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Gene therapy2021; 29(5); 236-246; doi: 10.1038/s41434-021-00279-1

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

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.
© 2021. The Author(s), under exclusive licence to Springer Nature Limited.
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Publication Date: 2021-07-19 PubMed ID: 34276046PubMed Central: 190884DOI: 10.1038/s41434-021-00279-1Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study explores a method of identifying gene doping in horses, using modern sequencing technology. It uses a two-step PCR method in parallel sequencing to detect multiple doping targets simultaneously, and tests its sensitivity through real-world applications.

Methodology

The researchers developed a screening method using a two-step Polymerase Chain Reaction (PCR) procedure. This is a DNA amplification technique that reproduces specific DNA sequences from the sample. Their method involves massively parallel sequencing, which allows them to screen for multiple doping targets at once. They pooled primer sets, sequences that start DNA replication, to provide scalability, using combinational indexing.

Quality Control and Data Processing

Reference standards with modified sequence were used as controls to ensure the accuracy and reliability of the experiment. They also employed custom software to generate transgene-specific amplicons, fragments of DNA or RNA that are produced by amplification, from any genome annotated in Ensembl, a database that provides DNA sequences and related information. This custom software allows for quick assay design. Additional scripts were used to process the data obtained from the experiments in batches. These scripts automatically filter sequences based on their quality and assign hits based on recognisable patterns.

Testing and Evaluation

To test this approach, they established a workflow with an initial set of 31 transgene and vector feature targets. A real-world setting was then simulated to evaluate the sensitivity of the parallel sequencing method. They administered an rAAV vector, a common gene therapy vector, intramuscularly into two horses and measured the detection sensitivity between this new method and real-time quantitative PCR (qPCR), a technique used to monitor the progress of the PCR reaction in real time. The vector was detected using both methods up to 79 hours post-administration, proving the potential of their new method for gene-doping detection.

Results

The results showed that the new method was effective in detecting the administered vector. However, detection became less frequent after 96 hours. This indicates that while the massively parallel sequencing PCR method can detect doping, it may be less effective for longer periods after administration. This should be considered when using this method for detection. However, it is a step forward in identifying gene doping in horses, providing a scalable and efficient means of testing.

Cite This Article

APA
Maniego J, Pesko B, Habershon-Butcher J, Huggett J, Taylor P, Scarth J, Ryder E. (2021). Screening for gene doping transgenes in horses via the use of massively parallel sequencing. Gene Ther, 29(5), 236-246. https://doi.org/10.1038/s41434-021-00279-1

Publication

Gene therapy
ISSN: 1476-5462
NlmUniqueID: 9421525
Country: England
Language: English
Volume: 29
Issue: 5
Pages: 236-246

Researcher Affiliations

Maniego, Jillian
  • Sport and Specialised Analytical Services, LGC, Fordham, Cambridgeshire, UK.
Pesko, Bogusia
  • Sport and Specialised Analytical Services, LGC, Fordham, Cambridgeshire, UK.
Habershon-Butcher, Jocelyn
  • British Horseracing Authority, London, UK.
Huggett, Jim
  • National Measurement Laboratory, LGC, Teddington, Middlesex, UK.
  • School of Biosciences & Medicine, Faculty of Health & Medical Sciences, University of Surrey, Guildford, Surrey, UK.
Taylor, Polly
  • Sport and Specialised Analytical Services, LGC, Fordham, Cambridgeshire, UK.
Scarth, James
  • Sport and Specialised Analytical Services, LGC, Fordham, Cambridgeshire, UK.
Ryder, Edward
  • Sport and Specialised Analytical Services, LGC, Fordham, Cambridgeshire, UK. edward.ryder@lgcgroup.com.

MeSH Terms

  • Animals
  • Doping in Sports / methods
  • Genetic Therapy
  • High-Throughput Nucleotide Sequencing
  • Horses
  • Real-Time Polymerase Chain Reaction / methods
  • Transgenes

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This article has been cited 6 times.
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