Compositions and methods for analyzing modified nucleotides
Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map 5m C in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally map 5hm C in a DNA.
1. A method, comprising:
(a) reacting 5-hydroxymethylcytosine ( 5hm C) in a nucleic acid sample with a glucosyl transferase in the absence of a Tet enzyme to form a nucleic acid product with a modified hmC;
(b) converting cytosine (C) to uracil and 5-methyl cytosine (5mC) to thymidine in the nucleic acid product from (a) with a protein that has at least 90% sequence homology to apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (Apobec);
(c) optionally amplifying the reaction product from (b); and
(d) hybridizing and/or sequencing the product from (b) or amplicon thereof.
2. The method according to claim 1 , further comprising: amplifying the product of step (b).
3. The method of claim 1 , wherein the nucleic acid sample is cell-free DNA isolated from a biological sample.
4. The method of claim 3 , wherein the biological sample comprises cells, tissues or bodily fluids.