Whole-Genome Bisulfite Sequencing (WGBS): An Improved “post-bisulfite” Conversion Library Construction Method from Low DNA Inputs

DNA methylation plays a role in a variety of biological processes including cell differentiation and disease on-set.  Genome-wide analysis of methyl-cytidine nucleotides is possible with whole-genome bisulfite sequencing (WGBS) which provides DNA methylation status at single nucleotide resolution. Conventional WGBS sample prep involves DNA shearing, ligation of methylated sequencing adaptors, bisulfite conversion of unmethylated cytidine nucleotides and sequencing on an Illumina sequencer.  However, a major challenge with conventional WGBS sample prep is that ~90% of the adaptor-tagged DNA is degraded during the bisulfite conversion step. As a consequence, a majority of DNA fragments contained in the adaptor-tagged sequencing library lose at least one flanking adaptor rendering them incapable of being sequenced.    We describe an improved “post-bisulfite” WGBS library construction method. Our “post-bisulfite” WGBS library construction method features bisulfite conversion of cytosines prior to ligation-free addition of the Illumina sequencing adaptors.  Thus DNA fragments, independent of size and position of bisulfite-mediated strand breaks, remain as viable templates for library construction and sequencing.   Our “post-bisulfite” WGBS library construction method exhibits increased sensitivity and efficiency and improved coverage for detecting methyl-cytidine nucleotides.