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Whole genome mutagenicity evaluation using Hawk-Seq™ demonstrates high inter-laboratory reproducibility and concordance with the transgenic rodent gene mutation assay
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Whole genome mutagenicity evaluation using Hawk-Seq™ demonstrates high inter-laboratory reproducibility and concordance with the transgenic rodent gene mutation assay
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Journal Article
Whole genome mutagenicity evaluation using Hawk-Seq™ demonstrates high inter-laboratory reproducibility and concordance with the transgenic rodent gene mutation assay
2025
Overview
Background
Error-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.
Results
The 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
2
) of the six types of BS frequencies exceeded 0.97 among the three laboratories for BP- or ENU-exposed samples. Thus, Hawk-Seq™ provides qualitatively and quantitatively reproducible results across laboratories. The OMFs in the Hawk-Seq™ analysis positively correlated (r
2
= 0.64) with
gpt
mutant frequencies (MFs). The fold induction of OMFs in the Hawk-Seq™ analysis of ENU- and MNU-exposed samples was at least 14.2 and 4.5, respectively, compared to 6.1 and 2.5 for
gpt
MFs. Meanwhile, the fold induction of OMFs in BP-exposed samples was ≤ 4.6, compared to 8.2 for
gpt
MFs. These observations suggest that Hawk-Seq™ demonstrates good concordance with the transgenic rodent (TGR) gene mutation assay, whereas the induction of mutation frequency by each mutagen might not directly correspond.
Conclusions
Hawk-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.
