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Abstract

Provided are methylcytosine-selective deaminases, compositions, kits, and methods employing them, including a sequencing method involving (a) contacting a DNA substrate comprising cytosine (C) and at least one methylcytosine nucleobase selected from 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) or comprising C and both 5mC and 5hmC, with a methylcytosine-selective deaminase to produce a deamination product, wherein the methylcytosine-selective deaminase (i) is capable of deaminating 5mC to thymidine (T) and/or 5hmC to hydroxymethyluridine (hmU) and (ii) preferentially deaminates the at least one methylcytosine nucleobase relative to cytosine (C); and (b) sequencing the deamination product, or amplifying the deamination product to produce an amplification product, and sequencing the amplification product, in each case, to produce sequence reads, wherein positions of Cs and position of the at least one methylcytosine nucleobase in the DNA substrate is determined based on the sequence reads.

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