Fig. 7 Effects of bisulfite treatment on 5mC and 5hmC in DNA and the sequencing results. (a)
Bisulfite-mediated deamination of cytosine. HSO 3
À adds across the 5,6-double bond of cytosine,
promoting deamination and conversion to 6-sulfonyluracil. 6-sulfonyluracil is desulfonated to
uracil (U) at higher pH. 5mC is also deaminated to thymine by bisulfite conversion, but the rate
is approximately two orders of magnitude slower than that of cytosine. Bisulfite quickly converts
5-hydroxymethylcytosine to form cytosine-5-methylenesulfonate (CMS), which does not readily
undergo deamination. (b) Shown are examples of Sanger sequencing traces of 5hmC-containing
DNA before and after bisulfite treatment. 5hmC resists bisulfite-induced deamination and is thus
read as cytosine. The control C-containing oligonucleotide, on the other hand, shows complete
conversion of all C’s in the top strand (highlighted sequences) to T’s (lower panel). Adapted from
Huang Y et al. (2010). The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS
One. Jan 26;5(1):e8888. doi: https://doi.org/10.1371/journal.pone.0008888. Used in accordance
with CC BY license (http://creativecommons.org/licenses/by/4.0/) [78]
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