Nanopore Sequencing Accurately Identifies the Mutagenic DNA Lesion O⁶‐Carboxymethyl Guanine and Reveals Its Behavior in Replication

Abstract

O⁶ -carboxymethylguanine (O⁶ -CMG) is a highly mutagenic alkylation product of DNA, triggering transition mutations relevant to gastrointestinal cancer. However, precise localization of a single O⁶ -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 O⁶ -CMG. A unique O⁶ -CMG signal was observed during NPS and a single-event call accuracy of >95 % was achieved. Moreover, O⁶ -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.