Whole genome bisulfite sequencing (WGBS) has been widely used to quantify cytosine DNA methylation frequency in an expanding array of cell and tissue types. Because of the denaturing conditions used, this method ultimately leads to the measurement of methylation frequencies at single cytosines. Hence, the methylation frequency of CpG dyads (two complementary CG dinucleotides) can only be indirectly inferred by overlaying the methylation frequency of two cytosines measured independently. Furthermore, hemi-methylated CpGs (hemiCpGs), a significant component of the DNA methylome, have not been previously analyzed in WGBS studies. We recently developed in silico Strand Annealing (iSA), a bioinformatics method applicable to WGBS data, to resolve the methylation status of CpG dyads into unmethylated, hemi-methylated, and methylated. HemiCpGs account for 4-20% of the DNA methylome in different cell types and some can be inherited across cell divisions, suggesting a role as a stable epigenetic mark. Therefore, it is important to resolve hemiCpGs from fully methylated CpGs in WGBS studies. The versatility of iSA enables its application downstream of other WGBS-related methods such as nasBS-seq, ChIP-BS-seq, TAB-seq, oxBS-seq, and fCAB-seq. iSA is also tunable for analyzing the single-fragment methylation status of cytosines in any sequence context. We exemplify its flexibility by uncovering the single-fragment non-CpG methylome. Here we provide this interactive shell script to be run from the Unix shell prompt in a terminal window. The running may take 2-7 hours, depending on the size of the dataset.

Please cite:

1. Xu, C. & Corces, V. G. Nascent DNA methylome mapping reveals inheritance of hemimethylation at CTCF/cohesin sites. Science 359, 1166-1170, doi:10.1126/science.aan5480 (2018).
2. Xu, C. & Corces, V. G. Resolution of the DNA methylation state of single CpG dyads using in silico Strand Annealing and WGBS data. Nat Protoc 14, 202-216, doi:10.1038/s41596-018-0090-x (2019).