METHODS FOR DIFFERENTIATING MODIFIED NUCLEOBASES
Disclosed herein, inter alia, are compositions, methods, and kits useful for detecting nucleobase modifications on one or both strands of a double-stranded nucleic acid fragment.
1 . A method of sequencing a nucleic acid molecule, wherein the nucleic acid molecule comprises, from 5′ to 3′, a first strand, a first primer binding sequence, a second strand comprising a cytosine nucleobase, and a second primer binding sequence, wherein the second strand is complementary to the first strand, the method comprising:
(a) annealing a blocking primer to the first primer binding sequence of the nucleic acid molecule and extending the blocking primer with a polymerase to form a blocking strand hybridized to the first strand;
(b) converting the cytosine nucleobase of the second strand to a uracil nucleobase, or uracil nucleobase analog; and
(c) sequencing the second strand to generate a sequencing read.
2 . The method of claim 1 , wherein the cytosine nucleobase of the second strand that is converted to the uracil nucleobase, or uracil nucleobase analog, is a modified cytosine nucleobase.
3 . The method of claim 2 , wherein the modified cytosine nucleobase of the second strand is a 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), or β-glucosyl-5-hydroxymethylcytosine (5gmC).
4 . The method of claim 3 , wherein the modified cytosine nucleobase is a 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC).
5 . The method of claim 1 , wherein converting the cytosine nucleobase of the second strand comprises contacting the cytosine nucleobase with a ten-eleven translocation (TET) enzyme, an apolipoprotein B mRNA editing enzyme, a catalytic polypeptide-like (APOBEC) enzyme, a borane-containing reducing agent, an oxidizing agent, a β-glucosyltransferase, or a combination thereof.
6 . The method of claim 1 , wherein converting the cytosine nucleobase of the second strand comprises i) contacting the cytosine nucleobase with a ten-eleven translocation (TET) enzyme to generate a 5-carboxylcytosine (5caC) nucleobase; and ii) contacting the 5caC nucleobase with a borane-containing reducing agent to generate a uracil nucleobase analog.
7 . The method of claim 1 , wherein converting the cytosine nucleobase of the second strand comprises i) contacting the cytosine nucleobase with a β-glucosyltransferase to generate a β-glucosyl-5-hydroxymethylcytosine (5gmC) nucleobase; ii) contacting the 5gmC nucleobase with a ten-eleven translocation (TET) enzyme to generate a 5-carboxylcytosine (5caC) nucleobase; and iii) contacting the 5caC nucleobase with a borane-containing reducing agent to generate a uracil nucleobase analog.
8 . The method of claim 1 , wherein converting the cytosine nucleobase of the second strand comprises i) contacting the cytosine nucleobase with an oxidizing agent to generate a 5-formyl cytosine (5fC) nucleobase; and ii) contacting the 5fC nucleobase with a borane-containing reducing agent to generate a uracil nucleobase analog, wherein the oxidizing agent is selected from the group consisting of potassium perruthenate (KRuO4), Cu(II)/TEMPO (copper(II) perchlorate and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)), potassium ruthenate, and manganese oxide.
9 . The method of claim 1 , wherein converting the cytosine nucleobase of the second strand comprises i) contacting the cytosine nucleobase with a ten-eleven translocation (TET) enzyme to generate a 5-carboxylcytosine (5caC) nucleobase; and ii) contacting the second template strand with an apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) enzyme to generate a uracil nucleobase.
10 . The method of claim 1 , wherein the cytosine nucleobase of the second strand that is converted to the uracil nucleobase, or uracil nucleobase analog, is an unmodified cytosine nucleobase.
11 . The method of claim 10 , wherein converting the unmodified cytosine nucleobase of the second strand comprises contacting the unmodified cytosine nucleobase with sodium bisulfite to generate a uracil nucleobase.
12 . The method of claim 1 , wherein the polymerase is a strand-displacing polymerase.
13 . The method of claim 1 , further comprising removing the blocking strand and sequencing the first strand.
14 . The method of claim 1 , wherein sequencing comprises sequencing by synthesis.
15 . The method of claim 1 , wherein sequencing comprises hybridizing a sequencing primer to the second primer binding sequence, incorporating one or more modified nucleotides into the sequencing primer with a polymerase to create an extension strand, and detecting the one or more incorporated nucleotides.
16 . The method of claim 1 , wherein sequencing comprises annealing a sequencing primer to the second primer binding sequence and contacting the sequencing primer with a sequencing solution comprising one or more modified nucleotides comprising a reversible terminator, and monitoring the sequential incorporation of complementary nucleotides to generate one or more sequencing reads, wherein the reversible terminator is removed prior to the introduction of the next complementary nucleotide.
17 .- 40 . (canceled)
41 . The method of claim 1 , wherein, prior to step (c), the nucleic acid molecule is amplified.
42 . The method of claim 1 , further comprising annealing a probe oligonucleotide to the displaced second strand and separating the probe-hybridized double-stranded nucleic acid from nucleic acids not hybridized to the probe.
43 . The method of claim 12 , wherein the extension of the blocking primer with the strand-displacing polymerase terminates at a terminating nucleotide between the second strand and the first strand.
44 . The method of claim 43 , wherein the terminating nucleotide comprises a removable group that blocks progression of the strand-displacing polymerase, and further wherein the terminating nucleotide is treated to release the removable group prior to sequencing.
45 . The method of claim 43 , wherein the terminating nucleotide is an RNA nucleotide.
46 . The method of claim 42 , wherein the probe oligonucleotide is covalently attached to a substrate.
47 . The method of claim 42 , wherein the probe oligonucleotide is labeled with a first member of a binding pair, and separating the probe-hybridized double-stranded nucleic acid from nucleic acids not hybridized to a probe comprises capturing the probe with a second member of the binding pair.
48 . The method of claim 47 , wherein (i) the first member of the binding pair is biotin and the second member of the binding pair is avidin or streptavidin, or (ii) the second member of the binding pair is biotin and the first member of the binding pair is avidin or streptavidin.
49 . The method of claim 1 , wherein the blocking primer comprises one or more locked nucleic acids (LNAs), 2-amino-deoxyadenosines (2-amino-dAs), trimethoxystilbene-functionalized oligonucleotides (TFOs), pyrene-functionalized oligonucleotides (PFOs), peptide nucleic acids (PNAs), or aminoethyl-phenoxazine-dCs (AP-dCs).