Reduced Bisulfite Sequencing: Quantitative Base-Resolution Sequencing of 5-Formylcytosine.

The generation of tools to study mammalian epigenetics is vital to understanding normal biological function and to identify how it is dysregulated in disease. The well-studied epigenetic DNA modification 5-methylcytosine can be enzymatically oxidized to 5-formylcytosine (5fC) in vivo. 5fC has been demonstrated to be an intermediate in demethylation, but recent evidence suggests that 5fC may have an epigenetic function of its own. We have developed reduced bisulfite sequencing (redBS-seq), which can quantitatively locate 5fC bases at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), 5fC is converted to uracil, as happens to unmodified cytosine (C), and thus cannot be discriminated from C. However, in redBS-seq, a specific reduction of 5fC to 5-hydroxymethylcytosine (5hmC) stops this conversion, allowing its discrimination from C. 5fC levels are inferred by comparison of a redBS-Seq run with a BS-seq run.