Compositions and methods for analyzing cell-free DNA in methylation partitioning assays
Provided herein is a DNA analysis method comprising partitioning a sample into at least a first subsample and a second subsample, wherein the first subsample comprises DNA (e.g., cell-free DNA) with a cytosine modification in a greater proportion; the first subsample undergoes a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample; and DNA is sequenced to distinguish the first nucleobase from the second nucleobase. Also provided is a combination comprising first and second populations of captured DNA, wherein the first population comprises or was derived from DNA with a cytosine modification in a greater proportion than the second population, and wherein the first population comprises a form of a first nucleobase originally present in the DNA with altered base pairing specificity and a second nucleobase without altered base pairing specificity.
1. 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.
2. The method of claim 1 , wherein the set of target-specific probes is configured to capture cfDNA corresponding to the sequence-variable target set with a greater capture yield than cfDNA corresponding to the epigenetic target set.
3. The method of claim 1 , comprising sequencing the cfDNA molecules corresponding to the sequence-variable target region set to a greater depth of sequencing than the cfDNA molecules corresponding to the epigenetic target region set.
4. The method of claim 3 , wherein the captured cfDNA molecules of the sequence-variable target set are sequenced to a 4-100 fold greater depth of sequencing than the captured cfDNA molecules of the epigenetic target region set.
5. The method of claim 1 , further comprising ligating barcode-containing adapters to the cfDNA before contacting the cfDNA with the set of target-specific probes.
6. The method of claim 1 , wherein the epigenetic target region set comprises at least one of the following: a hypermethylation variable target region set, a hypomethylation variable target region set, a methylation control target region set, or a fragmentation variable target region set.
7. The method of claim 6 , wherein the fragmentation variable target region set comprises transcription start site regions and/or CTCF binding regions.
8. The method of claim 1 , wherein (a) partitioning the sample into a plurality of subsamples comprises partitioning on the basis of methylation level; and/or (b) partitioning the sample into a plurality of subsamples comprises partitioning on the basis of binding to a protein.
9. The method of claim 8 , wherein (a) the partitioning step comprises contacting the cfDNA with a methyl binding reagent immobilized on a solid support and/or (b) the partitioning step comprises contacting the cfDNA with a binding reagent which is specific for the protein and is immobilized on a solid support.
10. The method of claim 9 , wherein the first subsample and second subsample are differentially tagged before subjecting the first subsample to the procedure that affects the first nucleobase in the DNA differently from the second nucleobase in the DNA of the first subsample.
11. The method of claim 1 , wherein the first subsample and second subsample are pooled after subjecting the first subsample to the procedure that affects the first nucleobase in the DNA differently from the second nucleobase in the DNA of the first sub sample.
12. 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.
13. The method of claim 12 , wherein the method further comprises differentially tagging the third subsample, so as to be distinguishable from the first subsample and second subsample.
14. The method of claim 13 , wherein the first, second, and third subsamples are combined after subjecting the first subsample to the procedure that affects the first nucleobase in the DNA differently from the second nucleobase in the DNA of the first sub sample.
15. The method of claim 1 , wherein the procedure to which the first subsample is subjected alters base-pairing specificity of the first nucleobase without substantially altering base-pairing specificity of the second nucleobase.
16. The method of claim 1 , wherein the first nucleobase comprises mC.
17. The method of claim 16 , wherein the second nucleobase comprises 5-hydroxymethylcytosine (hmC).
18. The method of claim 17 , wherein the procedure to which the first subsample is subjected comprises protection of 5 hmC.
19. The method of claim 18 , wherein the procedure to which the first subsample is subjected comprises Tet-assisted bisulfite conversion.
20. The method of claim 1 , further comprising, following step (f), (g) determining presence or absence of one or more sequence variants in (i) the captured cfDNA of the first subsample corresponding to the sequence-variable target set and (ii) the captured cfDNA of the second subsample corresponding to the sequence-variable target set; and (h) determining presence or absence of one or more epigenetic variants in (i) the captured cfDNA of the first subsample corresponding to the epigenetic target set and (ii) the captured cfDNA of the second subsample corresponding to the epigenetic target set.