captured sets.
  • the sequence-variable target region probe set has a footprint of at least 0.5 kb, e.g., at least 1 kb, at least 2 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, or at least 40 kb.
  • the epigenetic target region probe set has a footprint in the range of 0.5-100 kb, e.g., 0.5-2 kb, 2-10 kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50 kb, 50-60 kb, 60-70 kb, 70-80 kb, 80-90 kb, and 90-100 kb.
  • the sequence-variable target region probe set has a footprint of at least 50 kbp, e.g., at least 100 kbp, at least 200 kbp, at least 300 kbp, or at least 400 kbp.
  • the sequencevariable target region probe set has a footprint in the range of 100-2000 kbp, e.g., 100-200 kbp, 200-300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900-1,000 kbp, 1-1.5 Mbp or 1.5-2 Mbp. In some embodiments, the sequence-variable target region set has a footprint of at least 2 Mbp.
  • probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at 70 of the genes of Table 4.
  • probes specific for the sequencevariable target region set comprise probes specific for the at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 of the SNVs of Table 4.
  • probes specific for the sequence-variable target region set comprise probes specific for at least 1, at least 2, at least 3, at least 4, at least 5, or 6 of the fusions of Table 4. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 1, at least 2, or 3 of the indels of Table 4. In some embodiments, probes specific for the sequencevariable target region set comprise probes specific for at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the genes of Table 5.
  • probes specific for the sequence-variable target region set comprise probes specific for at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the SNVs of Table 5. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least 1, at least 2, at least 3, at least 4, at least 5, or 6 of the fusions of Table 5.
  • probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or 18 of the indels of Table 5.
  • probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 of the genes of Table 6.
  • Long-read sequencing technologies useful herein can include any suitable long-read sequencing methods, including, but not limited to, Pacific Biosciences (PacBio) singlemolecule 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.
  • 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, el al., “Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis,” FlOOOResearch, 6: 100, 2017).
  • next-generation sequencing methods detect augmented signals from a clonal population of amplified DNA fragments
  • SMRT sequencing captures a single DNA molecule, maintaining base modification during sequencing.
  • the error rate of raw PacBio SMRT sequencing-generated data is about 13-15%, as the signal-to-noise ratio from single DNA molecules not high.
  • this platform uses a circular DNA template by ligating hairpin adaptors to both ends of target double-stranded DNA.
  • the DNA template is sequenced multiple times to generate a continuous long read (CLR).
  • 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.
  • 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.
  • inter-pulse duration IPD
  • base modifications can be inferred from the comparison of a modified template to an in silica 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.
  • 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).
  • the sequencing comprises SMRT sequencing.
  • the end repair may be performed using dNTPs, which comprise 5-caC, 4mC, 5mC, 5hmC, 6mA, and/or 8oxoG.
  • reaction data can include both kinetics and other behavior of the enzyme and fluctuations in current through the nanopore.
  • 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 a part of the reaction data, is indicative of the presence of the modified base even where the modified base is not within the sensing portion of the nanopore.
  • Nanopore-based single molecule sequencing system is that commercialized by Oxford Nanopore Technologies (ONT).
  • ONT Oxford Nanopore Technologies
  • 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.
  • 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).
  • the sequencing comprises nanopore sequencing.
  • the end repair may be performed using dNTPs, which comprise 5-caC, 4mC, 5mC, 5hmC, 6mA, BrdU, FldU, IdU, and/or EdU.
  • 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.
  • 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 (Fiillgrabe, et al.
  • the construct is then split to separate the sense and antisense sample strands.
  • 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.
  • 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).
  • 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.
  • 5hmC has been shown to have value as a marker of biological states and disease which includes early cancer detection from cell-free DNA.
  • 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.
  • 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.
  • 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%.
  • 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, 200 or 500 different genes, or up to, for example, 5000, 2500, 1000, 500 or 100 different genes.
  • 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.
  • 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).
  • sequencing of epigenetic target regions requires a lesser depth of sequencing than sequencing of a sequencevariable target region, e.g. for analysis of mutations.
  • lesser sequencing depths may in some cases be adequate for the methods described herein.
  • sequencing DNA that was amplified using RCA provides sequence reads comprising multiple copies of the sequence of an original sample molecule or converted molecule and the copies are used to determine a consensus sequence of the original sample molecule or converted molecule.
  • nucleic acids corresponding to the sequence-variable target region set are sequenced to a greater depth of sequencing than nucleic acids corresponding to the epigenetic target region set.
  • nucleic acids corresponding to the hydroxymethylation-variable target region set are sequenced to a greater depth of sequencing than nucleic acids corresponding to at least one other target region set.
  • the depth of sequencing for nucleic acids corresponding to the sequence-variable and/or hydroxymethylationvariable target region sets may be at least 1 .25-, 1 .5-, 1 .75-, 2-, 2.25-, 2.5-, 2.75-, 3-, 3.5-, 4-,
  • said depth of sequencing is at least 2-fold greater.
  • said depth of sequencing is at least 5-fold greater.
  • said depth of sequencing is at least 10-fold greater.
  • said depth of sequencing is 4- to 10-fold greater.
  • said depth of sequencing is 4- to 100-fold greater.
  • the captured cfDNA corresponding to the sequencevariable target region set and the captured cfDNA corresponding to the epigenetic target region set are sequenced concurrently, e.g., in the same sequencing cell (such as the flow cell of an Illumina sequencer) and/or in the same composition, which may be a pooled composition resulting from recombining separately captured sets or a composition obtained by capturing the cfDNA corresponding to the sequence-variable target region set and the captured cfDNA corresponding to the epigenetic target region set in the same vessel.
  • the captured cfDNA corresponding to the hydroxymethylation variable target region set and the captured cfDNA corresponding to the at least one other target region set are sequenced concurrently, e.g., in the same sequencing cell (such as the flow cell of an Illumina sequencer) and/or in the same composition, which may be a pooled composition resulting from recombining separately captured sets or a composition obtained by capturing the cfDNA corresponding to the hydroxymethylation variable target region set and the captured cfDNA corresponding to the at least one other target region set in the same vessel.
  • methods herein comprise contacting DNA with a methylation-sensitive nuclease, thereby degrading DNA comprising unmethylated sequences or sequences having low levels of methylation.
  • the methylationsensitive nuclease is a methylation-sensitive restriction enzyme (MSRE), thereby degrading DNA comprising an unmethylated recognition site of the MSRE.
  • MSRE methylation-sensitive restriction enzyme
  • Methylation-sensitive nucleases can thus be used in methods herein comprising one or more steps that deplete unmodified or unmethylated sequences, such as those that are prevalent in cfDNA from a subject.
  • methods herein comprise contacting DNA with a methylation-dependent nuclease, thereby degrading DNA comprising methylated sequences or sequences having high levels of methylation.
  • the methylationdependent nuclease is a methylation-dependent restriction enzyme (MDRE), thereby degrading DNA comprising a methylated recognition site of the MSRE.
  • MDRE methylation-dependent restriction enzyme
  • Methylation-dependent nucleases can thus be used in methods herein comprising one or more steps that deplete modified or methylated sequences, such as those that are prevalent in cfDNA from a subject.
  • the contacting the DNA with the MSRE occurs after ligating one or more adapters to the end-repaired DNA molecules and/or before contacting the mCpG-bound DNA with the deaminase.
  • the one or more adapters is resistant to digestion by the MSRE.
  • the one or more adapters that is resistant to digestion by MSRE comprises one or more methylated nucleotides, one or more nucleotide analogs resistant to methylation sensitive restriction enzymes, or does not comprise a nucleotide sequence recognized by the MSRE.
  • the methylated nucleotides in the one or more adapters that is resistant to digestion by MSRE comprise 5-methylcytosine and/or 5-hydroxymethylcytosine.
  • partitioning procedures may result in imperfect sorting of DNA molecules among the subsamples.
  • the choice of a methylation-dependent nuclease or methylation-sensitive nuclease can be made so as to degrade nonspecifically partitioned DNA.
  • the second subsample can be contacted with a methylation-dependent nuclease, such as a methylation-dependent restriction enzyme. This can degrade nonspecifically partitioned DNA in the second subsample (e.g., methylated DNA) to produce a treated second subsample.
  • the first subsample can be contacted with a methylationsensitive endonuclease, such as a methylation-sensitive restriction enzyme, thereby degrading nonspecifically partitioned DNA in the first subsample to produce a treated first subsample.
  • a methylationsensitive endonuclease such as a methylation-sensitive restriction enzyme
  • Degradation of nonspecifically partitioned DNA in either or both of the first or second subsamples is proposed as an improvement to the performance of methods that rely on accurate partitioning of DNA on the basis of a cytosine modification, e.g., to detect the presence of aberrantly modified DNA in a sample, to determine the tissue of origin of DNA, and/or to determine whether a subject has cancer.
  • such degradation may provide improved sensitivity and/or simplify downstream analyses.
  • a methylation-dependent nuclease such as a methylation-dependent restriction enzyme
  • a methylation-sensitive nuclease such as a methylation-sensitive restriction enzyme
  • nucleases In contacting a subsample with a nuclease, one or more nucleases can be used. In some embodiments, a subsample is contacted with a plurality of nucleases. The subsample may be contacted with the nucleases sequentially or simultaneously. Simultaneous use of nucleases may be advantageous when the nucleases are active under similar conditions (e.g., buffer composition) to avoid unnecessary sample manipulation.
  • Contacting the second subsample with more than one methylation-dependent restriction enzyme can more completely degrade nonspecifically partitioned hypermethylated DNA.
  • contacting the first subsample with more than one methylation-sensitive restriction enzyme can more completely degrade nonspecifically partitioned hypomethylated and/or unmethylated DNA.
  • a methylation-dependent nuclease comprises one or more of MspJI, LpnPI, FspEI, or McrBC. In some embodiments, at least two methylation-dependent nucleases are used. In some embodiments, at least three methylation-dependent nucleases are used. In some embodiments, the methylation-dependent nuclease comprises FspEI. In some embodiments, the methylation-dependent nuclease comprises FspEI and MspJI, e.g., used sequentially.
  • a methylation-sensitive nuclease comprises one or more of Aatll, AccII, Acil, Aorl3HI, Aorl5HI, BspT104I, BssHII, BstUI, CfrlOI, Clal, Cpol, Eco52I, Haell, HapII, Hhal, Hin6I, Hpall, HpyCH4IV, Mlul, MspI, Nael, Notl, Nrul, Nsbl, PmaCI, Pspl406I, Pvul, SacII, Sall, Smal, and SnaBI. In some embodiments, at least two methylationsensitive nucleases are used.
