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

Screening for gene doping transgenes in horses via the use of massively parallel sequencing / Jillian Maniego, Bogusia Pesko, Jocelyn Habershon-Butcher, Jim Huggett, Polly Taylor, James Scarth, Edward Ryder. - (Gene Therapy (2021) 19 July)

  • PMID: 34276046
  • DOI: 10.1038/s41434-021-00279-1


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.


19 July 2021
Habershon-Butcher, Jocelyn
Huggett, Jim
Maniego, Jillian
Pesko, Bogusia
Ryder, Edward
Scarth, James
Taylor, Polly
United Kingdom
Equestrian (FEI) - International Equestrian Federation
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British Horseracing Authority (BHA)
University of Surrey
Analytical aspects
Animal doping
Testing method development
Doping classes
M3. Gene And Cell Doping
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Gene Therapy
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Date generated
31 August 2021
Date of last modification
1 September 2021
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