The primary mapping output of Bismark contains one line per read and shows a number of useful pieces of information such as mapping position, alignment strand, the bisulfite read sequence, its equivalent genomic sequence and a methylation call string (Supplementary Material). This mapping output can be subjected to post-processing (Supplementary Material) or can be used to extract the methylation information at individual cytosine positions. This secondary methylation-state output can be generated using a flexible methylation extractor component that accompanies Bismark. The methylation output discriminates between sequence context (CpG, CHG or CHH) and can be obtained in either a comprehensive (all alignment strands merged) or alignment strand-specific format. The latter can be very useful to study asymmetric methylation (hemi- or CHH methylation) in a strand-specific manner. The output of the methylation extractor will create one entry (or line) per cytosine, whereby the strand information is used to encode its methylation state: ‘+’ indicates a methylated and ‘−’ a non-methylated cytosine. This output can be converted into other alignment formats such as SAM/BAM, or imported into genome browsers, such as SeqMonk, where it can be visualized and further explored by the researcher without requiring additional computational expertise.
