Estimating somatic mutation rates by bottlenecked duplex sequencing in non-model organisms: Daphnia magna as a case study.

Somatic mutations are evolutionarily important as determinants of individual organismal fitness, as well as being a focus of clinical research on age-related disease, such as cancer. Identifying somatic mutations and quantifying mutation rates, however, is extremely challenging and genome-wide somatic mutation rates have only been reported for a few model organisms. Here, we describe the application of Duplex Sequencing on bottlenecked WGS libraries to quantify somatic nuclear genome-wide base substitution rates in . , historically an ecological model system, has more recently been the focus of mutation studies, in part because of its high germline mutation rates. Using our protocol and pipeline, we estimate a somatic mutation rate of 5.6 × 10 substitutions per site (in a genotype where the germline rate is 3.60 × 10 substitutions per site per generation). To obtain this estimate, we tested multiple dilution levels to maximize sequencing efficiency and developed bioinformatic filters needed to minimize false positives when a high-quality reference genome is not available. In addition to laying the groundwork for estimating genotypic variation in rates of somatic mutations within , we provide a framework for quantifying somatic mutations in other non-model systems, and also highlight recent innovations to single molecule sequencing that will help to further refine such estimates.