Methods and Compositions for the Simultaneous Identification and Mapping of DNA Methylation
Provided herein is a method for generating a strand of DNA. In some embodiments, this method may comprise: (a) ligating a hairpin adaptor to a double-stranded fragment of DNA to produce a ligation product; (b) enzymatically generating a free 3′ end in a double-stranded region of the hairpin adaptor in the ligation product; and (c) extending the free 3′ end in a dCTP-free reaction mix that comprises a strand-displacing or nick-translating polymerase, dGTP, dATP, dTTP and modified dCTP to generate a hairpin product that has an original strand and a neosynthesized strand that contains modified Cs.
1 . A method for generating a deamination-resistant strand of DNA, comprising
(a) ligating a hairpin adaptor to a double-stranded fragment of DNA to produce a ligation product;
(b) enzymatically generating a free 3′ end in a double-stranded region of the hairpin adaptor in the ligation products; and
(c) extending the free 3′ end in a dCTP-free reaction mix that comprises a strand-displacing or nick-translating polymerase, dGTP, dATP, dTTP and modified dCTP to generate a hairpin product that has an original strand and a neosynthesized strand that contains modified Cs.
2 . The method of claim 1 , further comprising
(d) deaminating the hairpin product or an adaptor-ligated product thereof, wherein the modified Cs protect the neosynthesized strand from deamination.
3 . The method of claim 2 , wherein the deaminating is done using bisulfite or using a cytosine deaminase, optionally after enzymatically protecting any modified Cs in the original strand from deamination.
4 . (canceled)
5 . The method of claim 3 , wherein the cytosine deaminase modifies a double-stranded or single-stranded substrate.
6 . The method of claim 2 , further comprising amplifying the deaminated product of step (d) thereby converting any deaminated Cs to Ts in the amplification product.
7 . The method of claim 6 , further comprising enriching for target molecules using a probe that is complementary to a sequence in the double-stranded fragment of (a).
8 . The method of claim 2 , further comprising sequencing the deaminated product, or an amplification product thereof, to produce sequence.
9 . The method of claim 8 , further comprising identifying a C in the sequence corresponding to the original strand, wherein the C corresponds to a modified cytosine.
10 . The method of claim 9 , further comprising mapping the modified cytosine to a site in a reference genome and annotating the site as being modified.
11 . The method of claim 1 , wherein the modified dCTP is dmCTP, pyrrolo-dCTP or N 4 -dmCTP.
12 . The method of claim 1 , wherein the double-stranded fragment of DNA is selected from a fragment of mammalian DNA and a molecule of cfDNA.
13 . (canceled)
14 . The method of claim 1 , further comprising enzymatically modifying the double-stranded fragment of DNA, the ligation product or hairpin product to protect any modified cytosines or hydroxymethylcytosines from deamination.
15 . The method claim 1 , wherein in step (a) both ends of the double-stranded fragment of DNA are ligated to the hairpin adaptor and in step (b) the top and bottom strands of the double-stranded fragment of DNA become separated.
16 . The method of claim 1 , wherein step (b) is done using USER, an endonuclease, a nicking endonuclease or an RNase.
17 . The method of claim 1 , wherein the hairpin adaptor has at least one modified C and no Cs.
18 . The method of claim 1 , wherein the modified C of the adaptor is mCTP, pyrrolo-CTP or N 4 -mCTP.
19 . A reaction mix comprising:
(a) a hairpin DNA that has a free 3′ end in a double-stranded region;
(b) a strand-displacing or nick-translating polymerase, and
(c) dGTP, dATP, dTTP, modified dCTP and no dCTP.
20 . The reaction mix of claim 19 , wherein the hairpin DNA comprises a fragment of mammalian DNA ligated to a hairpin adaptor or comprises a molecule of cfDNA ligated to a hairpin adaptor.
21 . (canceled)
22 . The reaction mix claim 19 , wherein the modified dCTP is dmCTP, pyrrolo-dCTP or N 4 -dmCTP.
23 . A nucleic acid molecule comprising, in order from 5′ to 3′:
a first sequence, a linker, and a second sequence, wherein:
(a) the first sequence is composed of Gs, As, Ts, Cs and modified Cs;
the second sequence is composed of Gs, As, Ts, modified Cs and no Cs; and
the first and second sequences are complementary; or
(b) the first sequence is composed of Gs, As, Ts, Us and modified Cs and
the second sequence is composed of Gs, As, Ts, modified Cs and no Cs; and
the first and second sequences are complementary except for the Us in the first sequence.
24 . (canceled)
25 . A kit for generating a deamination-resistant strand of DNA, comprising:
(a) a hairpin adaptor containing a U in a double-stranded region of the adaptor;
(b) one or more enzymes that create a nick at the site of the U;
(c) a modified dCTP; and
(d) a nick-translating or strand-displacing polymerase, optionally wherein the modified dCTP is dmCTP, pyrrolo-dCTP or N 4 -dmCTP.
26 . (canceled)
27 . The kit of claim 25 , wherein the adaptor contains modified Cs and no Cs, and optionally wherein the modified Cs of the adaptor are mCTP. pyrrolo-CTP or N 4 -mCTP.
28 . (canceled)
29 . The kit of claim 25 , further comprising a deaminase, wherein the modified Cs are deamination resistant.
30 . A method for generating a deamination-resistant strand of DNA, comprising:
(a) separating the strands of a double-stranded fragment of DNA to produce a single-stranded fragment;
(b) attaching a double-stranded adaptor to the 3′ end of the single-stranded fragment;
(c) extending the free 3′ end of an attached double-stranded adaptor in a dCTP-free reaction mix that comprises a strand-displacing or nick-translating polymerase; and dGTP, dATP, dTTP, and modified dCTP, to generate a double-stranded product, and
(d) attaching a hairpin adaptor to the 5′ end of the double-stranded product to generate a hairpin product that has an original strand and a neosynthesized strand that contains modified Cs.