MethPy: a new software for analyzing non-CpG methylation after bisulfite assay and Sanger sequencing

Despite the advent of “methylomic” analyses, bisulfite modification of genomic DNA followed by Sanger sequencing remains the most precise assay for investigating the methylation profile of specific sequences at single-cytosine resolution. We and others highlighted the possibility that cytosine methylation outside the canonical CpG dinucleotides—indicated as “non-CpG” or “CpH” methylation—can be significantly present, particularly in brain cells and tissues and that these non-CpG methyl-groups also display a functional role in modulating gene expression. The sequencing output obtained after bisulfite assay needs to be compared to the reference sequence to identify the modified cytosines. This task can be performed through software-assisted analysis but, so far, limitedly at the CpG moieties. To address this gap, the MethPy software has been developed to analyze non-CpG methylation profiles in an automated manner. MethPy was programmed in Python and enables the comparison of bisulfite-modified sequences with their corresponding genomic ones. The results are immediately showed with the option to show multiple output formats (text, tables, and graphs). To our knowledge, this is the first tool that allows the analysis of non-CpG methylation at single-base resolution and may contribute to streamlining and accelerating the analysis of Sanger sequencing of bisulfite PCR products.