Methods and systems to improve the signal to noise ratio of dna methylation partitioning assays

Abstract

In an aspect, the present disclosure provides a method for determining a methylation status comprises: providing a biological sample of nucleic acid molecules; partitioning at least a subset of the nucleic acid molecules in the biological sample based on the methylation status of the nucleic acid molecules into a plurality of partitioned sets; digesting at least a subset of the one or more partitioned sets in the plurality of partitioned sets with at least one methylation sensitive restriction enzyme; enriching at least a subset of the nucleic acid molecules in the plurality of partitioned sets for genomic regions of interest, wherein the at least a subset of the nucleic acid molecules comprises digested nucleic acid molecules in the one or more partitioned sets; and determining methylation status at one or more genetic loci of the nucleic acid molecules in at least one of the partitioned sets.

1 . (canceled) 
     
     
         2 . (canceled) 
     
     
         3 . A method of analyzing nucleic acid molecules in a biological sample, comprising:
 a) partitioning at least a subset of the nucleic acid molecules in the biological sample, based on the methylation status of the nucleic acid molecules into a plurality of partitioned sets, wherein the biological sample comprises methylated nucleic acid molecules and unmethylated nucleic acid molecules and the plurality of partitioned sets comprises a first partitioned set and a second partitioned set, wherein methylated nucleic acid molecules are overrepresented in the first partitioned set relative to the second partitioned set;   b) digesting at least a subset of the first partitioned set in the plurality of partitioned sets with at least one methylation sensitive restriction enzyme; and   c) capturing a first target region set comprising epigenetic target regions from at least a portion of a first partitioned set, and capturing a second target region set comprising epigenetic target regions from at least a portion of the second partitioned set.   
     
     
         4 . The method of  claim 3 , wherein capturing the first target region set comprises contacting the DNA of the first partitioned set with a first set of target-specific probes, and capturing the second target region set comprises contacting the DNA of the second partitioned set with a second set of target-specific probes. 
     
     
         5 . The method of  claim 3 , further comprising determining methylation status at one or more genetic loci of the nucleic acid molecules in at least one of the partitioned sets or target region sets. 
     
     
         6 . The method of  claim 3 , wherein the first target region set and/or the second target region set comprise sequence-variable target regions. 
     
     
         7 . The method of  claim 3 , further comprising, prior to the digesting step, attaching one or more adapters to at least one end of at least a portion of the nucleic acid molecules in the plurality of partitioned sets. 
     
     
         8 . (canceled) 
     
     
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         10 . (canceled) 
     
     
         11 . The method of  claim 3 , further comprising detecting presence or absence of cancer in the biological sample. 
     
     
         12 . (canceled) 
     
     
         13 . The method of  claim 3 , wherein determining the methylation status comprises sequencing at least a subset of the digested nucleic acid molecules. 
     
     
         14 . The method of  claim 7 , wherein the one or more adapters comprises at least one tag. 
     
     
         15 . The method of  claim 3 , wherein the methylation sensitive restriction enzyme selectively digests nucleic acid molecules that are unmethylated at the recognition site of the methylation sensitive restriction enzyme. 
     
     
         16 . The method of  claim 3 , wherein at least a portion of nucleic acid molecules are amplified and/or sequenced after the digesting step, and nucleic acid molecules that were digested by the methylation sensitive restriction enzyme are not amplified and/or are not sequenced. 
     
     
         17 . The method of  claim 3 , comprising digesting at least a subset of the one or more partitioned sets in the plurality of partitioned sets with at least two methylation sensitive restriction enzymes. 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 17 , wherein the methylation sensitive restriction enzymes comprise or consist of BstUI and HpaII. 
     
     
         20 . The method of  claim 17 , wherein the methylation sensitive restriction enzymes comprise or consist of HhaI and AccII. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 17 , wherein the methylation sensitive restriction enzymes comprise or consist of BstUI, HpaII and Hin6I. 
     
     
         23 . The method of  claim 3 , wherein the methylation sensitive restriction enzyme is selected from the group consisting of AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, and SnaBI. 
     
     
         24 . The method of  claim 7 , wherein the one or more adapters are resistant to digestion by the methylation sensitive restriction enzymes. 
     
     
         25 . The method of  claim 24 , wherein the one or more resistant adapters comprise one or more methylated nucleotides, optionally wherein the methylated nucleotides comprise 5-methylcytosine and/or 5-hydroxymethylcytosine. 
     
     
         26 . The method of  claim 24 , wherein the one or more resistant adapters comprise one or more nucleotide analogs resistant to methylation sensitive restriction enzymes. 
     
     
         27 . The method of  claim 24 , wherein the one or more resistant adapter comprises a nucleotide sequence not recognized by methylation sensitive restriction enzymes. 
     
     
         28 . The method of  claim 14 , wherein the tag comprises a molecular barcode. 
     
     
         29 . The method of  claim 28 , wherein the molecular barcodes attached to nucleic acid molecules in a first partitioned set of the plurality of partitioned sets are different from the molecular barcodes attached to nucleic acid molecules in a second partitioned set of the plurality of partitioned sets. 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . The method of  claim 3 , wherein the methylated nucleic acid molecules comprise 5-methylcytosine and/or 5-hydroxymethylcytosine. 
     
     
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         35 . The method of  claim 3 , wherein the biological sample is a cell-free DNA sample. 
     
     
         36 . The method of  claim 35 , wherein the cell-free DNA is between 1 ng and 500 ng. 
     
     
         37 . The method of  claim 3 , wherein the partitioning comprises partitioning the nucleic acid molecules based on a differential binding affinity of the nucleic acid molecules to a binding agent that preferentially binds to nucleic acid molecules comprising methylated nucleotides. 
     
     
         38 . The method of  claim 37 , wherein the binding agent is a methyl binding domain (MBD) protein. 
     
     
         39 . (canceled) 
     
     
         40 . The method of  claim 3 , wherein the epigenetic target regions comprise differentially methylated regions for cancer detection. 
     
     
         41 - 44 . (canceled)