Mirror Bisulfite Sequencing: A Method for Single-Base Resolution of Hydroxymethylcytosine

Although
the role of 5-methylcytosine has been well studied, the
biological role of 5-hydroxymethylcytosine still remains unclear because
of the limited methods available for single-base detection of 5-hydroxymethylcytosine
(5hmC). Here, we present mirror bisulfite sequencing for 5hmC detection
at a single CpG site by synthesizing a DNA strand to mirror the parental
strand. This semiconservative duplex is sequentially treated with
β-glucosyltransferase and M.SssI methylase. The glucosyl-5hmCpG
in the parental strand inhibits methylation of the mirroring CpG site,
and after bisulfite conversion, a thymine in the mirroring strand
indicates a 5hmCpG site in the parental strand, whereas a cytosine
indicates a non-5hmC site. Using this method, the 5hmC levels of various
human tissues and paired liver tissues were mapped genomewide.