Bisulfite-free, nano-scale analysis of 5-hydroxymethylcytosine at single base resolution

High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples represents a continuous challenge. Here, we present CATCH-seq, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. CATCH-seq is based on selective 5hmC oxidation, labeling and subsequent C-to-T transition during PCR. Applications of CATCH-seq to nano-scale DNA samples reveal previously underappreciated non-CG 5hmCs in the hESC genome and base-resolution hydroxymethylome in human cell-free DNA. We developed a method named "CATCH-Seq" , a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. Our method is based on: (1) selective oxidation of 5hmC to 5fC ; (2) subsequent labeling of the newly generated 5fC; and (3) 5fC labeling adduct caused C-to-T transition during DNA amplification.We then applied CATCH-seq to 100 ng, 50 ng and 10 ng genomic DNA isolated from hESCs, respectively. To further demonstrate the utility of CATCH-seq in analyzing precious clinical samples, we next applied this approach to sequence the hydroxymethylome of cell-free DNA (cfDNA).