Compositions and methods for analyzing cell-free DNA in methylation partitioning assays

A method of isolating cell-free DNA (cfDNA) from 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 cfDNA with a cytosine modification in a greater proportion than the second subsample and the second subsample comprises hypomethylated cfDNA, the hypomethylated cfDNA comprising unmethylated cfDNA;
• (b) subjecting the first subsample to a procedure that affects a first nucleobase in the cfDNA differently from a second nucleobase in the cfDNA of the first subsample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, the first nucleobase and the second nucleobase have the same base pairing specificity, and the first nucleobase is a modified or unmodified cytosine and the second nucleobase is a modified or unmodified cytosine;
• thereby providing converted cfDNA of the first subsample;
• (c) contacting the converted cfDNA of the first subsample and the cfDNA of the second subsample with a set of target-specific probes,
• wherein the set of target-specific probes comprises target-binding probes specific for a sequence-variable target set and target-binding probes specific for an epigenetic target set,
• whereby (i) complexes of target-specific probes and converted cfDNA of the first subsample and (ii) complexes of target-specific probes and cfDNA of the second subsample are formed;
• (d) separating the complexes of target-specific probes and converted cfDNA of the first subsample from converted cfDNA of the first subsample not bound to target-specific probes, thereby providing captured cfDNA of the first subsample corresponding to the sequence-variable target set and captured cfDNA of the first subsample corresponding to the epigenetic target set;
• (e) separating the complexes of target-specific probes and cfDNA of the second subsample from cfDNA of the second subsample not bound to target-specific probes, thereby providing captured cfDNA of the second subsample corresponding to the sequence-variable target set and captured cfDNA of the second subsample corresponding to the epigenetic target set;
• and
• (f) sequencing (i) the captured cfDNA of the first subsample corresponding to the epigenetic target set and the sequence-variable target set and (ii) the captured cfDNA of the second subsample corresponding to the epigenetic target set and the sequence-variable target set in a manner that distinguishes the first nucleobase from the second nucleobase in the captured cfDNA of the first subsample.