text: The first reported method of methylation analysis using bisulfite-treated DNA utilized PCR and standard dideoxynucleotide DNA sequencing to directly determine the nucleotides resistant to bisulfite conversion.  Primers are designed to be strand-specific as well as bisulfite-specific (i.e., primers containing non-CpG cytosines such that they are not complementary to non-bisulfite-treated DNA), flanking (but not involving) the methylation site of interest.  Therefore, it will amplify both methylated and unmethylated sequences, in contrast to methylation-specific PCR.  All sites of unmethylated cytosines are displayed as thymines in the resulting amplified sequence of the sense strand, and as adenines in the amplified antisense strand.  By incorporating high throughput sequencing adaptors into the PCR primers, PCR products can be sequenced with massively parallel sequencing. Alternatively, and labour-intensively, PCR product can be cloned and sequenced. Nested PCR methods can be used to enhance the product for sequencing.
  All subsequent DNA methylation analysis techniques using bisulfite-treated DNA is based on this report by Frommer et al. (Figure 2). Although most other modalities are not true sequencing-based techniques, the term "bisulfite sequencing" is often used to describe bisulfite-conversion DNA methylation analysis techniques in general.