Whole genome mutagenicity evaluation using Hawk-Seq™ demonstrates high inter-laboratory reproducibility and concordance with the transgenic rodent gene mutation assay.

backgroundError-corrected next-generation sequencing (ecNGS) enables the sensitive detection of chemically induced mutations. Matsumura et al. reported Hawk-Seq™, an ecNGS method, demonstrating its utility in clarifying mutagenicity both qualitatively and quantitatively. To further promote the adoption of ecNGS-based assays, it is important to evaluate their inter-laboratory transferability and reproducibility. Therefore, we evaluated the inter-laboratory reproducibility of Hawk-Seq™ and its concordance with the transgenic rodent mutation (TGR) assay.

resultsThe Hawk-Seq™ protocol was successfully transferred from the developer's laboratory (lab A) to two additional laboratories (labs B, C). Whole genomic mutations were analyzed independently using the same genomic DNA samples from the livers of gpt delta mice exposed to benzo[a]pyrene (BP), N-ethyl-N-nitrosourea (ENU), and N-methyl-N-nitrosourea (MNU). In all laboratories, clear dose-dependent increases in base substitution (BS) frequencies were observed, specific to each mutagen (e.g. G:C to T:A for BP). Statistically significant increases in overall mutation frequencies (OMFs) were identified at the same doses across all laboratories, suggesting high reproducibility in mutagenicity assessment. The correlation coefficient (r

conclusionsHawk-Seq™-based whole-genome mutagenicity evaluation demonstrated high inter-laboratory reproducibility and concordance with gpt assay results. Our results contribute to the growing evidence that ecNGS assays provide a suitable, or improved, alternative to the TGR assay.