Differential adenine methylation analysis reveals increased variability in 6mA in the absence of methyl-directed mismatch repair

Methylated adenines (6mA) are an important epigenetic modification in bacteria, but challenges in accurate and high-throughput detection of 6mA have previously limited the ability to conduct comparative studies. Nanopore sequencing has allowed for accurate and cost-effective characterization of a variety of DNA modifications; however, most bioinformatics tools for Nanopore data are designed for CpG methylation, and there are currently no tools designed for comparative 6mA methylomics studies. In this work we present CoMMA, a pipeline for analyzing differential methylation of 6mA modification at a GATC motif. We applied the CoMMA pipeline to Escherichia coli clones that were experimentally evolved for 2400 generations to show how bioinformatic 6mA characterization is complementary to genetic sequencing and informative on its own. We show that, in the absence of methyl-directed mismatch repair, E. coli GATC sites are overall hypomethylated and display increased methylation variability, indicative of reduced selection and increased drift. The CoMMA pipeline is a reliable tool to be used with the growing number of Nanopore methylation callers. TEASER A novel bacterial differential methylation pipeline reveals that methyl-directed mismatch repair in Escherichia coli maintains 6mA genome-wide.