Detection of methylation changes in DNA samples using restriction enzymes and high-throughput sequencing.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- NUCLEIX LTD
- Filing Date
- 2024-05-19
- Publication Date
- 2026-06-09
Smart Images
Figure 2026518676000018 
Figure 2026518676000019 
Figure 2026518676000020
Abstract
Claims
1. A method for profiling the genetic and epigenetic characteristics of cell-free DNA (cfDNA) samples from a subject, wherein the method is (a) To obtain restriction endonuclease-treated DNA in which the cell-free DNA sample is digested with at least one methylation-sensitive restriction endonuclease, in which the methylation sites are intact and the unmethylation sites are cleaved. (b) Preparing a sequencing library from restriction endonuclease-treated DNA while preserving the sequence information of the ends of the DNA molecules, wherein the preparation of the sequencing library includes ligating sequencing adapters to DNA molecules in the restriction endonuclease-treated DNA, and each adapter is capable of ligating both digested and undigested DNA molecules. (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) Determining from the sequencing data the methylation value of at least one restriction locus, and at least one additional genetic or epigenetic characteristic of the cell-free DNA sample selected from the group consisting of cfDNA size, cfDNA 3' and / or 5' end position, DNA sequence variation, copy number variation, and fragment positioning, A method wherein the amount of cell-free DNA containing 3000 haploid equivalents is sufficient for the method, the cell-free DNA sample is not subjected to amplification before library preparation, and the determination of the methylation value and the at least one additional genetic or epigenetic characteristic of the cell-free DNA sample is performed based on the same sequencing data.
2. A method for processing cell-free DNA samples to obtain sequencing data for genetic and epigenetic analysis, wherein the method is (a) To obtain restriction endonuclease-treated DNA in which the cell-free DNA sample is digested with at least one methylation-sensitive restriction endonuclease, in which the methylation sites are intact and the unmethylation sites are cleaved. (b) Preparing a sequencing library from restriction endonuclease-treated DNA while preserving the sequence information of the ends of the DNA molecules, wherein the preparation of the sequencing library includes ligating sequencing adapters to DNA molecules in the restriction endonuclease-treated DNA, and each adapter is capable of ligating both digested and undigested DNA molecules. (c) Sequence determination of the sequence determination library using a high-throughput sequence determination method to obtain sequence determination data, (d) Determining from the sequencing data the methylation value of at least one restriction locus, and at least one additional genetic or epigenetic characteristic of the cell-free DNA sample selected from the group consisting of cfDNA size, cfDNA 3' and / or 5' end position, DNA sequence variation, copy number variation, and fragment positioning, The amount of cell-free DNA containing 3000 haploid equivalents is sufficient to achieve at least one of the following: a unique mapping rate of at least 85%, copy number completeness characterized by at least 0.65 Pearson correlations compared to the undigested sample, and fragment positioning completeness characterized by at least 0.55 Pearson correlations compared to the undigested sample. A method in which genetic and epigenetic analyses are performed based on the same sequencing data.
3. The method according to claim 1 or 2, wherein the amount of cell-free DNA containing 6,000 haploid equivalents is sufficient for the method.
4. The method according to any one of claims 1 to 3, wherein the cell-free DNA is plasma cell-free DNA, and the amount of the cell-free DNA is the amount obtained from 9 to 10 ml of blood.
5. The method according to any one of claims 1 to 4, wherein the amount of cell-free DNA is 10 to 200 ng.
6. The method according to any one of claims 1 to 5, wherein the amount of cell-free DNA is 20 to 100 ng.
7. The method according to any one of claims 1 to 6, further comprising the method of providing the DNA treated with the restriction endonucleases to end repair prior to ligation of the sequencing adapter to obtain a DNA molecule having blunt ends, wherein the at least one methylation-sensitive restriction endonuclease generates a non-blunt end, and the method further comprises the method of providing the DNA treated with the restriction endonucleases to end repair prior to ligation of the sequencing adapter to obtain a DNA molecule having blunt ends.
