Assessment of the error-corrected sequencing-based genotoxicity evaluation method: JEMS/MMS collaborative study

Abstract

Genotoxicity of chemicals is evaluated using methods such as Ames test that detect mutations in indicator genes. Error-corrected sequencing (ECS) is a newly developed method that uses complementary strand-derived DNA sequences; it can reduce sequencing error frequency to ca. 1/10⁷ bp. ECS can directly detect mutations induced by mutagens; this could help overcome the limitations in genotoxicity evaluation. The Hawk-Seq^TM, an ECS developed by Kao, could sensitively detect the mutations induced by various mutagens conventional test models. Evaluating the utility of ECS-based assays in terms of sensitivity and reproducibility is important for their promotion. Therefore, we initiated a collaborative study in the Mammalian Mutagenicity Study Group in the Japanese Environmental Mutagen and Genome Society to evaluate the ECS methods. We aimed to evaluate the sensitivity and reproducibility of Hawk-Seq^TM in terms of mutation frequency and pattern. We evaluated the effects of the experimental device and sequencers on mutation analysis to set a common protocol. The DNA electrophoresis tools, Agilent 4200TapeStation and 2100Bioanalyzer, were both applicable to a quality control of Hawk-Seq^TM library preparation. The background error (e.g. C>G) frequencies obtained in Hawk-Seq^TM analysis varied up to two-fold between sequencers, while the mutation frequency and pattern induced by mutagens were similar. Therefore, we decided the protocol and initiated technical transfer. We will evaluate inter-laboratory reproducibility using mouse DNA samples, thereby evaluate utility of ECS-based methods.