Methods for the epigenetic analysis of DNA, particularly cell-free DNA
Methods are provided for the epigenetic analysis of cell-free DNA using organic boranes to convert oxidized 5-methylcytosine residues in the cell-free DNA to dihydrouracil (DHU) residues. Cell-free DNA is contacted with an organic borane selected to successively bring about reduction, deamination, and decarboxylation of oxidized 5-methylcytosine residues such as 5-carboxylcytosine and 5-formylcytosine, resulting in DHU residues in place thereof. Following amplification, the treated cell-free DNA is sequenced, with the DHU residues read as thymine residues. Reaction mixtures, kits and additional methods are also provided, as are related methods for the epigenetic analysis of DNA, including cell-free DNA.
1. A method for converting an oxidized 5-methylcytosine (5mC) residue selected from 5-carboxylcytosine (5caC) and 5-formylcytosine (5fC) to a dihydrouridine (DHU) residue in cell-free DNA (cfDNA), the method comprising contacting cfDNA containing at least one oxidized 5mC residue selected from 5caC, 5fC, and combinations thereof with pyridine borane.
2. A method for converting a 5-hydroxymethylcytosine (5hmC) residue in a nucleic acid to a DHU residue without affecting 5-methylcytosine (5mC) residues, the method comprising: (a) contacting the nucleic acid with an oxidizing reagent effective to convert at least one 5hmC residue in the nucleic acid to at least one oxidized 5hmC residue selected from 5caC, 5fC, and combinations thereof without affecting 5mC residues; and thereafter
(b) contacting the nucleic acid with pyridine borane to provide a DHU residue in place of the at least one 5caC and/or 5fC.
3. The method of claim 2 , wherein the nucleic acid comprises cfDNA.
4. The method of claim 3 , wherein the oxidizing reagent is a chemical oxidizing reagent.
5. The method of claim 4 , wherein the chemical oxidizing reagent comprises a perruthenate salt.
6. The method of claim 2 , wherein the method is carried out without isolation of any intermediates.
7. The method of claim 2 , wherein the method is carried out in the absence of bisulfite.
8. A method for identifying co-occurrence of 5mC and 5hmC in a single DNA fragment in a cell-free DNA sample, comprising:
(a) tagging 5hmC residues in a fragmented, adapter-ligated cell-free DNA sample with an affinity tag that enables removal of the tagged 5hmC-containing DNA fragments from the sample;
(b) removing the tagged 5hmC-containing DNA fragments from the sample;
(c) oxidizing 5mC in the removed, tagged 5hmC-containing DNA fragments to convert unmodified 5mC residues therein to oxidized 5mC residues selected from 5caC, 5fC, and combinations thereof;
(d) treating the removed fragments with pyridine borane to convert the oxidized 5mC to DHU; and
(e) sequencing the removed fragments and identifying any removed fragments containing DHU as template fragments containing both 5mC and 5hmC.
9. The method of claim 5 , wherein the perruthenate salt comprises potassium perruthenate.
10. The method of claim 5 , wherein the perruthenate salt comprises a tetraalkylammonium perruthenate.
11. The method of claim 4 , wherein the chemical oxidizing reagent comprises a polymer-supported perruthenate.
12. The method of claim 4 , wherein the chemical oxidizing reagent comprises an inorganic peroxo compound.