8. The method according to any one of claims 1 to 7, wherein the high-throughput sequencing is whole-genome high-throughput sequencing.
9. The method according to any one of claims 1 to 8, wherein the high-throughput sequencing is high-throughput sequencing targeting only the target.
10. Determining the methylation level of at least one restriction locus is necessary. (i) Selecting at least one restriction locus and determining the number of sequence readouts to cover a predetermined genomic region of at least 50 bp in length that contains the restriction locus, (ii) The method according to any one of claims 1 to 9, comprising calculating the methylation value of the at least one restriction locus based on the read count and reference read count determined in step (i).
11. The method according to claim 10, wherein step (i) comprises determining a number of sequence reads that cover a predetermined genomic region of at least 100 bp in length containing the restriction locus.
12. The method according to any one of claims 1 to 11, wherein the at least one restriction locus is a plurality of restriction loci.
13. The method according to any one of claims 1 to 12, wherein the at least one methylation-sensitive limiting endonuclease is a plurality of methylation-sensitive limiting endonucleases, and the digestion by the plurality of methylation-sensitive limiting endonucleases is simultaneous digestion.
14. The method according to claim 13, wherein the plurality of methylation-sensitive limiting endonucleases include HinP1I.
15. The method according to claim 13, wherein the plurality of methylation-sensitive limiting endonucleases include AciI.
16. The method according to claim 13, wherein the digestion is carried out using HinP1I and AciI.
17. The method according to any one of claims 1 to 16, wherein the step of subjecting the cell-free DNA sample to digestion with at least one methylation-sensitive limiting endonuclease further comprises determining the digestive efficacy and proceeding to the preparation of a sequencing library if the digestive efficacy exceeds a predetermined threshold.
18. A method for detecting cancer-related genetic and epigenetic changes in a cell-free DNA sample (cfDNA) from a subject, the method comprising: obtaining a genetic and epigenetic profile of the cfDNA sample by profiling the methylation of the cfDNA sample according to any one of claims 1 to 17 and optionally profiling at least one additional genetic and epigenetic characteristic; and detecting cancer-related genetic and epigenetic changes in the cfDNA sample by comparing the genetic and epigenetic profile of the cfDNA sample with one or more reference genetic and epigenetic profiles selected from cancer profiles and non-cancer profiles.
19. The method according to claim 18, wherein the cell-free DNA sample is from a subject suspected of having cancer or at risk of having cancer, and the method further comprises subjecting the subject to active cancer surveillance and follow-up examinations if cancer-related changes are detected, wherein the cancer surveillance and follow-up examinations include one or more of the following: blood tests, urine tests, cytology, imaging, endoscopy, and biopsy.
20. A method for evaluating the presence or absence of cancer in a subject, wherein the method is (a) To obtain restriction endonuclease-treated DNA in which the target cell-free DNA (cfDNA) sample is digested with at least one methylation-sensitive restriction endonuclease, and the methylation sites are intact and the unmethylation sites are cleaved. (b) The DNA treated with restriction endonuclease is sequenced by a high-throughput sequencing method, (c) Selecting at least one multi-omics genomic region comprising tumor hypermethylation restriction loci, tumor mutation loci within 150 bp of each other, and at least one additional genetic or epigenetic feature of the cell-free DNA sample selected from the group consisting of cfDNA size, cfDNA 3' and / or 5' end position, copy number variation, and fragment positioning, (d) A method comprising determining the likelihood that the subject has cancer based on an analysis of a sequence reading covering the at least one multi-omics region.
21. The method according to claim 20, wherein the at least one multi-omics region includes a tumor hypermethylation restriction locus and a tumor mutation locus that are within 100 bp of each other.