  • the methylation-sensitive nucleases comprise BstUI and Hpall. In some embodiments, the two methylation-sensitive nucleases comprise Hhal and AccII. In some embodiments, the methylation-sensitive nucleases comprise BstUI, Hpall and Hin6I. [00503] In some embodiments, FspEI is used for digesting the nucleic acid molecules in at least one subsample (e.g., a hypomethylated partition).
  • BstUI, Hpall and Hin6I are used for digesting the nucleic acid molecules in at least one subsample (e.g., a hypermethylated partition) and FspEI is used for digesting the nucleic acid molecules in at least one other subsample (e.g., a hypomethylated partition).
  • the nucleic acid molecules therein may be digested with a methylation-sensitive nuclease or a methylation-dependent nuclease.
  • the nucleic acid molecules in an intermediately methylated partition are digested with the same nuclease(s) as the hypermethylated partition.
  • the intermediately methylated partition may be pooled with the hypermethylated partition and then the pooled partitions may be subjected to digestion.
  • the nucleic acid molecules in an intermediately methylated partition are digested with the same nuclease(s) as the hypomethylated partition.
  • the intermediately methylated partition may be pooled with the hypomethylated partition and then the pooled partitions may be subjected to digestion.
  • nucleases that can be heat-inactivated at a relatively low temperature (e.g., 65°C or less, or 60°C or less) to avoid denaturing DNA, in that denaturation may interfere with subsequent ligation steps.
  • the third subsample is in some embodiments contacted with a methylation- sensitive nuclease.
  • a methylation-sensitive nuclease Such a step may have any of the features described elsewhere herein with respect to contacting steps, and may be performed before or after a step of tagging or attaching adapters as discussed above.
  • the first and third subsamples are combined before being contacted with a methylation-sensitive nuclease.
  • Such a step may have any of the features described elsewhere herein with respect to contacting steps, and may be performed before or after a step of tagging or attaching adapters as discussed above.
  • the first and third subsamples are differentially tagged before being combined.
  • the third subsample is in some embodiments contacted with a methylation-dependent nuclease.
  • a methylation-dependent nuclease is in some embodiments contacted with a methylation-dependent nuclease.
  • Such a step may have any of the features described elsewhere herein with respect to contacting steps, and may be performed before or after a step of tagging or attaching adapters as discussed above.
  • the second and third subsamples are combined before being contacted with a methylationdependent nuclease.
  • Such a step may have any of the features described elsewhere herein with respect to contacting steps, and may be performed before or after a step of tagging or attaching adapters as discussed above.
  • the second and third subsamples are differentially tagged before being combined.
  • the DNA is purified after being contacted with the nuclease, e.g., using SPRI beads.
  • SPRI beads Such purification may occur after heat inactivation of the nuclease.
  • purification can be omitted; thus, for example, a subsequent step such as amplification can be performed on the subsample containing heat-inactivated nuclease.
  • the contacting step can occur in the presence of a purification reagent such as SPRI beads, e g., to minimize losses associated with tube transfers.
  • the SPRI beads can be re-used for cleanup by adding molecular crowding reagents (e.g., PEG) and salt.
  • the present disclosure provides methods of selectively deaminating DNA in a sample.
  • DNA in the sample is contacted to an mCpG-binding protein, thereby providing mCpG-bound DNA.
  • mCpG-bound DNA is then contacted with a deaminase, thereby providing a converted sample in which unmethylated CpGs in the DNA are converted to UpGs.
  • the methods disclosed herein further comprise sequencing the selectively deaminated DNA and analyzing at least some of the sequence data to detect the presence or absence of base modifications and/or mutations present in the DNA sample.
  • analyzing the sequence data further comprises analyzing at least some of the sequence data corresponding to regions that are not identified as being synthesized during the end repair to detect the presence or absence of base modifications or mutations present in the DNA sample. In some embodiments of the disclosed methods, analyzing the sequence data further comprises filtering out one or more repaired regions of end-repaired DNA such that the one or more repaired regions are not used to determine the methylation status of cytosines in the DNA sample. In some embodiments, analyzing the sequence data further comprises classifying all base calls within the one or more repaired regions as not having double stranded support.
  • analyzing the sequence data further comprises classifying all base calls within the one or more repaired regions as not having double stranded support for detecting the single nucleotide variants (SNVs) in the DNA sample.
  • the method further comprises quantifying DNA damage in the DNA sample through the identification of the one or more regions of the end-repaired DNA that were synthesized during the end repair.
  • analyzing the sequence data comprises determining a level of measured artifacts in a sample.
  • the sample comprises cfDNA.
  • Genetic data can be used for characterizing a specific form of cancer. Cancers are often heterogeneous in both composition and staging. Genetic profile data may allow characterization of specific sub-types of cancer that may be useful in the diagnosis or treatment of that specific sub-type. This information may also provide a subject or practitioner clues regarding the prognosis of a specific type of cancer and allow either a subject or practitioner to adapt treatment options in accord with the progress of the disease. Some cancers progress, becoming more aggressive and genetically unstable. Other cancers may remain benign, inactive or dormant. The system and methods of this disclosure may be useful in determining disease progression.
  • the present methods are useful in determining the efficacy of a particular treatment option.
  • the present methods can also be used for detecting epigenetic variations in conditions other than cancer.
  • the methods of the disclosure may be used to characterize the heterogeneity of an abnormal condition in a subject, the method comprising generating a genetic profile of extracellular polynucleotides in the subject, wherein the genetic profile comprises a plurality of data resulting from epigenetic information (such as methylation profiling), and optionally copy number variation and rare mutation analyses.
  • a disease may be heterogeneous. Disease cells may not be identical.
  • some tumors are known to comprise different types of tumor cells, some cells in different stages of the cancer.
  • heterogeneity may comprise multiple foci of disease.
  • the present methods can thus be used to generate_or profile, fingerprint or set of data that is a summation of epigenetic, and optionally genetic, information derived from different cells in a heterogeneous disease.
  • This set of data may comprise epigenetic information, copy number variation, and/or rare mutation analyses alone or in combination.
  • the present disclosure provides methods of analyzing DNA.
  • the disclosed methods comprise analyzing DNA (such as DNA from a subject) to identify at least one cell type, cell cluster type, tissue type, and/or cancer type from which one or more type-specific epigenetic target regions and/or type-specific sequence-variable target regions originated.
  • methods comprise determining the level of one or more typespecific epigenetic target regions and/or type-specific sequence-variable target regions that originated from the at least one cell type, cell cluster type, tissue type, and/or cancer type.
  • detecting the presence, levels, or absence of DNA sequences and/or modifications facilitates disease diagnosis or identification of appropriate treatments.
  • the presence of or a change in the levels of one or more sequences and/or modifications is indicative of the presence or absence of a disease or disorder in a subject, such as cancer or precancer, or other disorder that causes changes in nucleic acids relative to a healthy subject.
  • Information and data generated by the methods disclosed herein can also be used for characterizing a specific form of cancer.
  • the methods disclosed herein may allow characterization of specific sub-types of cancer that may be important in the diagnosis or treatment of that specific sub-type. This information may also provide a subject or practitioner clues regarding the prognosis of a specific type of cancer and allow either a subject or practitioner to adapt treatment options in accord with the progress of the disease. Some cancers can progress to become more aggressive and genetically unstable. Other cancers may remain benign, inactive or dormant.
  • the system and methods of this disclosure may be useful in determining disease progression.
  • the methods of the disclosure may be used to characterize the heterogeneity of a condition in a subject.
  • Such methods can include, e.g., generating an aggregate profile of extracellular nucleic acids derived from the subject, wherein the aggregate profile comprises a plurality of data resulting from various nucleic acid analyses.
  • the aggregate profile comprises epigenetic and mutation analyses.
  • an aggregate profile comprises a summation of information derived from different cells in a heterogeneous disease. This summation may comprise structural variation identities and levels, copy number variation, epigenetic variation, or other mutation analyses.
  • An exemplary method for analyzing DNA comprises the following steps (e.g., in the order listed below), which is illustrated in FIG. 1:
  • Preparing an extracted DNA sample e.g., extracting DNA, such as cfDNA, from a human sample, such as a blood sample).
  • dNTPs a deaminase-resistant modified cytosine
  • dNTP(e.g., a methylated cytosine or unmethylated cytosine, depending on desired treatment of re-synthesized DNA) is incorporated into repaired regions of the end-repaired DNA molecules at one or more locations.
  • NGS next-generation sequencing
  • Another exemplary method for analyzing DNA comprises the following steps (e.g., in the order listed below), which is illustrated in FIG. 2:
  • NGS next-generation sequencing
  • Separating e.g., via elution
  • Separation e.g., elution
  • Separation parameters may be tuned to capture DNA having a desired level of methylation.
  • methods described herein comprise identifying or predicting the presence or absence of DNA produced by a tumor (or neoplastic cells, or cancer cells), determining the probability that a test subject has a tumor or cancer, and/or characterizing a tumor, neoplastic cells or cancer as described herein.
  • the present methods can be used to diagnose presence of a condition, e.g., cancer or precancer, in a subject, to characterize a condition (such as to determine a cancer stage or heterogeneity of a cancer), to monitor a subject’s response to receiving a treatment for a condition (such as a response to a chemotherapeutic or immunotherapeutic), assess prognosis of a subject (such as to predict a survival outcome in a subject having a cancer), to determine a subject’s risk of developing a condition, to predict a subsequent course of a condition in a subject, to determine metastasis or recurrence of a cancer in a subject (or a risk of cancer metastasis or recurrence), and/or to monitor a subject’s health as part of a preventative health monitoring program (such as to determine whether and/or when a subject is in need of further diagnostic screening).
  • a condition e.g., cancer or precancer
  • the present disclosure can also be useful in determining the efficacy of a particular treatment option.
  • Successful treatment options may increase the amount of rare mutations detected in subject's blood if the treatment is successful as more cancers may die and shed DNA. In other examples, this may not occur.
  • certain treatment options may be correlated with genetic profiles of cancers over time. This correlation may be useful in selecting a therapy.
  • target regions are analyzed to determine whether they show methylation characteristics of tumor cells or cells that do not ordinarily contribute significantly to cfDNA and/or target regions are analyzed to determine whether they show methylation characteristic of tumor cells or cells that do not ordinarily contribute significantly to cfDNA.
  • the present methods can be used to monitor the likelihood of residual disease or the likelihood of recurrence of disease.
  • the subject has used tobacco, e.g., for at least 1, 5, 10, or 15 years.
