Evaluation of a targeted enrichment panel for gene editing detection and assessment of population variation in...

Gene editing and genome manipulation offer great promise for treating diseases in both humans and animals. There is a danger, however, that this technology could be used for other purposes such as performance enhancement. To detect such 'gene doping' events, we evaluated a targeted enrichment panel and next-generation sequencing to assess its reproducibility, sensitivity, and capability of variant detection on a wide variety of samples and biological matrices. The panel was verified against existing data for the myostatin gene, a PCR-based SNP panel, and whole genome sequencing in a subset of samples. As successful detection of seamless edits will rely on a detailed understanding of the natural population, we also screened over 170 Thoroughbreds and catalogued numerous novel variants. These included several resulting in coding alterations, and a structural variant. Samples spiked with transgenic cDNA-based material to simulate gene doping events were detected down to 3.2% mosaicism, giving confidence that mosaic mutations resulting from embryonic introduction of gene editing reagents can be detected using these methods. The ability of software packages to detect gene doping events was also assessed, including multiple genome alignment tools, variant callers, and structural variant callers. Freebayes performed strongest at SNP-based editing detection, and Delly and Manta had complementary advantages depending on the mutation type. For routine testing, a multi-faceted approach to calling should be taken to maximise the detection capabilities.