Nanopore Sequencing Accurately Identifies the Mutagenic DNA Lesion O ⁶ ‐Carboxymethyl Guanine and Reveals Its Behavior in Replication

O-6-carboxymethylguanine (O-6-CMG) is a highly mutagenic alkylation product of DNA, triggering transition mutations relevant to gastrointestinal cancer. However, precise localization of a single O-6-CMG with conventional sequencing platforms is challenging. Here nanopore sequencing (NPS), which directly senses single DNA bases according to their physiochemical properties, was employed to detect O-6-CMG. A unique O-6-CMG signal was observed during NPS and a single-event call accuracy of >95% was achieved. Moreover, O-6-CMG was found to be a replication obstacle for Phi29 DNA polymerase (Phi29 DNAP), suggesting this lesion could cause DNA sequencing biases in next generation sequencing (NGS) approaches.