MethPy is a new software for the analysis of the sequence files generated by Sanger sequencing in PCR-amplified bisulphite-modified DNA samples. The sequencing output obtained after bisulphite 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. The MethPy software has been developed to analyze non-CpG methylation profiles in an automated manner. The unicity of MethPy is that it allows to automatically detect and mark methylated cytosines outside the CpG dinucleotides, i.e., the cytosines in the so called “non-CpG”, or “CpH” context (CpA, CpC, CpT).
Since non-CpG methylation has been largely ignored or underestimated, considering these moieties always non-methylated and therefore modified, softwares able to mark methylated non-CpGs were never developed. So far, the reconnaissance of these cytosines was “manually” performed by the researcher. MethPy covers this gap, in a moment in which the relevant and functional presence of non-CpG methylation is increasingly acknowledged.
MethPy was programmed in Python and enables the comparison of bisulphite-modified sequences with their corresponding reference sequences. The results are immediately showed with the option to show multiple output formats (text, tables, and graphs).
MethPy is free of use, but the source must be cited:
https://dmsp.web.uniroma1.it/it/methpy
Roiati M, Diniz Ferreira Borges L, Cattani A, Lucarelli M and Fuso A. MethPy: a new software for analyzing non-CpG methylation after bisulphite assay and Sanger sequencing”. 2025, in press.
When using MethPy, please cite the application and the related paper as follow:
"Roiati M, Borges LDF, Cattani A, Lucarelli M, Fuso A. MethPy: a new software for analyzing non-CpG methylation after bisulfite assay and Sanger sequencing. Sci Rep. 2025 Nov 26;15(1):42068. doi: 10.1038/s41598-025-26089-8"
