Ultra-Deep Sequencing of Bisulfite-Modified DNA

Ultra-deep sequencing of bisulfite-modified DNA is considered the gold standard method for assessment of DNA methylation across the genome. In this chapter, an introduction to whole-genome bisulfite sequencing and reduced representation bisulfite sequencing are presented, as well as their advantages and disadvantages. Specific topics include (i) sample preparation and study design; (ii) quality controls and other technical considerations for ultra-deep sequencing; (iii) testing methods for differentially methylated sites or regions, focusing on the beta-binomial model; (iv) annotation, visualization, and post hoc tests of results; and (v) experimental extensions to bisulfite sequencing. Specific applications of bisulfite sequencing to cancers, such as leukemia, and their findings are also presented.