  • the subject has a high BMI, e.g., a BMI of 25 or greater, 26 or greater, 27 or greater, 28 or greater, 29 or greater, or 30 or greater.
  • the subject is at least 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old.
  • the subject has poor nutrition, e.g., high consumption of one or more of red meat and/or processed meat, trans fat, saturated fat, and refined sugars, and/or low consumption of fruits and vegetables, complex carbohydrates, and/or unsaturated fats.
  • High and low consumption can be defined, e.g., as exceeding or falling below, respectively, recommendations in Dietary Guidelines for Americans 2020-2025, available at dietaryguidelines.gov/sites/default/files/2021- 03/Dietary _Guidelines_for_Americans-2020-2025.pdf .
  • the subject has high alcohol consumption, e.g., at least three, four, or five drinks per day on average (where a drink is about one ounce or 30 mb of 80-proof hard liquor or the equivalent).
  • the subject has a family history of cancer, e.g., at least one, two, or three blood relatives were previously diagnosed with cancer.
  • the relatives are at least third-degree relatives (e g., great-grandparent, great aunt or uncle, first cousin), at least second- degree relatives (e.g., grandparent, aunt or uncle, or half-sibling), or first-degree relatives (e.g., parent or full sibling).
  • the one or more methods described in the present disclosure may be used to assist in the treatment of a type of cancer.
  • the methods and systems disclosed herein may be used to identify customized or targeted therapies to treat a given disease or condition in patients based on the classification of a nucleic acid variant as being of somatic or germline origin.
  • the disease under consideration is a type of cancer.
  • the cancer is a type of cancer that is not a hematological cancer, e.g., a solid tumor cancer such as a carcinoma, adenocarcinoma, or sarcoma.
  • Type and/or stage of cancer can be detected from 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, such as 5mC and 5mC profiles.
  • the present methods can in some cases be used in combination with methods used to detect other genetic/epigenetic variations, e.g. in
  • a method described herein comprises identifying the presence of target regions and/or DNA produced by a tumor (or neoplastic cells, or cancer cells) or by precancer cells. In some embodiments, a method described herein comprises determining the level of target regions and/or identifying the presence of DNA produced by a tumor (or neoplastic cells, or cancer cells) or by precancer cells. In some embodiments, determining the level of target regions comprises determining either an increased level or decreased level of target regions, wherein the increased or decreased level of target regions is determined by comparing the level of target regions with a threshold level/value.
  • Genetic and/or epigenetic data can also be used for characterizing a specific form of cancer. Cancers are often heterogeneous in both composition and staging. Genetic and/or epigenetic profile data may allow characterization of specific sub-types of cancer that may be important in the diagnosis or treatment of that specific sub-type. This information may also provide a subject or practitioner clues regarding the prognosis of a specific type of cancer and allow either a subject or practitioner to adapt treatment options in accord with the progress of the disease. Some cancers can progress to become more aggressive and genetically unstable. Other cancers may remain benign, inactive or dormant. The system and methods of this disclosure may be useful in determining disease progression.
  • an abnormal condition is cancer, e.g. as described herein.
  • the abnormal condition may be one resulting in a heterogeneous genomic population.
  • some tumors are known to comprise tumor cells in different stages of the cancer.
  • the present methods can also be used to quantify levels of different cell types, such as immune cell types, including rare immune cell types, such as activated lymphocytes and myeloid cells at particular stages of differentiation. Such quantification can be based on the numbers of molecules corresponding to a given cell type in a sample.
  • Sequence information obtained in the present methods may comprise sequence reads of the nucleic acids generated by a nucleic acid sequencer.
  • the nucleic acid sequencer performs pyrosequencing, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-synthesis, 5-letter sequencing, 6-letter sequencing, sequencing-by-ligation or sequencing-by-hybridization on the nucleic acids to generate sequencing reads.
  • the method further comprises grouping the sequence reads into families of sequence reads, each family comprising sequence reads generated from a nucleic acid in the sample.
  • the methods comprise determining the likelihood that the subject from which the sample was obtained has cancer or precancer, or has a metastasis, that is related to changes in proportions of types of immune cells.
  • the present methods can be used to generate or profile, fingerprint or set of data that is a summation of genetic and/or epigenetic information derived from different cells in a heterogeneous disease. This set of data may comprise copy number variation, epigenetic variation, and mutation analyses alone or in combination.
  • the present methods can be used to diagnose, prognose, monitor or observe cancers, or other diseases.
  • the methods herein do not involve the diagnosing, prognosing or monitoring a fetus and as such are not directed to non-invasive prenatal testing.
  • these methodologies may be employed in a pregnant subject to diagnose, prognose, monitor or observe cancers or other diseases in an unborn subject whose DNA and other polynucleotides may co-circulate with maternal molecules.
  • Non-limiting examples of other genetic-based diseases, disorders, or conditions that are optionally evaluated using the methods and systems disclosed herein include achondroplasia, alpha- 1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Charcot-Marie-Tooth (CMT), cri du chat, Crohn's disease, cystic fibrosis, Dercum disease, down syndrome, Duane syndrome, Duchenne muscular dystrophy, Factor V Leiden thrombophilia, familial hypercholesterolemia, familial mediterranean fever, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, retin
  • the methods can provide a measure of the extent of DNA damage through the quantification of the methylated cytosines of CpG sites, the methods disclosed herein can also be used to quantify the level of DNA damage present in the original DNA sample.
  • the method further comprises calculating a synthesis index which is a quantitative measure of the regions synthesized in the end repair.
  • the synthesis index may be on a molecule level and/or a sample level.
  • the synthesis index may be the proportion of sequencing data which corresponds to synthesized regions.
  • the method further comprises comparing the synthesis index to one or more reference values to classify the DNA sample.
  • the classification may be whether the DNA sample derives from a subject with or without cancer.
  • the reference values may be derived from one or more control DNA samples which are known to have specific properties, such as being derived from a subject known to have cancer, e.g. a specific type of cancer.
  • the reference values may be obtained by performing the method used to obtain the synthesis index on control samples (i.e. using the same end repair, deamination, amplification, ligation and/or sequencing methods).
  • a method described herein comprises detecting a presence or absence of DNA originating or derived from a tumor cell at a preselected timepoint following a previous cancer treatment of a subject previously diagnosed with cancer using a set of sequence information obtained as described herein.
  • the method may further comprise determining a cancer recurrence score that is indicative of the presence or absence of the DNA originating or derived from the tumor cell for the subject.
  • a cancer recurrence score may further be used to determine a cancer recurrence status.
  • the cancer recurrence status may be at risk for cancer recurrence, e.g., when the cancer recurrence score is above a predetermined threshold.
  • the cancer recurrence status may be at low or lower risk for cancer recurrence, e.g., when the cancer recurrence score is above a predetermined threshold.
  • a cancer recurrence score equal to the predetermined threshold may result in a cancer recurrence status of either at risk for cancer recurrence or at low or lower risk for cancer recurrence.
  • a cancer recurrence score is compared with a predetermined cancer recurrence threshold, and the subject is classified as a candidate for a subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold or not a candidate for therapy when the cancer recurrence score is below the cancer recurrence threshold.
  • a cancer recurrence score equal to the cancer recurrence threshold may result in classification as either a candidate for a subsequent cancer treatment or not a candidate for therapy.
  • the present methods can also be used to quantify levels of different cell types, such as immune cell types, including rare immune cell types, such as activated lymphocytes and myeloid cells at particular stages of differentiation. Such quantification can be based on the numbers of molecules corresponding to a given cell type in a sample.
  • Sequence information obtained in the present methods may comprise sequence reads of the nucleic acids generated by a nucleic acid sequencer.
  • the nucleic acid sequencer performs pyrosequencing, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by -synthesis, 5-letter sequencing, 6-letter sequencing, sequencing-by-ligation or sequencing-by-hybridization on the nucleic acids to generate sequencing reads.
  • the method further comprises grouping the sequence reads into families of sequence reads, each family comprising sequence reads generated from a nucleic acid in the sample.
  • the methods comprise determining the likelihood that the subject from which the sample was obtained has cancer, precancer, an infection, transplant rejection, or other diseases or disorder that is related to changes in proportions of types of immune cells.
  • Comparisons of immune cell identities and/or immune cell quantities/proportions between two or more samples collected from a subject at two different time points can allow for monitoring of one or more aspects of a condition in the subject over time, such as a response of the subject to a treatment, the severity of the condition (such as a cancer stage) in the subject, a recurrence of the condition (such as a cancer), and/or the subject’s risk of developing the condition (such as a cancer).
  • the methods discussed above may further comprise any compatible feature or features set forth elsewhere herein, including in the section regarding methods of determining a risk of cancer recurrence in a subject and/or classifying a subject as being a candidate for a subsequent cancer treatment.
  • a method provided herein is or comprises a method of determining a risk of cancer recurrence in a subject. In some embodiments, a method provided herein is or comprises a method of detecting the presence of absence of a metastasis in a subject. In some embodiments, a method provided herein is or comprises a method of classifying a subject as being a candidate for a subsequent cancer treatment.
  • Any of such methods may comprise collecting a sample (such as DNA, such as DNA originating or derived from a tumor cell) from the subject diagnosed with the cancer at one or more preselected timepoints following one or more previous cancer treatments to the subject.
  • the subject may be any of the subjects described herein.
  • the sample may comprise chromatin, cfDNA, or other cell materials.
  • the sample, such as the DNA sample may be a tissue sample.
  • the DNA may be DNA, such as cfDNA, from a blood sample (e.g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample).
  • the DNA may comprise DNA obtained from a tissue sample.
  • the previous cancer treatment may comprise surgery, administration of a therapeutic composition, and/or chemotherapy.
  • Any of such methods may comprise sequencing the captured DNA molecules, whereby a set of sequence information is produced.
  • the captured DNA molecules of the sequence-variable target region set may be sequenced to a greater depth of sequencing than the captured DNA molecules of the epigenetic target region set.
  • Any of such methods may comprise detecting a presence or absence of DNA originating or derived from a tumor cell at a preselected timepoint using the set of sequence information.
  • the detection of the presence or absence of DNA, such as cfDNA, originating or derived from a tumor cell may be performed according to any of the embodiments thereof described elsewhere herein.
