To assess quality of BS/OXBS-Seq data, spike-in DNA controls were mixed with genomic DNA samples. We used CEGX_QC (https://bitbucket.org/cegx-bfx/cegxqc) package with default parameters to detect the conversion rate of 5mC and 5hmC. The conversion efficiency was determined on the basis of synthetic DNA controls from CEGX and was considered successful if C > 90% for BS and OXBS libraries, while hmC < 5% in BS libraries and >80% in OXBS libraries (https://insidedna.me/tool_page_assets/pdf_manual/cegxqc.pdf) (table S8A). Similarly, to determine the methylation efficiency of M.SssI in MAB-seq, we spiked in unmethylated CG lambda DNA (Promega, WI, USA) and observed almost complete methylation of unmethylated CGs (~97%) and high bisulfite conversion efficiency (~97%) (table S8B). We randomly selected 30 million reads from each trimmed sequence files, mapped them onto lambda genome using bsmap, and extracted methylation signals using command “methratio.py -t 2 -r -m 5 -z.” M.SssI enables methylation of Cs with high efficiency in the CG context, instead of the CH context, while the bisulfite treatment converts unmethylated Cs into Ts in both CG and CH contexts. Therefore, the M.SssI methylase efficiency was calculated by the methylated-CG% based on the cytosine methylation level in the CG context, while bisulfite conversion efficiency was identified by unmethylated-CH% based on the lack of cytosine methylation in the CH context. Because of the fact that MAB-seq allows us to confidentially determine 5fC/caC only in the CG context (30) and that 5hmC is almost exclusively located in CG dinucleotides (31), we focused our studies of 5mC and 5hmC in CG sites to generate comparable and comprehensive genome-wide maps for all three cytosine modifications.

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