DNA methylation detection at single base resolution using targeted next generation bisulfite sequencing and cross validation using capillary sequencing

Abstract

With a purpose of accurate and simultaneous determination of DNA methylation from multiple loci in multiple samples, here, we are demonstrating a method to aid rapid DNA methylation detection of genomic sequences. Using genomic DNA of peripheral blood from 14 healthy individuals, DNA methylation in 465 CpG sites from 12 loci of genes (ADAM22, ATF2, BCR, CD83, CREBBP, IL12B, IL17RA, MAP2K2, RBM38, TGFBR2, TGFBR3, and WNT5A) was analysed by targeted next generation bisulfite sequencing. Analysed region for three genes, BCR, IL17RA and RBM38 showed an absolute mean DNA methylation of 25.6%, 89.2% and 38.9% respectively. Other nine gene loci were unmethylated and exhibited <10% absolute mean DNA methylation. Two genes, IL17RA and RBM38 were technically validated using direct capillary sequencing and results were comparable with positive correlation (P=0.0088 & P<0.0001 respectively) in the CpG sites for DNA methylation. All CpG sites analysed from RBM38 genes locus displayed 95% limits of agreement for DNA methylation measurements from the two methods. The present approach provides a fast and reliable DNA methylation quantitative data at single base resolution with good coverage of the CpG sites under analysis in multiple loci and samples simultaneously. Use of targeted next generation bisulfite sequencing may provide an opportunity to explore genes in the discovery panel for biomarker identification and facilitate functional validation.

Keywords: Biomarker; CpG islands; DNA methylation; Gene discovery; Next generation sequencing (NGS).