22. Analysis of sequence readouts covering at least one multi-omics region, For each multi-omics domain, (i) The number of methylation mutation sequence readings covering the multi-omics region that include all nucleotides of the restriction locus and present the mutant genotype in the same alignment, (ii) The number of methylated wild-type sequence readouts covering the multi-omics region that include all nucleotides of the restriction locus and present the wild-type genotype in the same alignment, (iii) The number of unmethylated mutant sequence reads covering the multi-omics region that begin or end at a nucleotide within the restriction locus and present a mutant genotype in the same alignment, and (iv) Determining at least one of the number of unmethylated wild-type sequence reads covering the multi-omics region that begin or end at a nucleotide within the restriction locus and present the wild-type genotype in the same alignment, The method according to claim 20 or 21, comprising comparing the number of readings determined in (i) to (iv) with a reference value for cancer patients and / or healthy individuals in order to assess the likelihood that the subject has cancer.
23. A method for characterizing cell-free DNA (cfDNA) samples from subjects suspected of having cancer or at risk of having cancer, wherein the method is (a) To obtain restriction endonuclease-treated DNA in which the cell-free DNA sample is digested with at least one methylation-sensitive restriction endonuclease, in which the methylation sites are intact and the unmethylation sites are cleaved. (b) The DNA treated with restriction endonuclease is sequenced by a high-throughput sequencing method, (c) Selecting at least one multi-omics genomic region of the cell-free DNA sample that includes at least one additional genetic or epigenetic characteristic selected from the group consisting of cfDNA size, cfDNA 3' and / or 5' end position, copy number variation, and fragment positioning, (d) For each multi-omics region, determine the presence or absence of cancer-related hypermethylation and / or hypomethylation, (e) For each multi-omics domain, Presence or absence of cancer-related cfDNA size, Presence or absence of cancer-related cfDNA 3' and / or 5' terminal positions, The presence or absence of one or more cancer-related mutations, The presence or absence of cancer-related copy number variations, and This includes determining at least one, preferably at least two, more preferably at least three, and most preferably all additional genetic or epigenetic characteristics of the cell-free DNA sample, selected from the group consisting of the presence or absence of cancer-related nucleosome positioning. A method for characterizing the cell-free DNA sample.
24. A method for profiling the methylation of a DNA sample from a subject, wherein the method is (a) To obtain restriction endonuclease-treated DNA in which the methylated sites are intact and the unmethylated sites are cleaved, by subjecting the DNA sample to digestion with at least one methylation-sensitive restriction endonuclease, (b) Preparing a sequencing library from the restriction endonuclease-treated DNA, wherein the preparation of the sequencing library includes ligating sequencing adapters to the restriction endonuclease-treated DNA fragments, each adapter capable of ligating to both digested and undigested DNA molecules. (c) To obtain sequence readings by sequencing the sequencing library using a high-throughput sequencing method, (d) Selecting at least one restriction locus and determining the number of sequence readouts to cover a predetermined genomic region of at least 50 bp in length that contains the restriction locus, (d) Determining the presence or absence of cancer-related hypermethylation and / or hypomethylation in the predetermined genomic region, (e) With respect to the predetermined genome region, Presence or absence of cancer-related cfDNA size, Presence or absence of cancer-related cfDNA 3' and / or 5' terminal positions, The presence or absence of one or more cancer-related mutations, The presence or absence of cancer-related copy number variations, and This includes determining at least one, preferably at least two, more preferably at least three, and most preferably all additional genetic or epigenetic characteristics of the cell-free DNA sample, selected from the group consisting of the presence or absence of cancer-related nucleosome positioning. A method for profiling the methylation of the cell-free DNA sample.
25. The method according to claim 24, wherein the predetermined region covering the restriction locus begins at least 25 bp upstream of the cleavage site within the restriction locus and ends at least 25 bp downstream of the cleavage site within the restriction locus.
26. The method according to claim 25, wherein step (d) comprises determining a number of sequence reads that cover a predetermined genomic region of at least 100 bp in length containing the restriction locus.
27. The method according to claim 26, wherein the predetermined region covering the restriction locus begins at least 50 bp upstream of the cleavage site within the restriction locus and ends at least 50 bp downstream of the cleavage site within the restriction locus.