  • Methods of determining a risk of cancer recurrence in a subject may comprise determining a cancer recurrence score that is indicative of the presence or absence, or amount, of the DNA, such as genomic regions of interest and target regions, originating or derived from the tumor cell for the subject.
  • the cancer recurrence score may further be used to determine a cancer recurrence status.
  • the cancer recurrence status may be at risk for cancer recurrence, e.g., when the cancer recurrence score is above a predetermined threshold.
  • the cancer recurrence status may be at low or lower risk for cancer recurrence, e.g., when the cancer recurrence score is above a predetermined threshold.
  • a cancer recurrence score equal to the predetermined threshold may result in a cancer recurrence status of either at risk for cancer recurrence or at low or lower risk for cancer recurrence.
  • Methods of detecting the presence or absence of metastasis in a subject may comprise comparing the presence or level of a tissue-specific cell material to the presence or level of the tissue-specific cell material obtained from the subject at a different time, a reference level of the tissue-specific cell material, or to a comparator cell material. Methods herein may comprise additional steps to determine whether a metastasis is present.
  • Methods of classifying a subject as being a candidate for a subsequent cancer treatment may comprise comparing the cancer recurrence score of the subject with a predetermined cancer recurrence threshold, thereby classifying the subject as a candidate for the subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold or not a candidate for therapy when the cancer recurrence score is below the cancer recurrence threshold.
  • a cancer recurrence score equal to the cancer recurrence threshold may result in classification as either a candidate for a subsequent cancer treatment or not a candidate for therapy.
  • the subsequent cancer treatment comprises chemotherapy or administration of a therapeutic composition.
  • sequence-variable target region sequences are obtained, and determining the cancer recurrence score may comprise determining at least a first subscore indicative of the amount of the levels of particular immune cell types, SNVs, insertions/deletions, CNVs and/or fusions present in sequence-variable target region sequences.
  • a number of mutations in the sequence-variable target regions chosen from 1, 2, 3, 4, or 5 is sufficient for the first subscore to result in a cancer recurrence score classified as positive for cancer recurrence.
  • the number of mutations is chosen from 1, 2, or 3.
  • abnormal molecules i.e., molecules with an epigenetic state different from DNA found in a corresponding sample from a healthy subject
  • epigenetic changes associated with cancer such as with a metastasis
  • methylation of hypermethylation variable target regions and/or perturbed fragmentation of fragmentation variable target regions where “perturbed” means different from DNA found in a corresponding sample from a healthy subject.
  • a proportion of molecules corresponding to the hypermethylation variable target region set and/or fragmentation variable target region set that indicate hypermethylation in the hypermethylation variable target region set and/or abnormal fragmentation in the fragmentation variable target region set greater than or equal to a value in the range of 0.001%-10% is sufficient for the subscore to be classified as positive for cancer recurrence.
  • the range may be 0.001%-l%, 0.005%-l%, 0.01 %-5%, 0.01%-2%, or 0.01 %- 1%.
  • any of such methods may comprise determining a fraction of tumor DNA from the fraction of molecules in the set of sequence information that indicate one or more features indicative of origination from a tumor cell.
  • the fraction of tumor DNA may be determined based on a combination of molecules corresponding to epigenetic target regions and molecules corresponding to sequence-variable target regions.
  • a fraction of tumor DNA greater than or equal to a threshold in the range of 10 10 to I O 9 , 10 9 to 10 8 , 10 8 to I O 7 , 10 7 to I O 6 , 10 6 to I O 5 , I O 5 to I O 4 , 10 ⁇ to I O 3 , 10 3 to I O 2 , or 10 2 to 10 1 is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence.
  • the fraction of tumor DNA greater than a threshold of at least 10' 7 is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence.
  • a determination that a fraction of tumor DNA is greater than a threshold may be made based on a cumulative probability. For example, the sample was considered positive if the cumulative probability that the tumor fraction was greater than a threshold in any of the foregoing ranges exceeds a probability threshold of at least 0.5, 0.75, 0.9, 0.95, 0.98, 0.99, 0.995, or 0.999. In some embodiments, the probability threshold is at least 0.95, such as 0.99.
  • the set of sequence information comprises sequencevariable target region sequences and epigenetic target region sequences
  • determining the cancer recurrence score comprises determining a subscore indicative of the amount of SNVs, insertions/deletions, CNVs and/or fusions present in sequence-variable target region sequences and a subscore indicative of the amount of abnormal molecules in epigenetic target region sequences, and combining the subscores to provide the cancer recurrence score.
  • subscores may be combined by applying a threshold to each subscore independently (e.g., greater than a predetermined number of mutations (e.g., > 1) in sequencevariable target regions, and greater than a predetermined fraction of abnormal molecules (i.e., molecules with an epigenetic state different from the DNA found in a corresponding sample from a healthy subject; e g., tumor) in epigenetic target regions), or training a machine learning classifier to determine status based on a plurality of positive and negative training samples.
  • a threshold e.g., greater than a predetermined number of mutations (e.g., > 1) in sequencevariable target regions, and greater than a predetermined fraction of abnormal molecules (i.e., molecules with an epigenetic state different from the DNA found in a corresponding sample from a healthy subject; e g., tumor) in epigenetic target regions
  • a threshold e.g., greater than a predetermined number of mutations (e.g., > 1) in sequencevariable
  • subscores may be combined by applying a threshold to each subscore independently in sequence-variable target regions, respectively, and greater than a predetermined fraction of abnormal molecules (i.e., molecules with an epigenetic state different from the DNA found in a corresponding sample from a healthy subject; e.g., tumor) in epigenetic target regions), or training a machine learning classifier to determine status based on a plurality of positive and negative training samples.
  • a threshold i.e., molecules with an epigenetic state different from the DNA found in a corresponding sample from a healthy subject; e.g., tumor
  • a value for the combined score in the range of -4 to 2 or -3 to 1 is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence.
  • the cancer recurrence status of the subject may be at risk for cancer recurrence and/or the subject may be classified as a candidate for a subsequent cancer treatment.
  • the cancer is any one of the types of cancer described elsewhere herein, e.g., colorectal cancer.
  • the methods according to the present disclosure can be useful in predicting a subject’s response to a particular treatment option, such as over a period of time.
  • successful treatment options may increase the amount of cancer associated DNA sequences detected in a subject's blood, such as if the treatment is successful as more cancers may die and shed DNA.
  • certain treatment options may be correlated with genetic profiles of cancers over time. This correlation may be useful in selecting a therapy.
  • successful treatment options may result in an increase or decrease in the levels of different immune cell types (including rare immune cell types), and/or an increase or decrease in the levels of a specific protein or proteins and/or a specific DNA sequence (e.g., of a CDR3), such as in the blood, and an unsuccessful treatment may result in no change. In other examples, this may not occur.
  • quantities of each of a plurality of cell types are determined based on sequencing and analysis (such as determination of epigenetic and/or genomic signatures) of DNA isolated from at least one sample comprising cells (such as a tissue sample or a blood sample, e g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample) from a subject.
  • a tissue sample or a blood sample e g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample
  • differences in levels and/or presence of particular genetic and/or epigenetic signatures in DNA isolated from blood samples from a subject can be used to quantify cell types, such as immune cell types, within the sample.
  • a comparison of the disclosed genetic and/or epigenetic signatures in DNA isolated from blood samples collected from a subject at two or more time points can be used to monitor changes in cell type quantities in the subject under different conditions (such as prior to and after a treatment), or over time (e.g., as part of a preventative health monitoring program).
  • the disclosed methods can include evaluating (such as quantifying) and/or interpreting cell types (such as immune cell types) present in one or more samples (such as a tissue sample or a blood sample, e.g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample) collected from a subject at one or more timepoints in comparison to a selected baseline value or reference standard (or a selected set of baseline values or reference standards).
  • samples such as a tissue sample or a blood sample, e.g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample
  • a baseline value or reference standard may be a quantity of cell types measured in one or more samples (such as an average quantity or range of quantities of cell types present in at least two samples) collected from the subject at one or more time points, such as prior to receiving a treatment, prior to diagnosis of a condition (such as a cancer), or as part of a preventative health monitoring program.
  • a baseline value or reference standard may be a quantity of cell types measured in one or more samples (such as an average quantity or range of quantities of cell types present in at least two samples) collected at one or more timepoints from one or more subjects that do not have the condition (such as a healthy subject that does not have a cancer), one or more subjects that responded favorably to the treatment, or one or more subjects that have not received the treatment.
  • the baseline value or reference standard utilized is a standard or profde derived from a single reference subject. In other embodiments, the baseline value or reference standard utilized is a standard or profile derived from averaged data from multiple reference subjects.
  • the reference standard in various embodiments, can be a single value, a mean, an average, a numerical mean or range of numerical means, a numerical pattern, or a graphical pattern created from the cell type quantity data derived from a single reference subject or from multiple reference subjects. Selection of the particular baseline values or reference standards, or selection of the one or more reference subjects, depends upon the use to which the methods described herein are to be put by, for example, a research scientist or a clinician (such as a physician).
  • a sample collected at a first time point is a tissue sample and a sample collected at a subsequent time point (such as a second time point) is a blood sample.
  • a condition such as a cancer
  • a response of the subject to a treatment one or more characteristic of a condition (such as a cancer stage) in the subject, recurrence of a condition (such as a cancer), and/or a subject’s risk of developing a condition (such as a cancer).
  • methods are provided wherein quantities of cell types present in at least one sample (such as at least one tissue sample and/or at least one blood sample, e.g., a whole blood sample, buffy coat sample, leukapheresis sample, or PBMC sample) collected from a subject at one or more timepoints (such as prior to receiving a treatment) are compared to quantities of cell types present in at least one sample collected from the subject at one or more different time points (such as after receiving the treatment).
  • tissue sample such as at least one tissue sample and/or at least one blood sample, e.g., a whole blood sample, buffy coat sample, leukapheresis sample, or PBMC sample
  • the disclosed methods can allow for patient-specific monitoring, such that, for example, differences in cell type quantities between samples collected from the subject at different timepoints may indicate changes (such as presence or absence of a condition, response to a treatment, a prognosis, or the like) that are significant with respect to the subject but may yet fall within a normal range of a general healthy population.
