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

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.

Duplex sequencing provides detailed characterization of mutation frequencies and spectra in the bone marrow of MutaMouse males exposed to procarbazine hydrochloride.

Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with > 80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.

Duplex sequencing identifies genomic features that determine susceptibility to benzo(a)pyrene-induced in vivo mutations.

Exposure to environmental mutagens increases the risk of cancer and genetic disorders. We used Duplex Sequencing (DS), a high-accuracy error-corrected sequencing technology, to analyze mutation induction across twenty 2.4 kb intergenic and genic targets in the bone marrow of MutaMouse males exposed to benzo(a)pyrene (BaP), a widespread environmental pollutant. DS revealed a linear dose-related induction of mutations across all targets with low intra-group variability. Heterochromatic and intergenic regions exhibited the highest mutation frequencies (MF). C:G > A:T transversions at CCA, CCC and GCC trinucleotides were enriched in BaP-exposed mice consistent with the known etiology of BaP mutagenesis. However, GC-content had no effect on mutation susceptibility. A positive correlation was observed between DS and the "gold-standard" transgenic rodent gene mutation assay. Overall, we demonstrate that DS is a promising approach to study in vivo mutagenesis and yields critical insight into the genomic features governing mutation susceptibility, spectrum, and variability across the genome.