Hawk-Seq Shows High Reproducibility in Rodent Mutagenicity Testing

Researchers have demonstrated that Hawk-Seq, an error-corrected next-generation sequencing method, provides highly reproducible results for whole-genome mutagenicity evaluation across different laboratories. Published in the journal Genes and Environment, the study confirms that this sequencing approach yields consistent data when transferred between independent facilities. The findings suggest that Hawk-Seq can serve as a reliable alternative to traditional transgenic rodent gene mutation assays, offering a robust tool for assessing the mutagenic potential of chemical substances in genotoxicity research.

The study was conducted as a collaborative effort within the Japanese Environmental Mutagen and Genome Society Mammalian Mutagenicity Study Group. The primary objective was to evaluate the inter-laboratory transferability and reproducibility of the Hawk-Seq protocol. To achieve this, the developers of the method, located at Kao, transferred their experimental protocols to two additional laboratories: Teijin Pharma Limited and Eisai Co., Ltd. This multi-site design allowed the researchers to test whether the sequencing method could produce consistent mutation frequency data when performed by different teams using the same biological samples.

The experimental design utilized genomic DNA samples previously obtained from the livers of male inbred C57BL/6JJmsSlc-Tg (gpt delta) mice. These mice had been exposed to three distinct chemical mutagens: benzo[a]pyrene, N-ethyl-N-nitrosourea, and N-methyl-N-nitrosourea. The DNA samples were shipped to the three participating laboratories, where each team independently prepared sequencing libraries and analyzed the induced mutations. By using identical DNA samples, the researchers ensured that any differences in results could be attributed to the laboratory procedures rather than biological variability in the source material.

Hawk-Seq is an error-corrected next-generation sequencing technology that utilizes complementary strand information to significantly reduce sequencing errors. This capability enables the direct detection of rare mutations induced by mutagens in genomic DNA. In this study, the laboratories used the NovaSeq 6000 platform to sequence the prepared libraries, generating at least 50 million paired-end reads per sample. The data processing involved mapping the reads to the mouse reference genome and generating double-stranded DNA consensus sequences to accurately identify true mutations while filtering out background noise.

The results indicated that the Hawk-Seq protocol was successfully transferred to the two additional laboratories. In all three facilities, clear dose-dependent increases in base substitution frequencies were observed for each mutagen. For example, exposure to benzo[a]pyrene led to a specific increase in G:C to T:A mutations, a profile consistent with the known mutagenic mechanism of this compound. Statistically significant increases in overall mutation frequencies were identified at the same dose levels across all laboratories, demonstrating that the method can reliably detect mutagenic effects regardless of the laboratory environment.

Quantitative analysis revealed a high degree of correlation in mutation frequencies between the laboratories. The correlation coefficient for the six types of base substitution frequencies exceeded 0.97 among the three labs for samples exposed to benzo[a]pyrene or N-ethyl-N-nitrosourea. This strong correlation suggests that the between-laboratory reproducibility of Hawk-Seq is substantially high. Furthermore, the coefficient of variation for overall mutation frequencies between laboratories was found to be smaller than the variation observed between individual animals within the same dose group, indicating that the method minimizes technical variability.

The study also assessed the concordance between Hawk-Seq results and the traditional transgenic rodent gene mutation assay, often referred to as the gpt assay. The overall mutation frequencies obtained from Hawk-Seq analysis positively correlated with the gpt mutant frequencies. However, the magnitude of the response varied depending on the specific mutagen. For N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea, Hawk-Seq showed larger fold-inductions in mutation frequency compared to the gpt assay. In contrast, for benzo[a]pyrene, the gpt assay indicated a higher fold-induction than Hawk-Seq.

These discrepancies in sensitivity between the two methods highlight that the induction of mutation frequency by each mutagen does not directly correspond between the assays. The authors note that this may be a specific observation related to benzo[a]pyrene, as previous studies have suggested that exogenous bacterial genes, such as the gpt gene in the transgenic model, may be preferential targets for this type of mutagenesis. Additionally, the G:C content and sequence context of the genomic locus can influence whether a mutagen produces a stronger or weaker response in a representative genomic region compared to the whole genome.

Despite these differences in magnitude, the study concludes that Hawk-Seq provides qualitatively and quantitatively reproducible results across laboratories. The method demonstrated sufficient sensitivity to identify statistically significant increases in mutation frequencies at the same or lower doses compared to the gpt assay. This sensitivity is attributed to the relatively lower inter-animal variations in overall mutation frequencies obtained using Hawk-Seq. The ability to detect these changes with high precision supports the utility of Hawk-Seq as a sensitive mutagenicity test.

The findings contribute to the growing body of evidence supporting the use of error-corrected next-generation sequencing assays in genotoxicity research. By providing mutation data with substantial reproducibility and concordance to traditional transgenic rodent assays, Hawk-Seq offers a viable alternative that also provides additional information on mutagenic mechanisms. The study underscores the importance of standardizing and validating new methodologies to promote their adoption in the field of chemical genotoxicity.

The successful transfer of the Hawk-Seq protocol highlights the potential for broader implementation of error-corrected sequencing technologies in regulatory and research settings. The optimization of library preparation steps, such as the amount of DNA subjected to PCR enrichment, was crucial for achieving high efficiency in generating consensus reads.

Looking forward, the authors suggest that the widespread adoption of error-corrected next-generation sequencing methods could enhance the evaluation of mutagens in genotoxicity research. The accumulation of data from these methods will help refine the understanding of the relationship between mutagenicity and carcinogenicity. Further discussion within the expert community is needed to determine how to interpret specific findings, such as increases in particular mutation subtypes or in specific genomic regions. The study provides a strong foundation for these future discussions by establishing the reproducibility and concordance of Hawk-Seq with existing standards.

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

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

Article References: Matsumura, S., Hosoi, S., Hirose, T., Otsubo, Y., Saito, K., Miyazawa, M., Kawade, A., Hakura, A., Kakiuchi, D., Asakura, S., Koyama, N., Okada, Y., Chikura, S., Kimoto, T., Masumura, K., Suzuki, T., & Sugiyama, K.-I. (2025). Whole genome mutagenicity evaluation using Hawk-Seq™ demonstrates high inter-laboratory reproducibility and concordance with the transgenic rodent gene mutation assay. Genes and Environment, 47(1), Article 13. https://doi.org/10.1186/s41021-025-00336-w

Image Credits: AI Generated

DOI: 10.1186/s41021-025-00336-w

Keywords: Whole, genome, mutagenicity, evaluation, Hawk-Seq, demonstrates, high, inter-laboratory, reproducibility, concordance, transgenic, rodent