  • methods are provided for monitoring one or more aspects of a condition in a subject over time, such as but not limited to, a subject’s response to receiving a treatment for a condition (such as a response to a chemotherapeutic or immunotherapeutic).
  • a condition such as a response to a chemotherapeutic or immunotherapeutic.
  • one or more samples is collected from the subject at at least 1-10, at least 1-5, at least 2-5, or at least 1, at least 2, 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 15, or at least 20 time points prior to the subject receiving the treatment.
  • one or more samples is collected from a subject at least once per year, such as about 1-12 times or about 2-6 times, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per year. In other embodiments, one or more samples is collected from the subject less than once per year, such as about once every 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. In some embodiments, one or more samples is collected from the subject about once every 1-5 years or about once every 1-2 years, such as about every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 years.
  • the chemotherapy administered to a subject may comprise FOLFOX or FOLFIRI.
  • a therapy may be administered to a subject that comprises at least one PARP inhibitor.
  • the therapies are PARP inhibitors, such as Olaparib (LYNPARZA®), Rucaparib (RUBRACA®), Niraparib (ZEJULA®), and Talazoparib (TALZENNA®). These may be used for treating mutations in BRCA1, BRCA2, ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD5 IB, RAD51C, RAD5 ID and RAD54L alterations, and/or for genes associated Homologous Recombination Repair (HRR).
  • HRR Homologous Recombination Repair
  • therapy is customized based on the status of a nucleic acid variant as being of somatic or germline origin.
  • essentially any cancer therapy e.g., surgical therapy, radiation therapy, chemotherapy, immunotherapy, and/or the like
  • Customized therapies can include at least one immunotherapy (or an immunotherapeutic agent).
  • Immunotherapy refers generally to methods of enhancing an immune response against a given cancer type.
  • immunotherapy refers to methods of enhancing a T cell response against a tumor or cancer.
  • Immunotherapies are treatments with one or more agents that act to stimulate the immune system so as to kill or at least to inhibit growth of cancer cells, and preferably to reduce further growth of the cancer, reduce the size of the cancer and/or eliminate the cancer.
  • Some such agents bind to a target present on cancer cells; some bind to a target present on immune cells and not on cancer cells; some bind to a target present on both cancer cells and immune cells.
  • Such agents include, but are not limited to, checkpoint inhibitors and/or antibodies.
  • Checkpoint inhibitors are inhibitors of pathways of the immune system that maintain self-tolerance and modulate the duration and amplitude of physiological immune responses in peripheral tissues to minimize collateral tissue damage (see, e.g., Pardoll, Nature Reviews Cancer 12, 252-264 (2012)).
  • Exemplary agents include antibodies against any of PD-1, PD-2, PD-L1, PD-L2, CTLA-4, 0X40, B7.1, B7He, LAG3, CD137, KIR, CCR5, CD27, CD40, or CD47.
  • Other exemplary agents include proinflammatory cytokines, such as IL-ip, IL-6, and TNF-a.
  • Other exemplary agents are T-cells activated against a tumor, such as T-cells activated by expressing a chimeric antigen targeting a tumor antigen recognized by the T-cell.
  • anti-PD-1 or anti-PD-Ll therapies comprise pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), and cemiplimab (LIBTAYO®), atezolizumab (TECENTRIQ®), durvalumab (INFINZI®), and avelumab (BAVENCIO®). These therapies may be used to treat patients identified as having high microsatellite instability (MSI) status or high tumor mutational burden (TMB)
  • MSI microsatellite instability
  • TMB tumor mutational burden
  • the inhibitory immune checkpoint molecule is CTLA4 or PD-1.
  • the inhibitory immune checkpoint molecule is a ligand for PD-1, such as PD-L1 or PD-L2.
  • the inhibitory immune checkpoint molecule is a ligand for CTLA4, such as CD80 or CD86.
  • the inhibitory immune checkpoint molecule is lymphocyte activation gene 3 (LAG3), killer cell immunoglobulin like receptor (KIR), T cell membrane protein 3 (TIM3), galectin 9 (GAL9), or adenosine A2a receptor (A2aR).
  • the immunotherapy or immunotherapeutic agent is an antagonist of an inhibitory immune checkpoint molecule.
  • the inhibitory immune checkpoint molecule is PD-1.
  • the inhibitory immune checkpoint molecule is PD- Ll.
  • the antagonist of the inhibitory immune checkpoint molecule is an antibody (e.g., a monoclonal antibody).
  • the antibody or monoclonal antibody is an anti-CTLA4, anti-PD-1, anti-PD-Ll, or anti-PD-L2 antibody.
  • the therapies target mutated forms of the EGFR protein.
  • Such therapies can include osimertinib (TAGRISSO®), erlotinib (TARCEVA®), and gefinitib (IRESSA®).
  • Therapies can include one or more of treatments for target therapies, including abemaciclib (VERZENIO®), abiraterone acetate (ZYTIGA®), acalabrutinib (CALQUENCE®), adagrasib (KRAZATI®), ado-trastuzumab emtansine (KADCYLA®), afatinib dimaleate (GILOTRIF®), alectinib (ALCENSA®), alemtuzumab (CAMPATH®), alitretinoin (PANRETIN®), alpelisib (PIQRAY®), amivantamab- vmjw (RYBREVANT®), anastrozole (ARIMIDEX®), apalutamide (ERLEADA®), asciminib hydrochloride (SCEMBLIX®), atezolizumab (TECENTRIQ®), avapritinib (AYVAKIT®), aveluma
  • Table 7 provides an exemplary list of drugs used to treat cancers with mutations observed in target genes associated with certain cancer types.
  • the subject has a cancer of a type listed in Table 7 including a mutation in one or more target genes listed in Table 7 for that cancer type, and the therapy administered to the subject comprises the drug listed in Table 7 for that cancer type and mutation.
  • the methods described herein can be used to treat patients by (i) detecting one or more mutations in the one or more target genes listed in Table 7; and (ii) administering the corresponding one or more drugs listed in Table 7. In some embodiments, these therapies may be used alone or in combination with other therapies to treat a disease.
  • the immune checkpoint molecule is a co-stimulatory molecule that amplifies a signal involved in a T cell response to an antigen.
  • CD28 is a co-stimulatory receptor expressed on T cells.
  • CD28 When a T cell binds to antigen through its T cell receptor, CD28 binds to CD80 (aka B7.1) or CD86 (aka B7.2) on antigen-presenting cells to amplify T cell receptor signaling and promote T cell activation. Because CD28 binds to the same ligands (CD80 and CD86) as CTLA4, CTLA4 is able to counteract or regulate the co-stimulatory signaling mediated by CD28.
  • the immune checkpoint molecule is a co- stimulatory molecule selected from CD28, inducible T cell co-stimulator (ICOS), CD 137, 0X40, or CD27.
  • the agonist antibody or monoclonal antibody is an anti-CD80, anti-CD86, anti-B7RPl, anti-B7-H3, anti-B7-H4, anti-CD137L, anti- OX40L, or anti-CD70 antibody.
  • Such treatments may include small-molecule drugs or monoclonal antibodies.
  • the methods may also improve biomarker testing in individuals suffering from disease and help determine if the individual is a candidate for a certain drug or combination of drugs based on the presence or absence of the biomarker. Additionally, the methods can improve identification of mutations that contribute to the development of resistance to targeted therapy. Consequently, the analysis techniques may reduce unnecessary or untimely therapeutic interventions, patient suffering, and patient mortality.
  • the status of a nucleic acid variant from a sample from a subject as being of somatic or germline origin may be compared with a database of comparator results from a reference population to identify customized or targeted therapies for that subject.
  • the reference population includes patients with the same cancer or disease type as the subject and/or patients who are receiving, or who have received, the same therapy as the subject.
  • a customized or targeted therapy may be identified when the nucleic variant and the comparator results satisfy certain classification criteria (e.g., are a substantial or an approximate match).
  • the customized therapies described herein are typically administered parenterally (e.g., intravenously or subcutaneously).
  • compositions containing an immunotherapeutic agent are typically administered intravenously. Certain therapeutic agents are administered orally. However, customized therapies (e.g., immunotherapeutic agents, etc.) may also be administered by any method known in the art, for example, buccal, sublingual, rectal, vaginal, intraurethral, topical, intraocular, intranasal, and/or intraauricular, which administration may include tablets, capsules, granules, aqueous suspensions, gels, sprays, suppositories, salves, ointments, or the like.
  • therapies e.g., immunotherapeutic agents, etc.
  • any method known in the art for example, buccal, sublingual, rectal, vaginal, intraurethral, topical, intraocular, intranasal, and/or intraauricular, which administration may include tablets, capsules, granules, aqueous suspensions, gels, sprays, suppositories, salves, o
  • the present methods can be used to diagnose the presence of a condition, e.g., cancer or precancer, in a subject, to characterize a condition (such as to determine a cancer stage or heterogeneity of a cancer), to monitor a subject’s response to receiving a treatment for a condition (such as a response to a chemotherapeutic or immunotherapeutic), assess prognosis of a subject (such as to predict a survival outcome in a subject having a cancer), to determine a subject’s risk of developing a condition, to predict a subsequent course of a condition in a subject, to determine metastasis or recurrence of a cancer in a subject (or a risk of cancer metastasis or recurrence), and/or to monitor a subject’s health as part of a preventative health monitoring program (such as to determine whether and/or when a subject is in need of further diagnostic screening).
  • a condition e.g., cancer or precancer
  • the methods according to the present disclosure can also be useful in predicting a subject’s response to a particular treatment option.
  • Successful treatment options may increase the amount of copy number variation, rare mutations, and/or cancer-related epigenetic signatures (such as hypermethylated regions or hypom ethylated regions) detected in a subject's blood (such as in DNA isolated from a buffy coat sample or any other sample comprising cells, such as a blood sample (e.g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample) from the subject) if the treatment is successful as more cancer cells may die and shed DNA, or if a successful treatment results in an increase or decrease in the quantity of a specific immune cell type in the blood and an unsuccessful treatment results in no change.
  • a blood sample e.g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample
  • quantities of each of a plurality of cell types are determined based on sequencing and analysis (such as determination of epigenetic and/or genomic signatures) of DNA isolated from at least one sample comprising cells (such as blood sample (e.g., a whole blood sample, a huffy coat sample, a leukapheresis sample, or a PBMC sample) from a subject.
  • DNA sample e.g., a whole blood sample, a huffy coat sample, a leukapheresis sample, or a PBMC sample
  • differences in levels and/or presence of particular genetic and/or epigenetic signatures in DNA isolated from blood samples from a subject can be used to quantify cell types, such as immune cell types, within the sample.
  • a comparison of one or more genetic and/or epigenetic signatures in DNA isolated from blood samples collected from a subject at two or more time points can be used to monitor changes in the one or more signatures and/or the one or more cell type quantities in the subject under different conditions (such as prior to and after a treatment), or over time (e.g., as part of a preventative health monitoring program).
  • therapy is customized based on the status of a detected nucleic acid variant as being of somatic or germline origin.
  • essentially any cancer therapy e.g., surgical therapy, radiation therapy, chemotherapy, and/or the like
  • customized therapies include at least one immunotherapy (or an immunotherapeutic agent).
  • Immunotherapy refers generally to methods of enhancing an immune response against a given cancer type.
  • immunotherapy refers to methods of enhancing a T cell response against a tumor or cancer.
