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

Gene Therapy volume 29, pages 236–246 (2022)Cite this article

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Abstract

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.

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Fig. 1: Two-step PCR generation of sequencing-ready amplicons.
Fig. 2: Spiking of EPO transgene templates at different copy numbers within primer and reference gene block pools.
Fig. 3: Sequence motif hits for the EPO transgene (orange) and altered reference blocks (blue) for primer pool sets 1 and 2.
Fig. 4: VEGFA transgene spiking of plasma and urine samples, comparing qPCR Ct values from varying initial spiking quantities and MPS motif read counts (primer set 1).
Fig. 5: Detection of target rAAV6-GFP features from the two horses used in the administration study over time, comparing qPCR and parallel sequencing results.

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Code availability

Please contact the corresponding author if you wish to access the code used for the design and analysis scripts. Availability is subject to approval from the British Horseracing Authority.

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Acknowledgements

The British Horseracing Authority (BHA) for funding the analytical work carried out for this study and conducting the animal administrations of the rAAV vector. Staff and students at the Centre for Racehorse Studies (CRS), UK, are also acknowledged for their care and sampling of the horses involved in this work.

Funding

This work was funded by the British Horseracing Authority (BHA).

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Authors and Affiliations

  1. Sport and Specialised Analytical Services, LGC, Fordham, Cambridgeshire, UK

    Jillian Maniego, Bogusia Pesko, Polly Taylor, James Scarth & Edward Ryder

  2. British Horseracing Authority, London, UK

    Jocelyn Habershon-Butcher

  3. National Measurement Laboratory, LGC, Teddington, Middlesex, UK

    Jim Huggett

  4. School of Biosciences & Medicine, Faculty of Health & Medical Sciences, University of Surrey, Guildford, Surrey, UK

    Jim Huggett

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  1. Jillian Maniego
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Correspondence to Edward Ryder.

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Maniego, J., Pesko, B., Habershon-Butcher, J. et al. Screening for gene doping transgenes in horses via the use of massively parallel sequencing. Gene Ther 29, 236–246 (2022). https://doi.org/10.1038/s41434-021-00279-1

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