Methods and compositions for discrimination between cytosine and modifications thereof and for methylome analysis

1. A method for differentiating unmethylated cytosine (C) and 5-methylcytosine (5-mC) from 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), 5-carboxycytosine (5-CaC) or 5-glycosylated hydroxymethylcytosine (5-ghmC), comprising:

(a) reacting a polynucleotide optionally containing C, 5-mC, 5-hmC, 5-fC, 5-CaC and/or 5-ghmC, with a cytidine deaminase having an amino acid sequence that is at least 90% identical to SEQ ID NO:2, wherein 5-mC is converted to a thymine (T) and C is converted to a uracil (U); and

(b) amplifying or cleaving the polynucleotide to identify the location of at least one converted nucleotide in the polynucleotide.

2. A method according to claim 1 , further comprising differentiating 5-hydroxymethylcytosine (5-hmC), from unmethylated cytosine (C) by reacting the polynucleotide prior to (a) with an oxygenase so as to generate a sequence wherein only C is altered to uracil (U).

3. A method according to claim 2 , further comprising sequencing the polynucleotide in which unmethylated cytosine (C) is converted to uracil (U) and sequencing the polynucleotide obtained in (a) where C is converted to U and 5-methylcytosine (5-mC) is converted to thymine (T) and comparing the sequences to characterize 5-mC in the polynucleotide.

4. A method according to claim 1 , further comprising: constructing a methylome map.

5. A method according to claim 1 , wherein the polynucleotide in (a) is single-stranded.

6. A method according to claim 1 , further comprising, comparing the sequence of the deaminated polynucleotide from (a) with the sequence of the untreated polynucleotide.

7. A method according to claim 1 , wherein the cytidine deaminase has an amino acid sequence that is at least 95% identical to SEQ ID NO:2.

8. A method according to claim 1 , wherein cleaving the polynucleotide further comprises cleaving the polynucleotide with a glycosylase and endonuclease at a uracil (U) or cleaving the polynucleotide after DNA amplification, with a restriction endonuclease that recognizes a site after conversion of unmethylated cytosine (C) to thymine (T) in the polynucleotide.

9. A method for differentiating unmethylated cytosine (C) from 5-methylcytosine (5-mC), 5-hydroxymethyl cytosine (5-hmC), 5-formylcytosine (5-fC), 5-carboxycytosine (5-CaC) and/or 5-glycosylated hydroxymethylcytosine (5-ghmC) in a polynucleotide, comprising:

(a) reacting a first sample of the polynucleotide optionally containing C, 5-mC, 5-hmC, 5-fC, 5-CaC and/or 5-ghmC, with an oxygenase followed by a cytidine deaminase having an amino acid sequence that is at least 90% identical to SEQ ID NO:2, thereby converting C to uracil (U) and converting 5-mC to 5-hmC and 5-CaC; and

(b) amplifying or cleaving the polynucleotide to identify the location of the U in the polynucleotide.

10. A method according to claim 9 , further comprising: reacting a second sample of the polynucleotide with a cytidine deaminase in the absence of an oxygenase for converting an unmethylated cytosine (C) to uracil (U) and 5-methylcytosine (5-mC) to thymine (T).

11. A method according to claim 9 , wherein the polynucleotide from step (a) is single-stranded.

12. A method according to claim 10 , further comprising:

sequencing the first sample of the polynucleotide after the reaction with the oxygenase and cytidine deaminase to generate a first sequence;

sequencing the second sample of the polynucleotide after a reaction with cytidine deaminase but not with the oxygenase to generate a second sequence;

optionally sequencing a third sample of the polynucleotide absent a reaction with the cytidine deaminase or the oxygenase; and

comparing the first sample sequence and the second sample sequence and optionally the third sample sequence to detect and differentiate unmethylated cytosine (C) and 5-methylcytosine (5-mC).

13. A method according to claim 9 , wherein the oxygenase is a methylpyrimidine oxygenase or a 5-methylcytosine oxygenase.

14. A method according to claim 13 , wherein the 5-methylcytosine oxygenase is TET1.

15. A method according to claim 13 , wherein the methylpyrimidine oxygenase is a mYOX.

16. A method according to claim 9 , wherein the cytidine deaminase has an amino acid sequence that is at least 95% identical to SEQ ID NO:2.

17. A method for differentiating a 5-methylcytosine (5-mC) from an unmethylated cytosine (C), comprising:

reacting a first sample of a polynucleotide with sodium bisulfite sequencing reagents followed by a cytidine deaminase having an amino acid sequence that is at least 90% identical to SEQ ID NO:2, in the absence of an oxygenase, thereby converting 5-mC to thymine (T) and converting C to uracil (U); and

reacting a second sample of the polynucleotide with sodium bisulfite sequencing reagents without subsequent exposure to a cytidine deaminase, thereby converting C to U while retaining 5-mC as 5-mC.

18. A method according to claim 17 , further comprising amplifying the first and second samples of the polynucleotides for sequencing to identify the presence and location of at least one converted nucleotide in the polynucleotides.

19. A method according to claim 17 , wherein the cytidine deaminase has an amino acid sequence that is at least 95% identical to SEQ ID NO:2.

20. A method according to claim 17 , wherein the method is performed at a temperature less than 60° C.

21. A method according to claim 17 , wherein the polynucleotide has a length of greater than 1 Kb.

22. The method of claim 7 , wherein the cytidine deaminase has a mutation at one or more positions corresponding to positions 23, 25, 29, 45, 69, 104 and 123 in SEQ ID NO:2.

23. The method of claim 16 , wherein the cytidine deaminase has a mutation at one or more positions corresponding to positions 23, 25, 29, 45, 69, 104 and 123 in SEQ ID NO:2.

24. The method of claim 19 , wherein the cytidine deaminase has a mutation at one or more positions corresponding to positions 23, 25, 29, 45, 69, 104 and 123 in SEQ ID NO:2.