  • the status of a nucleic acid variant from a sample from a subject as being of somatic or germline origin may be compared with a database of comparator results from a reference population to identify customized or targeted therapies for that subject.
  • the reference population includes patients with the same cancer or disease type as the subject and/or patients who are receiving, or who have received, the same therapy as the subject.
  • a customized or targeted therapy (or therapies) may be identified when the nucleic variant and the comparator results satisfy certain classification criteria (e.g., are a substantial or an approximate match).
  • the disclosed methods can include evaluating (such as quantifying) and/or interpreting at least one cell material released from a potential metastasis site (such as at least one cell material in a sample from a subject) and/or cell types that contribute to DNA, such as cfDNA, in one or more samples collected from a subject at one or more timepoints in comparison to a selected baseline value or reference standard (or a selected set of baseline values or reference standards).
  • a baseline value or reference standard may be a presence or level of at least one cell material and/or a quantity of cell types measured in one or more samples (such as an average quantity or range of quantities of cell types present in at least two samples) collected from the subject at one or more time points, such as prior to receiving a treatment, prior to diagnosis of a condition (such as a cancer), or as part of a preventative health monitoring program.
  • a baseline value or reference standard may be a presence or level of at least one cell material and/or a quantity of cell types measured with respect to one or more samples (such as an average quantity or range of quantities of cell types present in at least two samples) collected at one or more timepoints from one or more subjects that do not have the condition (such as a healthy subject that does not have a cancer), one or more subjects that responded favorably to the treatment, or one or more subjects that have not received the treatment.
  • the baseline value or reference standard utilized is a standard or profile derived from a single reference subject. In other embodiments, the baseline value or reference standard utilized is a standard or profile derived from averaged data from multiple reference subjects.
  • the reference standard in various embodiments, can be a single value, a mean, an average, a numerical mean or range of numerical means, a numerical pattern, or a graphical pattern created from the cell type quantity data derived from a single reference subject or from multiple reference subjects. Selection of the particular baseline values or reference standards, or selection of the one or more reference subjects, depends upon the use to which the methods described herein are to be put by, for example, a research scientist or a clinician (such as a physician).
  • a baseline value or reference standard may be a quantity of copy number variation, rare mutations, cancer-related epigenetic signatures (such as hypermethylated regions or hypomethylated regions), and/or cell types measured in one or more samples (such as an average quantity or range of quantities of such signatures present in at least two samples) collected from the subject at one or more time points, such as prior to receiving a treatment, prior to diagnosis of a condition (such as a cancer), or as part of a preventative health monitoring program.
  • cancer-related epigenetic signatures such as hypermethylated regions or hypomethylated regions
  • cell types measured in one or more samples such as an average quantity or range of quantities of such signatures present in at least two samples
  • a baseline value or reference standard may be a quantity of, e.g., copy number variation, rare mutations, cancer-related epigenetic signatures (such as hypermethylated regions or hypomethylated regions), and/or cell types measured in one or more samples (such as an average quantity or range of quantities of such signatures and/or cell types present in at least two samples) collected at one or more timepoints from one or more subjects that do not have the condition (such as a healthy subject that does not have a cancer), one or more subjects that responded favorably to the treatment, or one or more subjects that have not received the treatment.
  • cancer-related epigenetic signatures such as hypermethylated regions or hypomethylated regions
  • cell types measured in one or more samples such as an average quantity or range of quantities of such signatures and/or cell types present in at least two samples
  • the baseline value or reference standard utilized is a standard or profile derived from a single reference subject. In other embodiments, the baseline value or reference standard utilized is a standard or profile derived from averaged data from multiple reference subjects.
  • the reference standard in various embodiments, can be a single value, a mean, an average, a numerical mean or range of numerical means, a numerical pattern, or a graphical pattern created from the genetic and/or epigenetic signature quantity data derived from a single reference subject or from multiple reference subjects. Selection of the particular baseline values or reference standards, or selection of the one or more reference subjects, depends upon the use to which the methods described herein are to be put by, for example, a research scientist or a clinician (such as a physician).
  • one or more samples comprising cells may be collected from a subject at two or more timepoints, to assess changes in cell types (such as changes in quantities of cell types) between the two timepoints.
  • a blood sample e.g., a whole blood sample, a leukapheresis sample, or a PBMC sample
  • changes in cell types such as changes in quantities of cell types
  • the present methods can be used, for example, to determine the presence or absence of a condition (such as a cancer), a response of the subject to a treatment, one or more characteristic of a condition (such as a cancer stage) in the subject, recurrence of a condition (such as a cancer), and/or a subject’s risk of developing a condition (such as a cancer).
  • a condition such as a cancer
  • a response of the subject to a treatment e.g., a response of the subject to a treatment
  • one or more characteristic of a condition such as a cancer stage
  • recurrence of a condition such as a cancer
  • a subject e.g., a subject’s risk of developing a condition (such as a cancer).
  • methods are provided wherein quantities of cell types present in at least one sample (such as at least one whole blood sample, buffy coat sample, leukapheresis sample, or PBMC sample) collected from a subject at one or more timepoints (such as prior to receiving a treatment) are compared to quantities of cell types present in at least one sample collected from the subject at one or more different time points (such as after receiving the treatment).
  • quantities of cell types present in at least one sample such as at least one whole blood sample, buffy coat sample, leukapheresis sample, or PBMC sample
  • methods are provided for monitoring a response (such as a change in disease state, such as a presence or absence of a metastasis in a subject, such as measured by assessing a presence or level of at least one cell material released from a potential metastasis site in a sample from the subject) of a subject to a treatment (such as a chemotherapy or an immunotherapy).
  • a treatment such as a chemotherapy or an immunotherapy.
  • one or more samples is collected from the subject at at least 1-10, at least 1 -5, at least 2-5, or at least 1, at least 2, 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 15, or at least 20 time points prior to the subject receiving the treatment.
  • one or more samples is collected from the subject at at least 1-10, at least 1-5, at least 2-5, or at least 1, at least 2, 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 15, or at least 20 time points after the subject has received the treatment.
  • Sample collection from a subject can be ongoing during and/or after treatment to monitor the subject’s response to the treatment.
  • samples are not collected from a subject prior to diagnosis of a condition (such as a cancer) or prior to receiving a treatment.
  • genetic and/or epigenetic signatures, and/or cell types are compared between samples taken at at least 2-10, at least 2-5, at least 3-6, or at least 2, such as 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 15, or at least 20 time points collected after the subject has been diagnosed and/or after the subject has received the treatment.
  • Sample collection from a subject can be ongoing during and/or after treatment to monitor the subject’s response to the treatment.
  • one or more samples is collected from a subject at least once per year, such as about 1-12 times or about 2-6 times, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per year. In other embodiments, one or more samples is collected from the subject less than once per year, such as about once every 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. In some embodiments, one or more samples is collected from the subject about once every 1-5 years or about once every 1-2 years, such as about every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 years.
  • one or more samples is collected from the subject every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months.
  • one or more samples is collected from the subject at least once per day, such as 1, 2, 3, 4, 5, or 6 times per day. Selection of the one or more sample collection timepoints (e.g., the frequency of sample collection), or of the number of samples to be collected at each timepoint, depends upon the use to which the methods described herein are to be put by, for example, a research scientist or a clinician (such as a physician).
  • kits for use in the methods as described herein comprises one or more conversion reagents.
  • the conversion reagents may comprise reagents for any combination of steps described herein, including but not limited to in the numbered embodiments above and in any one of the workflows shown in the figures.
  • the kit comprises adapters.
  • the kit comprises PCR primers, wherein the PCR primers anneal to a target region or to an adapter.
  • the kit comprises additional elements elsewhere herein.
  • the kit comprises instructions for performing a method described herein.
  • the sequencing adaptor can comprise 20-30, 20-40, 30-50, 30-60, 40-60, 40-70, 50-60, 50-70, bases from end to end. In a particular example, the sequencing adaptor can comprise 20-30 bases from end to end. In another example, the sequencing adaptor can comprise 50-60 bases from end to end.
  • a sequencing adaptor can comprise one or more barcodes.
  • a sequencing adaptor can comprise a sample barcode. The sample barcode can comprise a pre-determined sequence. The sample barcodes can be used to identify the source of the polynucleotides.
  • the computer system 301 includes a central processing unit (CPU, also "processor” and “computer processor” herein) 305, which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • the computer system 301 also includes memory or memory location 310 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 315 (e.g., hard disk), communication interface 320 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 325, such as cache, other memory, data storage, and/or electronic display adapters.
  • the CPU 305 can execute a sequence of machine-readable instructions, which can be embodied in a program or software.
  • the instructions may be stored in a memory location, such as the memory 310. Examples of operations performed by the CPU 305 can include fetch, decode, execute, and writeback.
  • the present disclosure provides a non-transitory computer-readable medium comprising computer-executable instructions which, when executed by at least one electronic processor, perform at least a portion of a method described herein.
  • the method may comprise: (a) contacting DNA in the sample with an mCpG-binding protein, thereby providing mCpG-bound DNA; and (b) contacting the mCpG-bound DNA with a deaminase, thereby providing a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La divulgation concerne l'enrichissement et l'analyse d'ADN méthylé par désamination sélective. Plus particulièrement, la divulgation concerne des procédés d'enrichissement d'ADN méthylé dans un échantillon par mise en contact de l'ADN avec une protéine de liaison à mCpG, ce qui permet de fournir de l'ADN lié à mCpG, et par mise en contact de l'ADN lié à mCpG avec une désaminase, ce qui permet d'obtenir un échantillon converti dans lequel au moins une partie des CpG non méthylés dans l'ADN sont convertis en UpG.
PCT/US2025/021812 2024-03-28 2025-03-27 Procédés de désamination sélective utilisant des protéines de liaison à cpg Pending WO2025207924A1 (fr)

