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-carboxymethylcytosine 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 identifying 5hmC locations in adapter-ligated target DNA in a cell-free DNA sample, the method comprising:
(a) oxidizing 5hmC in the target DNA with an oxidizing reagent to yield DNA containing oxidized 5hmC without affecting 5mC, wherein the oxidized 5hmC is selected from 5caC, 5fC, and combinations thereof;
(b) reacting the DNA containing oxidized 5hmC with pyridine borane to reduce, deaminate, and either decarboxylate or deformylate the oxidized 5hmC, thereby providing modified DNA containing DHU in place of the oxidized 5hmC;
(c) amplifying and sequencing the modified DNA to provide 5hmC-indicative sequence reads; and
(d) comparing the 5hmC-indicative sequence reads to standard sequence reads obtained for the target molecule, wherein a change from C in the standard sequence reads to a T in the 5hmC-indicative sequence reads indicates a 5hmC location.
2. The method of claim 1 , wherein steps (a) through (c) are carried out without isolation of any intermediates.
3. The method of claim 1 , wherein steps (a) through (c) are carried out in the absence of bisulfite.
4. The method of claim 1 , wherein step (a) is carried out using a chemical oxidizing reagent.
5. The method of claim 4 , wherein the chemical oxidizing reagent is a perruthenate salt.
6. The method of claim 1 , wherein the adapter-ligated DNA comprises adapters containing a sample identifier sequence and at least one additional molecular barcode selected from a fragment identifier sequence and a strand identifier sequence.
7. The method of claim 6 , wherein the adapter-ligated DNA comprises both a fragment identifier sequence and a strand identifier sequence.
8. The method of claim 1 , wherein the cell-free DNA comprises double-stranded DNA.
9. The method of claim 1 , wherein the cell-free DNA comprises single-stranded DNA.