Analysis of methylated dna comprising methylation-sensitive or methylation-dependent restrictions

Abstract

The present disclosure provides compositions and methods related to analyzing DNA, such as cell-free DNA. In some embodiments, the cell-free DNA is from a subject having or suspected of having cancer and/or the cell-free DNA includes DNA from cancer cells. In some embodiments, the DNA is partitioned into a first subsample and a second subsample, wherein the first subsample comprises DNA with a nucleotide modification (e.g., a cytosine modification) in a greater proportion than the second subsample, and the second subsample is contacted with a methylation-dependent nuclease.

1 . A method of analyzing DNA in a sample, the method comprising:
 a) partitioning the sample into a plurality of subsamples, including a first subsample and a second subsample, wherein the first subsample comprises DNA with a cytosine modification in a greater proportion than the second subsample;   b) contacting the second subsample with a methylation-dependent nuclease, thereby degrading nonspecifically partitioned DNA in the second subsample to produce a treated second subsample and optionally contacting the first subsample with a methylation-sensitive endonuclease, thereby degrading nonspecifically partitioned DNA in the first subsample to produce a treated first subsample; and   c) capturing a first target region set comprising epigenetic target regions from at least a portion of the first subsample or the treated first subsample, and capturing a second target region set comprising epigenetic target regions from at least a portion of the treated second subsample.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , further comprising quantifying epigenetic target regions captured from or present in one or more of the first subsample, the treated first subsample, or the treated second subsample. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , further comprising sequencing DNA in the first target region set and the second target region set or in the treated second subsample. 
     
     
         6 . The method of  claim 5 , wherein DNA in the treated second subsample and DNA in the treated first subsample is sequenced. 
     
     
         7 . The method of  claim 1 , wherein the epigenetic target regions comprise a hypomethylation variable target region set. 
     
     
         8 . The method of  claim 7 , wherein the hypomethylation variable target region set comprises regions having a lower degree of methylation in at least one type of tissue than the degree of methylation in cell-free DNA from a healthy subject. 
     
     
         9 . The method of  claim 8 , wherein the method further comprises determining a presence, absence, or likelihood of cancer based at least in part on sequences or quantities of regions in the hypomethylation variable target region set. 
     
     
         10 . The method of  claim 7 , further comprising quantifying tumor DNA in the sample based at least in part on sequences or quantities of regions in the hypomethylation variable target region set. 
     
     
         11 - 15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein the DNA comprises cell-free DNA (cfDNA) obtained from a test subject. 
     
     
         17 . The method of  claim 1 , wherein the cytosine modification is methylation. 
     
     
         18 . The method of  claim 1 , wherein the cytosine modification is methylation at the 5 position of cytosine. 
     
     
         19 . The method of  claim 1 , wherein the first subsample is contacted with a methylation-sensitive endonuclease and the methylation-sensitive endonuclease cleaves an unmethylated CpG sequence. 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . The method of  claim 1 , wherein the methylation-dependent endonuclease cleaves a methylated CpG sequence. 
     
     
         24 . The method of  claim 1 , wherein the methylation-dependent endonuclease is one or more of MspJI, LpnPI, FspEI, or McrBC. 
     
     
         25 . The method of  claim 1 , wherein the first target region set comprises a hypermethylation variable target region set that comprises regions having a higher degree of methylation in at least one type of tissue than the degree of methylation in cell-free DNA from a healthy subject. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 25 , wherein the method further comprises determining a presence, absence, or likelihood of cancer based at least in part on sequences or quantities of regions in the hypermethylation variable target region set. 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 1 , wherein the first and/or second epigenetic target region set comprise a fragmentation variable target region set. 
     
     
         31 . The method of  claim 30 , wherein the fragmentation variable target region set comprises transcription start site regions and/or CTCF binding regions. 
     
     
         32 . (canceled) 
     
     
         33 . The method of  claim 1 , wherein the first target region set and/or second target region set further comprises sequence-variable target regions. 
     
     
         34 - 47 . (canceled) 
     
     
         48 . The method of  claim 1 , comprising differentially tagging the first subsample and second subsample, the treated first subsample and the second subsample, the first subsample and the treated second subsample, or the treated first subsample and the treated second subsample, wherein the treated first subsample and the second subsample, the first subsample and the treated second subsample, or the treated first subsample and the treated second subsample are pooled after contacting the first subsample with the methylation-sensitive endonuclease and/or contacting the second subsample with a methylation-dependent nuclease. 
     
     
         49 . (canceled) 
     
     
         50 . (canceled) 
     
     
         51 . The method of  claim 1 , wherein the plurality of subsamples comprises a third subsample, which comprises DNA with a cytosine modification in a greater proportion than the second subsample but in a lesser proportion than the first subsample. 
     
     
         52 . The method of  claim 51 , wherein the method further comprises differentially tagging the third subsample, and the first, second, and third subsamples are combined after contacting the first subsample with the methylation-sensitive endonuclease and/or contacting the second subsample with a methylation-dependent nuclease, optionally wherein DNA from the first, second, and third subsamples is sequenced in the same sequencing cell. 
     
     
         53 . (canceled) 
     
     
         54 . The method of  claim 51 , wherein the third subsample is contacted with a methylation-sensitive endonuclease. 
     
     
         55 . The method of  claim 52 , wherein the third subsample is combined with the first subsample, and the combined first and third subsamples are contacted with a methylation-sensitive endonuclease. 
     
     
         56 - 90 . (canceled)