28. The method according to any one of claims 24 to 27, wherein the at least one restriction locus is located within a CG island.
29. The method according to any one of claims 24 to 28, wherein the reference read count is a read count determined for a predetermined genomic region of at least 50 bp in length containing the restriction locus in an undigested control DNA sample, and is optionally corrected for differences in sequencing depth.
30. The method according to any one of claims 24 to 28, wherein the reference read count is a read count determined using a reference region of at least 50 bp in length containing a reference locus that is not cleaved by the restriction endonuclease.
31. The method according to any one of claims 24 to 28, wherein the reference read count is an average read count determined using a plurality of reference regions, each at least 50 bp in length, containing a reference locus that is not cleaved by the restriction endonuclease.
32. The method according to any one of claims 24 to 31, wherein calculating the methylation value includes normalizing the read count determined in step (d) with respect to the median read count of the DNA sample to obtain a normalized read count, and calculating the ratio of the normalized read count to a normalized reference read count.
33. A method for genetic and epigenetic profiling of a DNA sample, the method comprising: determining the methylation value of at least one restriction locus as described in any one of claims 24 to 32; and further determining at least one additional genetic or epigenetic characteristic of the DNA sample selected from sequencing data, from DNA sequence variations, copy number variations, and fragment positioning.
34. The method according to any one of claims 24 to 33, wherein the DNA is cell-free DNA extracted from a biological fluid sample.
35. The method according to any one of claims 24 to 33, wherein the DNA is DNA extracted from a tumor sample.
36. A method for identifying a genomic region differentially methylated between a first and a second DNA source, wherein the method is A first DNA methylation profile is obtained by profiling the methylation of at least one DNA sample from the first source according to the method of any one of claims 24 to 33, A second DNA methylation profile is obtained by profiling the methylation of at least one DNA sample from the second source according to the method of any one of claims 24 to 33, A method comprising comparing the first and second DNA methylation profiles to identify genomic regions that are differentially methylated between the first and second DNA sources.
37. The method according to claim 36, wherein the first DNA source is cancer DNA and the second DNA source is non-cancer DNA.
38. The method according to claim 36, wherein the first DNA source is cell-free plasma DNA from a cancer patient, and the second DNA source is cell-free plasma DNA from one or more healthy individuals.
39. The method according to claim 36, wherein the first and second DNA sources are from different stages of cancer.
40. A method for processing cell-free DNA (cfDNA) fragments in a sample, The cfDNA fragment is digested with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment. (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting a plurality of genome and / or epigenome feature scores from the sequencing data, wherein at least one extracted feature score is at least partially derived from the cleavage status of one or more recognition sites for MARE present in the cfDNA fragment.
41. The method according to claim 40, wherein one, two, three, four, five or more epigenomic feature scores in the plurality of genome and / or epigenomic feature scores are scores for features selected from the group consisting of hitspan Y (HSY), hitspan 100 (HS100), hitspan 4 (HS4), normalized hitspan Y (NHSY), normalized hitspan 100 (NHS100), normalized hitspan 4 (NHS4), start, end, continuity edge, local normalization start, local normalization end, local normalization continuity edge, nucleosome normalization start, nucleosome normalization end, nucleosome normalization continuity edge, methylation state, histone modification and / or spacing, nucleosome occupancy and / or positioning, alignment frequency, cfDNA fragment length, and cfDNA fragment terminal motif.
42. The method according to claim 40 or 41, wherein the sequence information of the ends of the restriction endonuclease-digested DNA fragment is preserved, thereby preserving sequence information from the fragment ends that existed before MARE digestion and from the fragment ends that result from MARE digestion.
43. The method according to any one of claims 40 to 42, wherein a plurality of MAREs are used in the method.
44. The method according to any one of claims 40 to 43, wherein the MARE comprises one, two, three, four, or more methylation-sensitive restriction enzymes (MSREs).
