A next-generation sequencing method for gene doping detection that distinguishes low levels of plasmid DNA against a background of genomic DNA

• Published in: Gene therapy Vol. 26; no. 7-8; pp. 338 - 346
• Main Authors: de Boer, Eddy N., van der Wouden, Petra E., Johansson, Lennart F., van Diemen, Cleo C., Haisma, Hidde J.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-08-2019; Nature Publishing Group
• Subjects: 42; 42/44; 45; 45/23; 631/1647/1513; 631/1647/2300/1514; 631/61/2300/1514; Biomedical and Life Sciences; Biomedicine; Cell Biology; Deoxyribonucleic acid; DNA; Doping in Sports - methods; Erythropoietin - genetics; Erythropoietin - metabolism; Exons; Gene Expression; Gene Therapy; Genes; Genetic engineering; Genetic Testing - methods; Genetic Testing - standards; Genome, Human; Genomics; High-Throughput Nucleotide Sequencing - methods; High-Throughput Nucleotide Sequencing - standards; Human Genetics; Humans; Insulin-like growth factor I; Insulin-like growth factor II; Intercellular Signaling Peptides and Proteins - genetics; Intercellular Signaling Peptides and Proteins - metabolism; Nanotechnology; Next-generation sequencing; Nucleotide sequence; Plasmids - genetics; Plasmids - metabolism; Polymerase chain reaction; Reproducibility of Results; Sensitivity and Specificity; Sequence Analysis, DNA - methods; Sequence Analysis, DNA - standards; Transgenes
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/s41434-019-0091-6

Gene doping confers health risks for athletes and is a threat to fair competition in sports. Therefore the anti-doping community has given attention on its detection. Previously published polymerase chain reaction-based methodologies for gene doping detection are targeting exon–exon junctions in the intron-less transgene. However, because these junctions are known, it would be relatively easy to evade detection by tampering with the copyDNA sequences. We have developed a targeted next-generation sequencing based assay for the detection of all exon–exon junctions of the potential doping genes,
EPO
,
IGF1
,
IGF2
,
GH1,
and
GH2
, which is resistant to tampering. Using this assay, all exon–exon junctions of copyDNA of doping genes could be detected with a sensitivity of 1296 copyDNA copies in 1000 ng of genomic DNA. In addition, promotor regions and plasmid-derived sequences are readily detectable in our sequence data. While we show the reliability of our method for a selection of genes, expanding the panel to detect other genes would be straightforward. As we were able to detect plasmid-derived sequences, we expect that genes with manipulated junctions, promotor regions, and plasmid or virus-derived sequences will also be readily detected.