The three main steps in the workflow of BS-Seeker2. (1) Index-building. Indexes for RRBS and WGBS are built separately from a three-letter converted genome. Four index instances are built to account for the asymmetric bisulfite-conversion of the two strands and properties of non-directional libraries. (2) Aligning reads to the indexes. Both WGBS and RRBS reads are converted to three-letters prior to mapping. For RRBS, adapters should be removed first. Converted reads are mapped onto four index instances for non-directional libraries (two instances for directional libraries), and mapping to each index instance will report two best hits. Multiple hits and mismatch numbers are checked before being reported as alignment results. The C-to-T match is regarded as a mismatch in this step, and is checked by the mismatch criteria. (3) Calling methylation level for each site. The user can decide whether to filter the un-converted reads in this step. BS-Seeker2 provides detailed outputs (BAM/SAM, wiggle, CGmap and ATCGmap files). Both the wiggle file and the BAM file can be directly imported in a genome browser, such as IGV. BS-Seeker2 is also integrated into the Galaxy web interface platform.