Methods for distinguishing 5-hydroxymethylcytosine from 5-methylcytosine
Provided herein are methods and kits for distinguishing 5-hydroxymethylcytosine from 5-methylcytosine.
1. A method comprising:
(a) obtaining a sample comprising a nucleic acid sequence, wherein said nucleic acid sequence comprises a 5-hydroxymethylcytosine and a 5-methylcytosine;
(b) labeling said 5-hydroxymethylcytosine;
(c) sequencing said nucleic acid sequence; and
(d) distinguishing said 5-hydroxymethylcytosine from said 5-methylcytosine.
2. The method of claim 1 , wherein said nucleic acid sequence is a mammalian nucleic acid sequence.
3. The method of claim 1 , wherein said sample comprises genomic DNA.
4. The method of claim 1 , wherein said sample comprises an extracellular fluid.
5. The method of claim 1 , wherein said sequencing is high-throughput sequencing.
6. The method of claim 1 , wherein said sample is from a subject having or suspected of having cancer.
7. The method of claim 1 , wherein said distinguishing comprises detecting said 5-hydroxymethylcytosine.
8. The method of claim 1 , wherein said distinguishing comprises detecting said 5-methylcytosine.
9. The method of claim 1 , wherein said labeling further comprises glycosylating said 5-hydroxymethylcytosine.
10. The method of claim 9 , comprising associating a label with a glucose added to said 5-hydroxymethylcytosine by said glycosylating.
11. The method of claim 9 , further comprising contacting said nucleic acid sequence with a dioxygenase or catalytically active fragment thereof to convert a methylated cytosine in said nucleic acid sequence to a modified base, wherein said dioxygenase or said catalytically active fragment thereof comprises TET1, TET2, TET3, CXXC4, a catalytically active fragment of any of these, or any combination thereof.
12. The method of claim 11 , further comprising contacting said nucleic acid sequence with sodium bisulfite.
13. The method of claim 1 , further comprising associating a label with an epigenetically modified cytosine of said nucleic acid sequence.
14. The method of claim 13 , wherein said epigenetically modified cytosine is said 5-methylcytosine or said 5-hydroxymethylcytosine.
15. The method of claim 14 , wherein said label comprises a bead.
16. The method of claim 13 , wherein said associating a label comprises glycosylating a hydroxymethylated cytosine, wherein said glycosylating comprises employing an alpha-glucosyltransferase, a beta-glucosyltransferase, or a beta-glucosyl-alpha-glucosyl-transferase.