Figure 1

Bisulfite amplicon sequencing (BSAS) method schematic. Genomic DNA is bisulfite converted and subjected to bisulfite-specific PCR, using primers specific for bisulfite converted DNA (bs-F and R red lines). Amplicons are subjected to Nextera XT library preparation including dual indexing. Final libraries consist of a random insert of bisulfite converted, amplified DNA, capture probes (black and gray) and specific index sequences (orange, magenta, green, pink). These libraries are multiplexed and sequenced on the Illumina MiSeq. Demultiplexing separates the dual indexed reads from each sample (orange and magenta are one sample, green and pink represent the other sample). These reads are aligned to an in silico converted reference sequence, and variant calling is used to identify the percentage of 5-mC.