Wu, H. et al. Nat. Biotechnol. 32, 1231–1240 (2014).

During DNA demethylation, 5-methylcytosine (5-mC) sites are progressively oxidized before removal by repair enzymes. Methods exist to profile the final two rare intermediates, 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), but most approaches involve enrichment steps and cannot provide single-base resolution or absolute quantification. Bisulfite sequencing converts unmodified cytosine as well as 5-fC and 5-caC to thymine, reading out positions of 5-mC and another intermediate, 5-hydroxymethylcytosine, at single-base resolution, but not 5-fC and 5-caC. Hu et al. introduce M.SssI methylase–assisted bisulfite sequencing (MAB-seq), which uses the bacterial methyltransferase M.SssI to methylate unmodified cytosine within CpG dinucleotides, protecting it from bisulfite conversion. When traditional bisulfite sequence data and MAB-seq data from the same sample are compared, 5-fC and 5-caC levels can be measured at single-base resolution, thus shedding light on the dynamics of DNA methylation in the genome.