Screening for gene doping transgenes in horses via the use of massively parallel sequencing.
Journal
Gene therapy
ISSN: 1476-5462
Titre abrégé: Gene Ther
Pays: England
ID NLM: 9421525
Informations de publication
Date de publication:
05 2022
05 2022
Historique:
received:
29
12
2020
accepted:
06
07
2021
revised:
02
07
2021
pubmed:
20
7
2021
medline:
7
6
2022
entrez:
19
7
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 34276046
doi: 10.1038/s41434-021-00279-1
pii: 10.1038/s41434-021-00279-1
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
236-246Informations de copyright
© 2021. The Author(s), under exclusive licence to Springer Nature Limited.
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