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Active DNA demethylation is mediated by ten-eleven translocation (TET) proteins that progressively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). We have developed a methylation-assisted bisulfite sequencing (MAB-seq) method that enables direct genome-scale mapping and quantification of 5fC and 5caC marks together at single-base resolution. In bisulfite sequencing (BS), unmethylated cytosine residues (Cs), 5fCs and 5caCs, are converted to uracil and cannot be discriminated from each other. The pretreatment of the DNA with the CpG methylation enzyme M.SssI, which converts only the Cs to 5mCs, protects Cs but not 5fCs and 5caCs, which enables direct detection of 5fCs and 5caCs as uracils. Here we also describe an adapted version of the protocol to perform reduced-representation MAB-seq (RRMAB-seq) that provides increased coverage on CpG-rich regions, thus reducing the execution costs and increasing the feasibility of the technique. The main advantage of MAB-seq is to reduce the number of chemical/enzymatic DNA treatments required before bisulfite treatment and to avoid the need for prohibitive sequencing coverage, thus making it more reliable and affordable than subtractive approaches. The method presented here is the ideal tool for studying DNA demethylation dynamics in any biological system. Overall timing is ∼3 d for library preparation.

Methylation-assisted bisulfite sequencing to simultaneously map 5fC and 5caC on a genome-wide scale for DNA demethylation analysis