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. Whereas gene transfer-based doping affects only the individual animal, alteration of the genome at the embryonic (or earlier) stage could result in any event being inherited by subsequent generations. In Thoroughbreds or other closed studbooks, this presents a clear threat to the integrity of the breed.
The nature of these alterations includes genomic integration of transgenes, small sequence changes, or larger structural variants, none of which will be known in advance. It is therefore imperative that any detection system is robust in its detection capability. Determining natural sequence variation from genuine gene doping events offers a significant challenge, and thus a thorough assessment of genetic variation in the thoroughbred population is paramount.
To this end, a targeted enrichment panel and next-generation sequencing was evaluated to assess its reproducibility, sensitivity and capability of variant detection on a wide variety of samples and biological matrices. Different analysis parameters and workflows were also investigated. The panel was verified against existing whole genome data, SNP data, and long read sequencing of the myostatin gene.
Validation of panels for testing will require suitable reference material, and experimental design / metadata collection which follows established methods and procedures. Subsequent confirmatory testing will also require bespoke solutions depending on the nature of the variants detected. The challenges these issues pose, and the approaches taken in our laboratory for panel validation are discussed, including a mock-confirmatory assay design for a novel structural variant discovered during population assessment.