Locus-Specific DNA Methylation Analysis by Targeted Deep Bisulfite Sequencing.

DNA methylation, i.e., the methylation of cytosine at carbon atom C5, has an important role in the regulation of gene expression. The methylation status of each cytosine in a specific genomic region can be determined by targeted deep bisulfite sequencing at single-molecule resolution. Here we describe the design of PCR primers that are used to amplify specific sequences from bisulfite-converted DNA, the preparation of sequencing libraries, the sequencing of these libraries on the MiSeq system, as well as the analysis of the sequence reads. Using appropriate software tools such as amplikyzer2, it is easy to analyze complex multiplexed samples with several regions of interest, to determine the mean methylation values of all CpG dinucleotides in a region or of each CpG dinucleotide across all or selected reads, and to compare these values between different samples and between different alleles within a sample.