Processing of whole-genome bisulfite sequencing data
This protocol is extracted from research article:
No evidence for DNA N6-methyladenine in mammals
Sci Adv, Mar 18, 2020; DOI: 10.1126/sciadv.aay3335

Bisulfite sequencing data (416 million 150-bp paired-end reads) were obtained from the National Center for Biotechnology Information short read archive (accession: SRX5716730 and SRX5716740) and aligned to a bisulfite-converted hg38 index with Bismark (bismark --N 1). Subsequently, methylation levels of cytosines in both CpG and non-CpG contexts were extracted (bismark_methylation_extractor --p --comprehensive --bedgraph). Summary methylation files were then postprocessed to remove all sites with less than 5× coverage. CpG sites with greater than 20% methylation were considered as “methylated.” The distribution of mCpGs was overlapped with the published genomic positions of 6mdA sites from the indicated studies using the R programming language. As we lacked information on the strandedness of the SMRT-seq reads, the overlapping analysis was not done in a strand-specific manner, resulting in the symmetrical nature of the plots.

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