Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Oct;65(8):234-242.
doi: 10.1002/em.22619. Epub 2024 Sep 12.

Power analyses to inform Duplex Sequencing study designs for MutaMouse liver and bone marrow

Elena Esina  1 , Annette E Dodge  2 , Andrew Williams  2 , David M Schuster  1 , Danielle P M LeBlanc  2 , Francesco Marchetti  2   3 , Carole L Yauk  1
Affiliations

Power analyses to inform Duplex Sequencing study designs for MutaMouse liver and bone marrow

Elena Esina et al. Environ Mol Mutagen. 2024 Oct.

Abstract

Regulatory genetic toxicology testing is essential for identifying potentially mutagenic hazards. Duplex Sequencing (DS) is an error-corrected next-generation sequencing technology that provides substantial advantages for mutation analysis over conventional mutagenicity assays including: improved accuracy of mutation detection, ability to measure changes in mutation spectrum, and applicability across diverse biological models. To apply DS for regulatory toxicology testing, power analyses are required to determine suitable sample sizes and study designs. In this study, we explored study designs to achieve sufficient power for various effect sizes in chemical mutagenicity assessment. We collected data from MutaMouse bone marrow and liver samples that were analyzed by DS using TwinStrand's Mouse Mutagenesis Panel. Average duplex reads achieved in two separates studies on liver and bone marrow were 8.4 × 108 (± 7.4 × 107) and 9.5 × 108 (± 1.0 × 108), respectively. Baseline mean mutation frequencies (MF) were 4.6 × 10-8 (± 6.7 × 10-9) and 4.6 × 10-8 (± 1.1 × 10-8), with estimated standard deviations for the animal-to-animal random effect of 0.15 and 0.20, for liver and bone marrow, respectively. We conducted simulation analyses based on these empirically derived parameters. We found that a sample size of four animals per group is sufficient to obtain over 80% power to detect a two-fold change in MF relative to baseline. In addition, we estimated the minimal total number of informative duplex bases sequenced with different sample sizes required to retain power for various effect sizes. Our work provides foundational data for establishing suitable study designs for mutagenicity testing using DS.

Keywords: duplex sequencing; error‐corrected sequencing; mutation frequency; power analysis; study design.

PubMed Disclaimer

References

REFERENCES

    1. Beal, M.A., Meier, M.J., LeBlanc, D.P., Maurice, C., O'Brien, J.M., Yauk, C.L. et al. (2020) Chemically induced mutations in a MutaMouse reporter gene inform mechanisms underlying human cancer mutational signatures. Communications Biology, 3(1), 1–11. Available from: https://doi.org/10.1038/s42003-020-01174-y
    1. Bercu, J.P., Zhang, S., Sobol, Z., Escobar, P.A., Van, P. & Schuler, M. (2023) Comparison of the transgenic rodent mutation assay, error corrected next generation duplex sequencing, and the alkaline comet assay to detect dose‐related mutations following exposure to N‐nitrosodiethylamine. Mutation Research, Genetic Toxicology and Environmental Mutagenesis, 891, 503685. Available from: https://doi.org/10.1016/j.mrgentox.2023.503685
    1. Cho, E., Swartz, C.D., Williams, A., Rivas, M., Recio, L., Witt, K.L. et al. (2023) Error‐corrected duplex sequencing enables direct detection and quantification of mutations in human TK6 cells with remarkable inter‐laboratory consistency. bioRxiv, p. 529418. Available from: https://doi.org/10.1101/2023.02.22.529418
    1. Dertinger, S.D., Li, D., Beevers, C., Douglas, G.R., Heflich, R.H., Lovell, D.P. et al. (2023) Assessing the quality and making appropriate use of historical negative control data: a report of the international workshop on Genotoxicity testing (IWGT). Environmental and Molecular Mutagenesis. Available from: https://doi.org/10.1002/em.22541
    1. Dodge, A.E., LeBlanc, D.P.M., Zhou, G., Williams, A., Meier, M.J., Van, P. et al. (2023) Duplex sequencing provides detailed characterization of mutation frequencies and spectra in the bone marrow of MutaMouse males exposed to procarbazine hydrochloride. Archives of Toxicology, 97(8), 2245–2259. Available from: https://doi.org/10.1007/s00204-023-03527-y

LinkOut - more resources