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
US202463570986P 2024-03-28 2024-03-28
US63/570,986 2024-03-28
US202463667401P 2024-07-03 2024-07-03
US63/667,401 2024-07-03
US202463669078P 2024-07-09 2024-07-09
US202463669082P 2024-07-09 2024-07-09
US63/669,078 2024-07-09
US63/669,082 2024-07-09
US202463670035P 2024-07-11 2024-07-11
US202463670034P 2024-07-11 2024-07-11
US63/670,035 2024-07-11
US63/670,034 2024-07-11

Publications (1)

Publication Number Publication Date
WO2025207924A1 true WO2025207924A1 (fr) 2025-10-02

Family

ID=95399281

Family Applications (6)

Application Number Title Priority Date Filing Date
PCT/US2025/021808 Pending WO2025207921A1 (fr) 2024-03-28 2025-03-27 Procédés d'enrichissement de méthylation faisant appel à une désamination spécifique de cpg
PCT/US2025/021843 Pending WO2025207939A1 (fr) 2024-03-28 2025-03-27 Procédés de séparation d'adn méthylé par désamination sensible au méthyle et liaison de protéines se liant au cpg
PCT/US2025/021845 Pending WO2025207941A1 (fr) 2024-03-28 2025-03-27 Procédés de séparation d'adn riche en cpg par liaison de protéines se liant au cpg et désamination sensible au méthyle
PCT/US2025/021813 Pending WO2025207925A1 (fr) 2024-03-28 2025-03-27 Procédés d'enrichissement par méthylation par l'utilisation d'une ligature préférentielle d'adaptateurs
PCT/US2025/021812 Pending WO2025207924A1 (fr) 2024-03-28 2025-03-27 Procédés de désamination sélective utilisant des protéines de liaison à cpg
PCT/US2025/021817 Pending WO2025207926A1 (fr) 2024-03-28 2025-03-27 Procédés de désamination sélective utilisant des désaminases sensibles au méthyle

Family Applications Before (4)

Application Number Title Priority Date Filing Date
PCT/US2025/021808 Pending WO2025207921A1 (fr) 2024-03-28 2025-03-27 Procédés d'enrichissement de méthylation faisant appel à une désamination spécifique de cpg
PCT/US2025/021843 Pending WO2025207939A1 (fr) 2024-03-28 2025-03-27 Procédés de séparation d'adn méthylé par désamination sensible au méthyle et liaison de protéines se liant au cpg
PCT/US2025/021845 Pending WO2025207941A1 (fr) 2024-03-28 2025-03-27 Procédés de séparation d'adn riche en cpg par liaison de protéines se liant au cpg et désamination sensible au méthyle
PCT/US2025/021813 Pending WO2025207925A1 (fr) 2024-03-28 2025-03-27 Procédés d'enrichissement par méthylation par l'utilisation d'une ligature préférentielle d'adaptateurs

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/US2025/021817 Pending WO2025207926A1 (fr) 2024-03-28 2025-03-27 Procédés de désamination sélective utilisant des désaminases sensibles au méthyle

Country Status (1)

Country Link
WO (6) WO2025207921A1 (fr)

Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010053519A1 (en) 1990-12-06 2001-12-20 Fodor Stephen P.A. Oligonucleotides
US20030152490A1 (en) 1994-02-10 2003-08-14 Mark Trulson Method and apparatus for imaging a sample on a device
EP1568786A2 (fr) * 2004-02-13 2005-08-31 Affymetrix, Inc. (A US Entity) Analyse de la méthylation utilisant des matrices d'acides nucléiques
WO2007010004A1 (fr) * 2005-07-19 2007-01-25 Epigenomics Ag Procede pour etudier des methylations de cytosine dans de l'adn
US7537898B2 (en) 2001-11-28 2009-05-26 Applied Biosystems, Llc Compositions and methods of selective nucleic acid isolation
US20110160078A1 (en) 2009-12-15 2011-06-30 Affymetrix, Inc. Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Labels
US20130040343A1 (en) * 2008-07-24 2013-02-14 Brookhaven Science Associates, Llc Methods for Detection of Methyl-CpG Dinucleotides
US9598731B2 (en) 2012-09-04 2017-03-21 Guardant Health, Inc. Systems and methods to detect rare mutations and copy number variation
US9738894B2 (en) 2003-03-21 2017-08-22 Roche Innovation Center Copenhagen A/S Short interfering RNA (siRNA) analogues
US9850523B1 (en) 2016-09-30 2017-12-26 Guardant Health, Inc. Methods for multi-resolution analysis of cell-free nucleic acids
US9902992B2 (en) 2012-09-04 2018-02-27 Guardant Helath, Inc. Systems and methods to detect rare mutations and copy number variation
WO2018119452A2 (fr) 2016-12-22 2018-06-28 Guardant Health, Inc. Procédés et systèmes pour analyser des molécules d'acide nucléique
US10260088B2 (en) 2015-10-30 2019-04-16 New England Biolabs, Inc. Compositions and methods for analyzing modified nucleotides
WO2020160414A1 (fr) 2019-01-31 2020-08-06 Guardant Health, Inc. Compositions et méthodes pour isoler de l'adn acellulaire
US10961525B2 (en) 2017-07-05 2021-03-30 The Trustees Of The University Of Pennsylvania Hyperactive AID/APOBEC and hmC dominant TET enzymes
WO2022087309A1 (fr) * 2020-10-23 2022-04-28 Guardant Health, Inc. Compositions et procédés d'analyse d'adn par division et conversion de base
WO2022147420A1 (fr) * 2020-12-30 2022-07-07 Guardant Health, Inc. Détection d'état épigénétique à l'aide d'une dégradation spécifique à une séquence
CN116287152A (zh) * 2023-02-22 2023-06-23 青岛大学 基于甲基化敏感的胞苷脱氨酶建立的基因甲基化检测方法
WO2024020573A1 (fr) * 2022-07-21 2024-01-25 Guardant Health, Inc. Procédés de détection et de réduction des artefacts de méthylation induits par la préparation des échantillons
WO2024073043A1 (fr) 2022-09-30 2024-04-04 Illumina, Inc. Procédés d'utilisation de protéines de liaison cpg dans la cartographie de nucléotides cytosine modifiés

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10331107B3 (de) * 2003-07-04 2004-12-02 Epigenomics Ag Verfahren zum Nachweis von Cytosin-Methylierungen in DNA mittels Cytidin-Deaminasen
US20150099670A1 (en) * 2013-10-07 2015-04-09 Weiwei Li Method of preparing post-bisulfite conversion DNA library
US10155939B1 (en) * 2017-06-15 2018-12-18 New England Biolabs, Inc. Method for performing multiple enzyme reactions in a single tube
IL315876A (en) * 2022-04-07 2024-11-01 Illumina Inc Modification of cytidine deaminases and methods of use
WO2024137880A2 (fr) * 2022-12-22 2024-06-27 Guardant Health, Inc. Procédés recourant à une amplification préservant la méthylation avec correction des erreurs
EP4646491A1 (fr) * 2023-01-06 2025-11-12 Illumina, Inc. Réduction des uraciles par polymérase

