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

Abstract

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.

Frequencies and spectra of aflatoxin B1-induced mutations in liver genomes of NEIL1-deficient mice as revealed by duplex sequencing

“…Comparison of our results with the published studies in which the same TwinStrand mouse mutagenesis panel was used, revealed both similarities and differences. Analogous to results of the current investigation, targets on chr13 and chr19 accumulated relatively low levels of mutations in bone marrow of MutaMouse following exposure to either benzo(a)pyrene ( 49 ) or procarbazine hydrochloride ( 56 ). The intergenic target on chr14 was heavily loaded with mutations in all three cases.…”

Frequency and spectrum of mutations in human sperm measured using duplex sequencing correlate with trio-basedde novomutation analyses

“…In this analysis, we report unique mutations only; mutations appearing more than once in the same sample were assumed to have arisen from clonal expansion and were thus counted as one mutation, which may guard against over-estimating the MF. 49 Of note, 96% of all SNV mutations (with a VAF < 1%) and 70% of non-SNV mutations were single count (non-clonal) mutations, respectively.…”

Dose-related Mutagenic and Clastogenic Effects of Benzo[b]fluoranthene in Mouse Somatic Tissues Detected by Duplex Sequencing and the Micronucleus Assay

“…Under this assumption, all identical mutations within a sample were counted as one single mutation to calculate the minimum mutation frequency (MFmin). We also counted all mutations to obtain the maximum mutation frequency (MFmax) (25). The variant allele fraction was set to 0.01 to exclude mosaicism and germline mutations.…”

“…Modern next-generation sequencing (NGS) technologies paired with error-correcting processes provide opportunities to comprehensively characterize mutagenesis (22). Duplex Sequencing (DS) is a targeted error-corrected NGS technology that allows the detection of rare mutations (23,24,25,26). It reduces the technical error rate of conventional NGS by uniquely tagging both strands of the original DNA fragments during library building, enabling the development of consensus sequences for each strand to eliminate PCR and sequencing errors (22,27,28).…”