Nanopore Sequencing Identification of DNA Aristolochic Acid Adducts

Abstract Aristolochic acid (AA) is a Group I carcinogen that forms covalent DNA adducts following metabolic activation, inducing characteristic mutational signatures and driving carcinogenesis. Liquid chromatography–mass spectrometry (LC–MS/MS) provides highly sensitive and specific detection of AA-derived DNA adducts, but commonly relies on enzymatically digested DNA, which does not retain positional and sequence-context information along individual DNA molecules. Here, we explored nanopore sequencing as a novel strategy for characterizing aristolactam II–deoxyadenosine DNA adducts (AL-II-dA) in a defined synthetic DNA context via the detection of intrinsic signal deviations. Using chemically synthesized AL-II-dA incorporated at defined sites, we show that the adduct induces consistent and localized alterations in normalized signal levels and dwell time across two independent sequencing platforms. These deviations were accompanied by changes in base-calling outcomes, including elevated error-specific base (ESB) values, increased odds ratios, and reduced base-calling quality scores (Q-scores), collectively forming a multidimensional signature associated with adduct presence. Building on these observations, we further explored a signal-based analytical approach to identify characteristic signal changes associated with the adduct directly from normalized signal levels. Our results show that nanopore sequencing can detect and localize a site-specifically incorporated AL-II-dA adduct in a defined sequence context through characteristic deviations in normalized signal levels, dwell time, and base-calling features. This study supports the potential of nanopore sequencing as a new approach for investigating DNA-adduct-associated signal characteristics.