Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing

Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineer-ing of bacterial species. Systematic identification of DNA methylation in R-M systems, including N6-methyladenine (6mA), 5-methylcytosine (5mC) and N4-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing tech-nology is capable of detecting 4mC directly for any bacterial species regardless of whether an as-sembled genome exists or not, it is not as scal-able to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assem-bled reference genome. In 4mC-TAB-seq, both cy-tosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We ap-plied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellu-losiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. In combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bac-teria in the future