METHYLATED DNA FRAGMENT ENRICHMENT, METHODS, COMPOSITIONS AND KITS

1 . A method of processing nucleic acid fragments, the method comprising:

providing an input sample comprising nucleic acid fragments, wherein in at least a portion of the nucleic acid fragments each fragment comprises one or more methylated cytosines;

converting unmethylated cytosines of nucleic acid fragments of the input sample to uracils, yielding converted fragments;

copying the converted fragments using a mixture of nucleotides, the mixture comprising a mixture of:

binding moiety-modified cytosines and binding moiety-lacking cytosines;

binding moiety-modified guanines and binding moiety-lacking guanines; or

binding moiety-modified cytosines, binding moiety-lacking cytosines, binding moiety-modified guanines, and binding moiety-lacking guanines;

wherein the copying yields a mixture of binding moiety-modified fragments and unmodified fragments;

binding at least some of the binding moiety-modified fragments to a substrate, yielding bound fragments and unbound supernatant fragments.

2 - 4 . (canceled)

5 . The method according to claim 1 , wherein the method further comprises separating the bound fragments from the unbound supernatant fragments, yielding the bound fragments enriched for fragments with one or more methylated cytosines.

6 . (canceled)

7 . The method according to claim 1 , wherein the input sample is enriched for targets prior to the converting step.

8 . The method according to claim 7 , wherein the targets are selected for a methylation assay for cancer, cancer type, cancer tissue of origin, cancer stage, or combinations of the foregoing.

9 . (canceled)

10 . The method according to claim 1 , wherein the input sample comprises DNA isolated from a bodily fluid.

11 . The method according to claim 1 , wherein the input sample comprises DNA from a cfDNA sample.

12 . The method according to claim 1 , wherein the input sample comprises fragmented genomic DNA.

13 . The method according to claim 1 , wherein the converting comprises selectively deaminating the unmethylated cytosines.

14 . (canceled)

15 . The method according to claim 1 , wherein the binding moiety-modified cytosines comprise biotin-modified cytosines, and the binding moiety-modified guanines comprise biotin-modified guanines.

16 . (canceled)

17 . The method according to claim 1 , wherein the substrate comprises beads.

18 . The method according to claim 1 , wherein the substrate comprises wells.

19 - 24 . (canceled)

25 . The method according to claim 1 , wherein providing the input sample comprises obtaining from a sample, and including in the input sample, nucleic acid fragments potentially comprising 1 or more CpG sites.

26 - 30 . (canceled)

31 . The method according to claim 1 , wherein the mixture of nucleotides comprises from 1 to 20 percent binding moiety-modified cytosines with the remainder of the cytosines lacking the binding moiety.

32 . The method according to claim 1 , wherein the mixture of nucleotides comprises from 2.5 to 10 percent binding moiety-modified cytosines with the remainder of the cytosines lacking the binding moiety.

33 . The method according to claim 1 , wherein the mixture of nucleotides comprises from 1 to 20 percent binding moiety-modified guanines with the remainder of the guanines lacking the binding moiety.

34 . The method according to claim 1 , wherein the mixture of nucleotides comprises from 2.5 to 10 percent binding moiety-modified guanines with the remainder of the guanines lacking the binding moiety.

35 . The method according to claim 1 , wherein the mixture of nucleotides comprises from 1 to 20 percent binding moiety-modified cytosines and guanines with the remainder of the cytosines and guanines lacking the binding moiety.

36 . The method according to claim 1 , wherein the mixture of nucleotides comprises from 2.5 to 10 percent binding moiety-modified cytosines and guanines with the remainder of the cytosines and guanines lacking the binding moiety.

37 . The method according to claim 5 , wherein the separating yields bound fragments enriched, relative to the input sample, for informative fragments for a methylation assay.

38 . (canceled)

39 . The method according to claim 1 , further comprising eluting the bound fragments to yield a fragment library enriched, relative to the input sample, for informative fragments for a methylation assay.

40 . (canceled)

41 . The method according to claim 39 , further comprising preparing a sequencing library from the fragment library.

42 . The method according to claim 41 further comprising sequencing the sequencing library.

43 . The method according to claim 42 wherein the sequencing is performed to a sequencing depth ranging from 5 to 20 million reads.

44 - 53 . (canceled)

54 . A composition comprising adenines, thymines, cytosines and guanines wherein the cytosines, guanines, or both cytosines and guanines are included in a mixture of binding moiety-modified nucleotides and binding moiety-lacking nucleotides.

55 - 61 . (canceled)

62 . A kit comprising the composition comprising:

the composition according to claim 54 ; and

instructions for using the composition.

63 - 68 . (canceled)