Dramatic improvements and falling costs of high throughput sequencing have made bisulfite sequencing (BS-Seq) a viable option for the global analysis of DNA methylation (Bock et al, 2011; Li et al, 2010; Lister et al, 2009; Lister et al, 2011; Meissner et al, 2008; Stadler et al, 2011; Xie et al, 2012). The analysis of methylation obtained from BS-Seq is relatively straight forward, but care should be taken for initial quality control, trimming and suitable alignment of BS-Seq libraries since these are susceptible to a variety of errors or biases that one could probably get away with in other sequencing applications (discussed in (Krueger et al, 2012)).
This protocol will take you through each of the individual steps we routinely take for BS-Seq or Reduced Representation Bisulfite-Seq (RRBS) data [in our brief guide to RRBS we discuss some additional points that are specifically relevant for RRBS-type experiments (RRBS_Guide)]. Sequencing files that come fresh from the sequencer first undergo (1) initial quality control, are then subjected to (2) quality- and adapter-trimming before (3) the bisulfite reads are aligned to a genome. Optionally, results may be (4) filtered after the alignments have been performed. This procedure yields a final set of methylation data that can be analysed to answer your biological questions of interest.
Bioinformatics Group, The Babraham Institute, Cambridge, CB22 3AT, United Kingdom
Corresponding author: Felix Krueger & Simon R Andrews