Routine Digital Methylation Profiling of Population Cohorts with Bisulfite Padlock Probes

Routine digital methylation profiling of population cohorts with bisulfite padlock probes.

DNA methylation at C(5) cytosine plays an essential role in the regulation of gene expression. We recently developed a second-generation bisulfite padlock probe (BSPP) method for specific and parallel digital quantification of DNA methylation. Specifically, we developed a new probe design algorithm to more efficiently generate bisulfite padlock probe sets, a new library-free protocol that dramatically reduce the time and cost of sample preparation and is compatible with automation, and bioinformatics pipeline called bisReadMapper that efficiently obtain both methylation levels and SNP genotypes from targeted or whole genome bisulfite sequencing. We have designed and synthesized ~330,000 padlock probes with high capture efficiency (~85% mapping rate and~96% on-target capture), which cover a set of carefully selected and highly informative genomic regions across the human genome (232,976 regions). The improved protocol used 96-multiplexed primers with 6-bp barcodes that can directly amplify the capture DNA after padlock circularization and ready for Illumina sequencing with minimal liquid handling steps. This improvement enables us to process samples in large cohorts up to 1,000-10,000 samples. Methylation measurements are highly consistent within a single batch or between separate batches (the Pearson’s R = 0.97-0.98). Approximately 500,000 CpG sites can be quantified with ~4Gb of total sequencing per sample. Additionally, we can call ~20,000 SNPs for each sample at an accuracy of 96% or better. The SNP calling feature also allows us to analyze allele-specific methylation and uniquely track samples, which is a critical feature in large-scale study. Examples from a number of recent studies, including mouse and human induced pluripotent stem cells, human peripheral blood and other primary tissue types, will be discussed.