Alignment choices for bisulphite treated data. Biased and unbiased alignments of bisulphite treated data. Bisulphite treatment converts unmethylated cytosines to uracil, which are sequenced as thymine. In a biased alignment, sequenced thymines are treated as ambiguously cytosine/thymine (ambiguity code Y). In case (b), of a unmethylated read, this ambiguity allows the converted read to align to two separate locations on the reference genome (blue), while in case (c), which consists of the same nucleotide sequence but contains methylated cytosines, the read aligns to a single location. This results in a greater confidence in the alignment of the methylated read. In the case of an unbiased alignment, all cytosines on both the sequenced reads and the reference genome are converted to thymines, and the sequences in both (b) and (c) align to the same locations on the genome, with no additional confidence in the alignment of the methylated read. In case (a), the read aligns to a single location in both a biased and unbiased alignment; however, in the unbiased case less information is available to make this alignment.