This video is about ASHG Workshop - Pt1
DNA methylation is essential for mammalian development and is often dysregulated in disease. Understanding the dynamics of DNA methylation can thus provide insight into disease etiology and serve as a biomarker. Base-pair resolution analysis of DNA methylation is facilitated by sequencing of bisulfite treated DNA, but this process can be laborious and expensive. Reduced representation bisulfite sequencing (RRBS) uses restriction enzymes to enrich parts of the genome where CpG dinucleotides occur, thus decreasing sequencing costs. To profile rare immune cell subsets, RRBS was further optimized for low input samples using a short-adaptor strategy with the KAPA Hyper Prep Kit and HiFi Uracil+ DNA Polymerase. Titration of the adaptor concentration in DNA limiting conditions (10ng DNA) improved conversion efficiency of the reaction and indicated an optimal adaptor-to-DNA molar ratio of more than >500X. In contrast, no improved reaction efficiency was observed above 50X for non-limiting input concentrations (500ng). Assay specificity and DNA methylation calls were indistinguishable in DNA-limiting conditions as compared to non-limiting conditions. These data provide a robust methodology for RRBS analysis in sample limiting conditions using the Kapa Biosystems efficient chemistry. Resultantly, we analyzed progressive stages in lymphocyte differentiation revealing a dynamic DNA hypomethylation and highlighting the utility of RRBS.