Identification of Methylation sites on pUC19 using Oxford Nanopore Sequencing

Nanopore sequencing allows direct sequencing of single-stranded DNA molecules in real time. To explore whether Nanopore sequencing can discriminate methylated bases, we sequenced two plasmids with and without methylation at the dam and dcm methylation sites (GmeATC and CmeCWGG motifs respectively). By analyzing the resistance of 5-mers occupying a pore we observed significant differences between methylated and unmethylated sequences at the sites of both types of methylation, presumably due to the difference in the obstruction of current flow across the pore by the methyl groups. This finding indicates that Nanopore sequencing has the potential to robustly detect methylated bases, and that the current basecalling model would be improved by incorporating models of modified bases. Additionally, we observed a large fraction of 5-mers that significantly deviate from the default METRICHOR basecalling models, indicating that re-calibration is necessary to correct systematic biases in those models and improve overall sequencing accuracy.