Screening for gene doping transgenes in horses via the use of massively parallel sequencing

Throughout the history of horse racing, doping techniques to suppress or enhance performance have expanded to match the technology available. The next frontier in doping, both in the equine and human sports areas, is predicted to be genetic manipulation; either by prohibited use of genome editing, or gene therapy via transgenes. By using massively-parallel sequencing via a two-step PCR method we can screen for multiple doping targets at once in pooled primer sets. This method has the advantages of high scalability through combinational indexing, and the use of reference standards with altered sequences as controls. Custom software produces transgene-specific amplicons from any Ensembl-annotated genome to facilitate rapid assay design. Additional scripts batch-process FASTQ data from experiments, automatically quality-filtering sequences and assigning hits based on discriminatory motifs. We report here our experiences in establishing the workflow with an initial 31 transgene and vector feature targets. To evaluate the sensitivity of parallel sequencing in a real-world setting, we performed an intramuscular (IM) administration of a control rAAV vector into two horses and compared the detection sensitivity between parallel sequencing and real-time qPCR. Vector was detected by all assays on both methods up to 79 h post-administration, becoming sporadic after 96 h.

Automated summary

Genetic manipulation is predicted to be the next frontier in doping, with massively-parallel sequencing allowing for screening of multiple doping targets simultaneously. The study demonstrated the high scalability and sensitivity of parallel sequencing by comparing its detection sensitivity with real-time qPCR in a real-world setting involving intramuscular administration of a control rAAV vector into two horses. The results showed that both methods detected the vector up to 79 hours post-administration, with sporadic detection after 96 hours.