Development of an error-corrected sequencing methodology and its application to chemical-genotoxicity evaluation

Abstract

Genotoxicity has been evaluated via tests utilizing indicator genes, such as the Ames test. However, these methods do not provide sufficient qualitative and quantitative mutation data for each mutagen. The newly developed error-corrected sequencings (ECSs) enable direct detection of chemically-induced mutations by reducing the genome-sequencing error rate to ca. 1/10⁷ bp. ECSs can improve our understanding of chemical mutagenicity by generating high-resolution mutation data. We had previously developed an original ECS method (Hawk-Seq^TM) and demonstrated its efficiency in detecting mutations. Hawk-Seq^TM could detect mutations caused by various mutagens in diverse genotoxicity test models (e.g., S. typhimurium, TG mice, and cultured human cells). The mutation spectra generated by Hawk-Seq^TM reflected the structural features of each mutagen, leading to a systematic understanding of mutagenicity. Moreover, we identified that the errors derived from single DNA strand overhangs were not eliminated from the Hawk-Seq^TM data. Therefore, we developed a new method, Jade-Seq^TM, which provided a 10-fold reduction in error frequency compared to Hawk-Seq^TM by utilizing a single-strand specific nuclease. This method will help elucidate rare mutations or improve mutation analysis robustness. Finally, we started a collaborative study to evaluate the sensitivity and reproducibility of Hawk-Seq^TM in the Mammalian Mutagenicity Study Group in the Japanese Environmental Mutagen and Genome Society. Our research will promote the application of ECS to the genotoxicity evaluation of chemicals.