45. The method according to any one of claims 40 to 44, wherein the MARE comprises one, two, three, four, or more methylation-dependent restriction enzymes (MDREs).
46. The method according to any one of claims 43 to 45, wherein the plurality of MAREs are used in a single common reaction mixture to obtain the restriction endonuclease-digested DNA fragment.
47. The method according to any one of claims 40 to 46, wherein step (d) is to map the sequencing data to a reference genome to generate a set of aligned sequence reads, and to extract the plurality of genome and / or epigenome feature scores from the aligned sequence reads.
48. The method according to claim 47, wherein step (d) further comprises extracting a plurality of genomic and / or epigenomic feature scores from the aligned sequence readings of individual genomic locations, and step (d) is repeated for a plurality of individual genomic locations represented in the set of aligned sequence readings.
49. The method according to claim 47 or 48, wherein step (d) further comprises extracting a plurality of genomic and / or epigenomic feature scores from the aligned sequence reads of individual cfDNA fragments, and step (d) is repeated for a plurality of individual cfDNA fragments.
50. The method according to any one of claims 40 to 49, wherein a classifier is trained using the data obtained for the plurality of genome and / or epigenome feature scores.
51. The method according to any one of claims 40 to 50, wherein the data obtained for the plurality of genome and / or epigenome feature scores are used in tissue origin classification, disease diagnosis, and / or disease or treatment monitoring applications.
52. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising identifying a subset of the cfDNA fragments that have been cleaved by MARE from the sequencing data.
53. The method according to claim 52, further comprising one or more genome and / or epigenome feature scores from sequencing data corresponding to the subset of the cfDNA fragments.
54. The method according to claim 53, wherein the one or more genome and / or epigenome feature scores are a plurality of genome and / or epigenome feature scores.
55. The method according to claim 53 or 54, wherein the one or more genome and / or epigenome feature scores include one, two, three, four, or more scores for features selected from the group consisting of hitspan Y (HSY), hitspan 100 (HS100), hitspan 4 (HS4), normalized hitspan Y (NHSY), normalized hitspan 100 (NHS100), normalized hitspan 4 (NHS4), start, end, continuity edge, local normalization start, local normalization end, local normalization continuity edge, nucleosome normalization start, nucleosome normalization end, nucleosome normalization continuity edge, methylation state, histone modification and / or spacing, nucleosome occupancy and / or positioning, alignment frequency, cfDNA fragment length, and cfDNA fragment terminal motifs.
56. The method according to any one of claims 52 to 55, wherein the step of preparing a sequencing library from the restriction endonuclease-treated DNA fragment preserves the sequence information of the ends of the restriction endonuclease-digested DNA fragment.
57. The method according to claim 56, wherein the sequence information of the ends of the restriction endonuclease-digested DNA fragment is preserved, wherein the sequence information from the fragment ends that existed before MARE digestion and the fragment ends that result from MARE digestion is preserved.
58. The method according to any one of claims 52 to 57, wherein a plurality of MAREs are used in the method.
59. The method according to any one of claims 52 to 58, wherein the MARE comprises one, two, three, four, or more methylation-sensitive restriction enzymes (MSREs).
60. The method according to any one of claims 52 to 59, wherein the MARE comprises one, two, three, four, or more methylation-dependent restriction enzymes (MDREs).
61. The method according to any one of claims 58 to 60, wherein the plurality of MAREs are used in a single common reaction mixture to obtain the restriction endonuclease-digested DNA fragment.
62. The method according to any one of claims 52 to 61, wherein step (d) includes mapping the sequencing data to a reference genome to generate a set of aligned sequence reads.
63. The method according to any one of claims 52 to 62, further comprising step (d) extracting a plurality of genomic and / or epigenomic feature scores from the aligned sequence reads of individual cfDNA fragments.
64. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, d) A method comprising extracting one, two, three, four, five, or more epigenomic feature scores selected from the group consisting of hitspan Y (HSY), hitspan 100 (HS100), hitspan 4 (HS4), normalized hitspan Y (NHSY), normalized hitspan 100 (NHS100), normalized hitspan 4 (NHS4), start, end, continuum edge, local normalization start, local normalization end, local normalization continuum edge, nucleosome normalization start, nucleosome normalization end, nucleosome normalization continuum edge, methylation status, histone modification and / or spacing, nucleosome occupancy and / or positioning, alignment frequency, cfDNA fragment length, and cfDNA fragment terminal motifs from the sequencing data.
65. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more hit span Y (HSY) feature scores from the sequence determination data.
66. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more hit span 100 (HS100) feature scores from the sequence determination data.
67. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more hit span 4 (HS4) feature scores from the sequence determination data.
68. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more normalized hit span Y (NHSY) feature scores from the sequence determination data.
69. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more normalized hit span 100 (NHS100) feature scores from the sequence determination data.
70. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more normalized hit span 4 (NHS4) feature scores from the sequence determination data.
71. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more start feature scores from the sequence determination data.
72. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more termination feature scores from the sequence determination data.
73. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more consecutive edge feature scores from the sequence determination data.
74. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more local normalization start feature scores from the sequencing data.
75. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more locally normalized termination feature scores from the sequencing data.
76. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more locally normalized continuous edge feature scores from the sequence determination data.
77. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more nucleosome normalization start feature scores from the sequencing data.
78. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more nucleosome normalization termination feature scores from the sequencing data.
79. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more nucleosome-normalized continuous edge feature scores from the sequencing data.
80. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more methylation state feature scores from the sequencing data.
81. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting histone modification and / or spacing feature scores from the sequencing data.
82. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more nucleosome occupancy and / or positioning feature scores from the sequencing data.
83. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more alignment frequency feature scores from the sequencing data.
84. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more cfDNA fragment length feature scores from the sequencing data.
85. A method for processing cell-free DNA (cfDNA) fragments in a sample, (a) Digesting the cfDNA fragment with one, two, three, four, or more methylation-recognition restriction endonucleases (MAREs) to obtain a restriction endonuclease-digested DNA fragment, (b) Preparing a sequencing library from restriction endonuclease-digested DNA fragments while preserving the sequence information of the ends of the restriction endonuclease-digested DNA fragments, (c) To sequence the sequence determination library using a high-throughput sequence determination method and provide sequence determination data, (d) A method comprising extracting one or more cfDNA fragment motif feature scores from the sequencing data.
86. The method according to any one of claims 64 to 85, further comprising training a classifier using data obtained for one or more epigenetic feature scores.
87. The method according to any one of claims 64 to 86, further comprising using data obtained for one or more epigenetic feature scores in applications of tissue origin classification, disease diagnosis, and / or disease or treatment monitoring.
88. The method according to claim 87, wherein the disease is cancer, and optionally the cancer is lung cancer.
89. The method according to any one of claims 64 to 88, wherein multiple epigenetic feature scores are obtained from the same sequencing data.
90. The method according to any one of claims 64 to 89, wherein the cfDNA is plasma cell-free DNA, and the amount of the cfDNA is the amount obtained from 9 to 10 ml of blood.
91. The method according to any one of claims 64 to 90, wherein the amount of cell-free DNA is 10 to 200 ng.
92. The method according to any one of claims 64 to 91, further comprising the method of subjecting the restriction endonuclease-treated DNA to end repair prior to ligation of the sequencing adapter to obtain a DNA molecule having a blunt end.
93. The method according to any one of claims 64 to 92, wherein the high-throughput sequencing is whole-genome high-throughput sequencing.
94. The method according to any one of claims 64 to 92, wherein the high-throughput sequencing is high-throughput sequencing targeting only the target.
95. The method according to any one of claims 64 to 94, wherein the MARE includes HinP1I.
96. The method according to any one of claims 64 to 95, wherein the MARE includes AciI.