Fig. 1

The workflow of conventional bisulphite (Bis), oxidative bisulphite (OxBis) and TET-assisted bisulphite (TAB) approaches for the detection of 5mC and 5hmC. Bisulphite treatment alone results in the conversion of unmethylated cytosine into uracil that will be read as thymine after PCR amplification, with both 5mC and 5hmC being read as cytosine. The readout of Bis is denoted as 5modC (5mC + 5hmC). The addition of an oxidation step prior to bisulphite treatment results in the conversion of 5hmC to 5fC that will be converted to uracil together with an unmethylated cytosine. Thus, the readout of OxBis is 5mC. In TAB, the first step involves β-glucosyltransferase-mediated protection of 5hmC with a glucose moiety, followed by TET-mediated oxidation of 5mC to 5caC, which will be converted to uracil. Thus, the readout of TAB is 5hmC