Deconstructing the Cancer Epigenome using Reduced Representation Bisulfite Sequencing (RRBS)

Reduced Representation Bisulfite Sequencing (RRBS) represents a relatively inexpensive methodology for investigating the influence of DNA methylation on the development and progression of diseases including cancer. RRBS is a targeted approach which combines the use of restriction enzyme selection, bisulfite conversion, and high throughput sequencing technologies. The computational processing and analysis of RRBS data therefore requires modified approaches compared to typical DNA sequencing, due to deviations from standard laboratory protocols. This chapter presents a step-by-step pipeline for the processing of raw RRBS reads including quality control, trimming, alignment, methylation extraction, differential methylation analysis, annotation, and downstream analysis. This pipeline is designed to ensure optimal processing for accurate results and to provide downstream analysis tools that can extract biological insights from the data.