Enzymatic conversion of methylated nucleic acids for sequencing

A method, wherein the method comprises the following steps:

• (a) contacting a Taq DNA ligase with a double-stranded DNA (dsDNA), wherein the dsDNA comprises at least one nick, at least one cytosine, and at least one methylcytosine, wherein the methylcytosine is selected from 5-methylcytosine and 5-hydroxymethylcytosine, thereby repairing the at least one nick;
• (b) enzymatically converting, with a TET methylcytosine dioxygenase 2 (TET), the at least one methylcytosine to one of 5-hydroxymethylcytosine and 5-carboxycytosine;
• (c) enzymatically converting, with a T4-phage beta-glucosyltransferase (T4-BGT) the 5-hydroxymethylcytosine to 5-(β-glucosyloxymethyl)cytosine;
• and
• (d) enzymatically converting, with an apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A), the at least one cytosine to uracil.

A method, wherein the method comprises the following steps:

• (a) contacting a first enzyme possessing methyltransferase activity with a double-stranded DNA (dsDNA), wherein the dsDNA comprises at least one hemimethylated CpG site, at least one additional cytosine, and at least one methylcytosine, wherein the methylcytosine is selected from 5-methylcytosine and 5-hydroxymethylcytosine, thereby methylating the hemimethylated CpG site, wherein the at least one additional cytosine remains unmethylated;
• (b) converting the at least one methylcytosine to a modified methylcytosine capable of base-pairing with a guanine;
• and
• (c) converting the at least one additional cytosine to uracil.

A method, wherein the method comprises the following steps:

• (a) ligating an adapter to a double-stranded DNA (dsDNA), wherein the dsDNA comprises at least one cytosine and at least one methylcytosine, wherein the methylcytosine is selected from 5-methylcytosine and 5-hydroxymethylcytosine, wherein the adapter comprisingcomprises a nucleic acid sequencingsequence comprising at least one methylcytosine, wherein each methylcytosine present in the adapter is a 5-hydroxymethylcytosine;
• (b) converting the at least one methylcytosine present in the dsDNA, and the at least one methylcytosine present in the adapter, to a modified methylcytosine capable of base-pairing with a guanine;
• and
• (c) converting the at least one additional cytosine to uracil.

A method, wherein the method comprises the following steps:

• (a) ligating an adapter to a double-stranded DNA (dsDNA), wherein the dsDNA comprises at least one cytosine and at least one methylcytosine selected from 5-methylcytosine and 5-hydroxymethylcytosine, the adapter comprising a nucleic acid sequence comprising at least one modified methylcytosine, wherein each modified methylcytosine present in the adaptor is a 5-(β-glucosyloxymethyl)cytosine;
• (b) converting the at least one methylcytosine present in the dsDNA to a modified methylcytosine capable of base-pairing with a guanine;
• and
• (c) converting the at least one additional cytosine to uracil.

A method, wherein the method comprises the following steps:

• (a) combining:
• (i) a double-stranded DNA (dsDNA) comprising, wherein the dsDNA comprises at least one cytosine, and at least one methylcytosine selected from 5-methylcytosine and 5-hydroxymethylcytosine, (ii) a cytidine deaminase, and (iii) at least one of a single-strand DNA-binding protein and a helicase;
• (b) converting the at least one methylcytosine to a modified methylcytosine capable of base-pairing with a guanine;
• and
• (c) converting, with the cytidine deaminase, the at least one cytosine to uracil.

A method, wherein the method comprises the following steps:

• (a) combining:
• (i) a double-stranded DNA (dsDNA), wherein the dsDNA comprises at least one cytosine, and at least one methylcytosine selected from 5-methylcytosine and 5-hydroxymethylcytosine, (ii) a cytidine deaminase, and (iii) at least one of a single-strand DNA-binding protein and a helicase
• (b) enzymatically converting, with a TET methylcytosine dioxygenase 2 (TET), the at least one methylcytosine to one of 5-hydroxmethylcytosine and 5-carboxycytosine;
• (c) enzymatically converting, with a T4-phage beta-glucosyltransferase (T4-BGT) 5-hydroxymethylcytosine, to 5-(β-glucosyloxymethyl)cytosine;
• and
• (d) enzymatically converting, with an apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A), the at least one cytosine to uracil.

A composition, wherein the composition comprises the following:

• (a) a double-stranded DNA (dsDNA) comprising at least one cytosine, and at least one modified cytosine selected from 5-carboxycytosine and 5-(β-glucosyloxymethyl)cytosine;
• (b) a buffer;
• and
• (c) a cytidine deaminase,
• wherein the amount of the dsDNA is from about 1 pg to about 10 ng,
• wherein the concentration of the cytidine deaminase is from about 0.05 μM to about 0.5 μM, and
• wherein the total volume of the composition is less than about 100 μL.

A method, wherein the method comprises the following steps:

• (a) providing a first composition, wherein the first composition comprises a double-stranded DNA (dsDNA) comprising at least one cytosine, and at least one methylcytosine, wherein the methylcytosine is selected from 5-methylcytosine and 5-hydroxymethylcytosine;
• (b) enzymatically converting, with a TET methylcytosine dioxygenase 2 (TET), the at least one methylcytosine to one of 5-hydroxymethylcytosine and 5-carboxycytosine, thereby forming a first dsDNA product;
• (c) enzymatically converting, with a T4-phage beta-glucosyltransferase (T4-BGT) 5-hydroxymethylcytosine, in the first dsDNA product to 5-(β-glucosyloxymethyl)cytosine, thereby forming a second dsDNA product;
• (d) enzymatically converting, with an apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A), the at least one cytosine in the second dsDNA product to uracil, thereby forming a methyl-sequencing product composition comprising the APOBEC3A and a third dsDNA product;
• (e) combining the methyl-sequencing product composition with an amplification composition;
• and
• (f) amplifying, by polymerase chain reaction, the third dsDNA product, wherein the amplifying occurs in the presence of the APOBEC3A.

A method, wherein the method comprises the following steps:

• (a) combining:
• a first enzyme wherein the first enzyme possesses nick repair activity,
• a second enzyme wherein the second enzyme possesses 5′ to 3′ polymerase activity, the second enzyme lacking 5′ to 3′ exonuclease activity,
• a third enzyme possessing, wherein the third enzyme possesses apurinic/apyrimidinic endonuclease activity,
• a double-stranded DNA (dsDNA) wherein the dsDNA comprises at least one single-stranded discontinuity selected from a nick and a gap, at least one cytosine, and at least one methylcytosine selected from 5-methylcytosine and 5-hydroxymethylcytosine, and
• a plurality of deoxynucleoside triphosphates (dNTPs);
• (b) repairing the at least one discontinuity in the dsDNA;
• (c) converting the at least one methylcytosine to a modified methylcytosine capable of base-pairing with a guanine;
• and
• (d) converting the at least one cytosine to uracil.