Patent Citations (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6582908B2 (en) 1990-12-06 2003-06-24 Affymetrix, Inc. Oligonucleotides
US20010053519A1 (en) 1990-12-06 2001-12-20 Fodor Stephen P.A. Oligonucleotides
US20030152490A1 (en) 1994-02-10 2003-08-14 Mark Trulson Method and apparatus for imaging a sample on a device
US7537898B2 (en) 2001-11-28 2009-05-26 Applied Biosystems, Llc Compositions and methods of selective nucleic acid isolation
US9738894B2 (en) 2003-03-21 2017-08-22 Roche Innovation Center Copenhagen A/S Short interfering RNA (siRNA) analogues
EP1568786A2 (fr) * 2004-02-13 2005-08-31 Affymetrix, Inc. (A US Entity) Analyse de la méthylation utilisant des matrices d'acides nucléiques
WO2007010004A1 (fr) * 2005-07-19 2007-01-25 Epigenomics Ag Procede pour etudier des methylations de cytosine dans de l'adn
US20130040343A1 (en) * 2008-07-24 2013-02-14 Brookhaven Science Associates, Llc Methods for Detection of Methyl-CpG Dinucleotides
US20110160078A1 (en) 2009-12-15 2011-06-30 Affymetrix, Inc. Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Labels
US9598731B2 (en) 2012-09-04 2017-03-21 Guardant Health, Inc. Systems and methods to detect rare mutations and copy number variation
US9902992B2 (en) 2012-09-04 2018-02-27 Guardant Helath, Inc. Systems and methods to detect rare mutations and copy number variation
US10260088B2 (en) 2015-10-30 2019-04-16 New England Biolabs, Inc. Compositions and methods for analyzing modified nucleotides
US9850523B1 (en) 2016-09-30 2017-12-26 Guardant Health, Inc. Methods for multi-resolution analysis of cell-free nucleic acids
WO2018119452A2 (fr) 2016-12-22 2018-06-28 Guardant Health, Inc. Procédés et systèmes pour analyser des molécules d'acide nucléique
US10961525B2 (en) 2017-07-05 2021-03-30 The Trustees Of The University Of Pennsylvania Hyperactive AID/APOBEC and hmC dominant TET enzymes
WO2020160414A1 (fr) 2019-01-31 2020-08-06 Guardant Health, Inc. Compositions et méthodes pour isoler de l'adn acellulaire
WO2022087309A1 (fr) * 2020-10-23 2022-04-28 Guardant Health, Inc. Compositions et procédés d'analyse d'adn par division et conversion de base
WO2022147420A1 (fr) * 2020-12-30 2022-07-07 Guardant Health, Inc. Détection d'état épigénétique à l'aide d'une dégradation spécifique à une séquence
WO2024020573A1 (fr) * 2022-07-21 2024-01-25 Guardant Health, Inc. Procédés de détection et de réduction des artefacts de méthylation induits par la préparation des échantillons
WO2024073043A1 (fr) 2022-09-30 2024-04-04 Illumina, Inc. Procédés d'utilisation de protéines de liaison cpg dans la cartographie de nucléotides cytosine modifiés
CN116287152A (zh) * 2023-02-22 2023-06-23 青岛大学 基于甲基化敏感的胞苷脱氨酶建立的基因甲基化检测方法

Non-Patent Citations (50)

* Cited by examiner, † Cited by third party
Title
"GeneBank", Database accession no. tig00001818-10-11279730497
"MethBank3.0: a database of DNA methylomes across a variety of species", NUCLEIC ACIDS RES, 2018
"MGnify", Database accession no. MGYP000056170311
"UniParc", Database accession no. UPIO001BOE918
"UniProt", Database accession no. A0A6B3W 113 |A0A6B3W 113_9BACI
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
BLANCO ET AL., J. BIOL. CHEM., vol. 264, 1989, pages 8935 - 8940
BOCK ET AL., NAT BIOTECH, vol. 28, 2010, pages 1106 - 1114
CORONEL: "Database Systems: Design, Implementation, & Management", 2014
DU ET AL.: "Methyl-CpG-binding domain proteins: readers of the epigenome", EPIGENOMICS, vol. 7, no. 6, 2015, pages 1051 - 73, XP093109738, DOI: 10.2217/epi.15.39
ELMASRI: "Fundamentals of Database Systems", 2010
FREIER ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 4429 - 4443
FULLGRABE ET AL., BIORXIV, 2022
GAJULA ET AL., NUCLEIC ACIDS RES., vol. 42, no. 15, September 2014 (2014-09-01), pages 9964 - 75
GALE ET AL., PLOS ONE, vol. 13, 2018, pages 0194630
GANSAUGE ET AL., NATURE PROTOCOLS, vol. 8, 2013, pages 737 - 748
GOUILKENIRY, ESSAYS IN BIOCHEMISTRY, vol. 63, 2019, pages 639 - 648
HENNION ET AL., GENOME BIOLOGY, vol. 21, 2020
IURLARO ET AL., GENOME BIOL., vol. 14, 2013, pages 119
JANG ET AL., GENES (BASEL)., vol. 8, no. 6, June 2017 (2017-06-01), pages 148
KINDE ET AL., PROC NAT'L ACAD SCI USA, vol. 108, 2011, pages 9530 - 9535
KO ET AL., NATURE, vol. 468, 2010, pages 839 - 843
KOU ET AL., PL S ONE, vol. 11, 2016, pages 0146638
KUROSE: "Computer Networking: A Top-Down Approach", 2016
KUTYAVIN, BIOCHEMISTRY, vol. 47, no. 51, 2008, pages 13666 - 1367
LIU ET AL., NAT CHEM BIOL, vol. 13, 2017, pages 181 - 187
LIU ET AL., NAT CHEM BIOL., vol. 13, no. 2, February 2017 (2017-02-01), pages 181 - 187
LIZARD ET AL., NAT. GENETICS, vol. 19, 1998, pages 225 - 232
LOU ET AL., PROC. NATL. ACAD. SCI., vol. 110, no. 49, 2013, pages 19872 - 19877
MULLER ET AL., NATURE METHODS, vol. 16, 2019, pages 429 - 436
NOTOMI ET AL., NUC. ACIDS RES., vol. 28, 2000, pages 63
PARDOLL, NATURE REVIEWS CANCER, vol. 12, 2012, pages 252 - 264
PETERSON: "Computer Networks: A Systems Approach, Morgan Kaufmann", 2011, RECURSIVE PRESS
ROBERTSON ET AL., NUCLEIC ACIDS RES., vol. 39, 2011, pages 8740 - 8751
SCHUTSKY ET AL., NATURE BIOTECHNOLOGY, vol. 36, 2018, pages 1083 - 1090
SCHUTSKY ET AL., NUCLEIC ACIDS RES., vol. 45, no. 13, 27 July 2017 (2017-07-27), pages 7655 - 7665
SCOTT, C.A.DURYEA, J.D.MACKAY, H. ET AL.: "Identification of cell type-specific methylation signals in bulk whole genome bisulfite sequencing data", GENOME BIOL, vol. 21, 2020, pages 156
SONG ET AL., NAT BIOTECH, vol. 29, 2011, pages 68 - 72
SONG ET AL., NAT. BIOTECH., vol. 29, 2010, pages 68 - 72
TROLL ET AL., BMC GENOMICS, vol. 20, 2019, pages 1023, Retrieved from the Internet <URL:https://doi.org/10.1186/s12864-019-6355-0>
TUCKER: "Programming Languages", 2006, MCGRAW-HILL SCIENCE/ENGINEERING/MATH
VAISVILA ET AL., MOL CELL, vol. 84, no. 5, 7 March 2024 (2024-03-07), pages 854 - 866
VAISVILA ET AL.: "Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA", BIORXIV, 2023, Retrieved from the Internet <URL:https://wwwbiorxiv.org/content/10.1101/2023.06.29.547047v1>
VAISVILA R: "EM-seq: Detection of DNA methylation at single base resolution from picograms of DNA", BIOLZRIN, 2019, Retrieved from the Internet <URL:www.biorxiv.org/content/10.1101/2019.12.20.884692v1>
VAISVILA ROMUALDAS ET AL: "Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA", BIORXIV, 19 December 2023 (2023-12-19), XP093237351, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2023.06.29.547047v2.full.pdf> DOI: 10.1101/2023.06.29.547047 *
WEIRATHER ET AL., F LOOORESEARCH, vol. 6, 2017, pages 100
WEIRATHER JL ET AL., F1000RESEARCH, vol. 6, 2017, pages 100
WEIRATHER JL ET AL.: "Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis", F LOOORESEARCH, vol. 6, 2017, pages 100
YANG ET AL., BIO-PROTOCOL, vol. 12, no. 17, 2023, pages 4496
YU ET AL., CELL, vol. 149, 2012, pages 1368 - 80

Also Published As

Publication number Publication date
WO2025207941A1 (fr) 2025-10-02
WO2025207921A1 (fr) 2025-10-02
WO2025207925A1 (fr) 2025-10-02
WO2025207939A1 (fr) 2025-10-02
WO2025207926A1 (fr) 2025-10-02

Similar Documents

Publication Publication Date Title
US20240191290A1 (en) Methods for detection and reduction of sample preparation-induced methylation artifacts
US20250084464A1 (en) Compositions and methods for synthesis and use of probes targeting nucleic acid rearrangements
US20240263241A1 (en) Methods and compositions for copy-number informed tissue-of-origin analysis
EP4638781A2 (fr) Procédés recourant à une amplification préservant la méthylation avec correction des erreurs
EP4547857A1 (fr) Enrichissement d&#39;adn méthylé de manière aberrante
WO2025090956A1 (fr) Procédés de détection de variants d&#39;acide nucléique à l&#39;aide de sondes de capture
US20250101494A1 (en) Methods for analyzing cytosine methylation and hydroxymethylation
WO2025029475A1 (fr) Procédés d&#39;enrichissement de variants nucléotidiques par sélection négative
US20240093292A1 (en) Quality control method
WO2024229143A1 (fr) Procédé de contrôle qualité pour les procédures de conversion enzymatique
WO2024159053A1 (fr) Procédé pour établir le profil de méthylation d&#39;acides nucléiques
WO2024264065A1 (fr) Procédés et compositions pour quantifier des acides nucléiques de cellules immunitaires
WO2025207924A1 (fr) Procédés de désamination sélective utilisant des protéines de liaison à cpg
WO2025090954A1 (fr) Procédé de détection de variants d&#39;acide nucléique
WO2025160433A1 (fr) Procédés d&#39;analyse de lectures de séquençage
WO2025235889A1 (fr) Procédés impliquant une pcr groupée multiplexée
WO2025038399A1 (fr) Procédés d&#39;enrichissement méthylé pour séquençage génétique et épigénétique à molécule unique
WO2024163478A1 (fr) Surveillance non invasive d&#39;altérations génomiques induites par des thérapies d&#39;édition génique
WO2025155895A1 (fr) Procédé de profilage de modification d&#39;acide nucléique
WO2025137620A1 (fr) Procédés de séquençage de méthylation de haute qualité et de haute précision
WO2024138180A2 (fr) Flux de travail ciblés et intégrés de séquençage de génome somatique entier et de méthylation d&#39;adn
WO2024229433A1 (fr) Procédés d&#39;analyse de la méthylation de l&#39;adn
WO2024073508A2 (fr) Procédés et compositions de quantification d&#39;adn de cellules immunitaires

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25720195

Country of ref document: EP

Kind code of ref document: A1