Compositions and methods for analyzing modified nucleotides
View Patent ↗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 an aliquot of a nucleic acid sample with a methylcytosine dioxygenase and a glucosyltransferase in a single reaction;
(b) reacting an aliquot of the product of step (a) with a cytidine deaminase; and
(c) determining the methylation status of a sequence in the nucleic acid sample by sequencing the reaction product of step (b) to determine which cytosine nucleotides have been converted to Uracil (U).
2. The method of claim 1 , wherein the nucleic acid sample contains fragments that are linked, in cis, to a gene in a transcriptionally active or inactive state, and the results of step (c) are compared to results obtained from a reference sample, wherein the reference sample comprises the same fragments that are linked, in cis, to the same gene in an opposite transcriptionally active or inactive state, to determine an altered pattern of cytosine methylation associated with transcriptional activity.
3. The method of claim 2 , wherein the altered pattern of cytosine methylation associated with transcriptional activity of the gene in an ex vivo cell or tissue is correlated with a disease or condition.
4. The method of claim 1 , wherein the cytidine deaminase is APOBEC3A.
5. The method according to claim 1 , wherein the methylcytosine dioxygenase has an amino acid sequence that is at least 90% identical to SEQ ID NO:1.
6. The method of claim 1 , wherein the methylcytosine dioxygenase has an amino acid sequence that is at least 90% identical to SEQ ID NO:3.
7. A method comprising:
(a) reacting an aliquot of a nucleic acid sample with a cytidine deaminase, wherein the nucleic acid sample is a library of DNA fragments wherein the DNA fragments are ligated to nucleic acid adaptors in which cytosine in the adaptors is replaced by pyrollo-dC; and
(b) determining the methylation status of a sequence in the nucleic acid sample by sequencing the reaction product to determine which cytosine nucleotides have been converted to Uracil (U).