Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control

Tozaki, Teruaki; Ohnuma, Aoi; Takasu, Masaki; Nakamura, Kotono; Kikuchi, Mio; Ishige, Taichiro; Kakoi, Hironaga; Hirora, Kei‐ichi; Tamura, Norihisa; Kusano, Kanichi; Nagata, Shun‐ichi

doi:10.1038/s41434-020-00185-y

Cited by 17 publications

(24 citation statements)

References 32 publications

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“…The number of acquired reads was 686,558,582, with 652,488,758 passing quality control and being mapped to the reference genome ( Table 3). The available reads were similar to those in our previous study [23]. Within the single horse that was sequenced, 6,064,956 SNPs and 727,565 INDELs were detected ( Table 3).…”

Section: Whole Genome Resequencingsupporting

confidence: 70%

“…). The available reads were similar to those in our previous study [ 23 ]. Within the single horse that was sequenced, 6,064,956 SNPs and 727,565 INDELs were detected ( Table 3 ).…”

Section: Resultsmentioning

confidence: 57%

“…SNPs and INDELs were detected by mapping to the horse reference genome sequence EquCab3.0 (GenBank assembly accession: GCA_002863925.1, 2,506,966,135 bp) using the Reseq analysis pipeline (Amelieff Corp., Tokyo, Japan), which employed the QCleaner software (Amelieff Corp.), Burrows-Wheeler Aligner (BWA, version 0.7.17) [ 14 ], Picard (version 2.13.2; https://sourceforge.net/apps/mediawiki/picard/), GATK (version 4.0.8.1; https://software.broadinstitute.org/gatk/best-practices/), and SnpEff (version v4_0) [ 16 ]. Parameters for all analyses are described in our previous study [ 23 ].…”

Section: Methodsmentioning

confidence: 99%

“…broadinstitute.org/gatk/best-practices/), and SnpEff (version v4_0) [16]. Parameters for all analyses are described in our previous study [23].…”

Section: Whole Genome Resequencingmentioning

confidence: 99%

“…For detection of inserted transgenes, primers are designed to anneal to different exons, and a hydrolysis probe is designed on their exon/exon junction. Recently, a less specific genome-wide detection method using whole genome resequencing (WGR) was developed to detect genome editing and inserted transgenes through the identification of DNA variation, including single nucleotide polymorphisms (SNPs), insertions/deletions (INDELs), and structural variants (SVs) [ 23 ].…”

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confidence: 99%

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Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports

Tozaki

Ohnuma

Kikuchi

et al. 2020

JES

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Gene doping is prohibited in horseracing and equestrian sports. In previous studies, we developed non-targeted transgene and genome editing detection methods based on whole genome resequencing (WGR) using genomic DNA extracted from whole blood. In this study, we aimed to develop a WGR method using DNA extracts from hair roots. Hair roots are a preferred substrate because their collection is less invasive than blood collection. Hair is also easier to store for long periods of time. Although almost all genomic DNA extracted from hair root samples stored for years at room temperature was degraded, the quality of genomic DNA from samples stored for years at refrigerated temperatures (4–8°C) was maintained. High-molecular-weight genomic DNA was isolated from hair roots using a magnetic silica beads method of extraction, enabling WGR from horsehair root extracts. Nucleotide sequencing results and numbers of single-nucleotide polymorphisms and insertions/deletions concurred with those previously reported for WGR of DNA extracted from whole blood. Therefore, we consider that storing hair samples at refrigerated temperatures prevents degradation of DNA, allowing the detection of gene doping in these samples based on WGR. It is likely this finding will also have a deterrent effect, as it is now possible to test horses with archived samples even if they or their parents are deceased. To our knowledge, this is the first report employing WGR on horsehair roots stored for a long term.

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Section: Whole Genome Resequencingsupporting

confidence: 70%

“…). The available reads were similar to those in our previous study [ 23 ]. Within the single horse that was sequenced, 6,064,956 SNPs and 727,565 INDELs were detected ( Table 3 ).…”

Section: Resultsmentioning

confidence: 57%

Section: Methodsmentioning

confidence: 99%

“…broadinstitute.org/gatk/best-practices/), and SnpEff (version v4_0) [16]. Parameters for all analyses are described in our previous study [23].…”

Section: Whole Genome Resequencingmentioning

confidence: 99%

mentioning

confidence: 99%

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Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports

Tozaki

Ohnuma

Kikuchi

et al. 2020

JES

Self Cite

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EPO transgene detection in dried blood spots for antidoping application

Marchand

Roulland

Semence

et al. 2021

Drug Testing and Analysis

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The modification of gene expression to treat diseases is a field of research with exponential growth. As doping in sport closely follows emerging therapies, a surveillance of the modification of gene expression to enhance performance is needed. The gene coding for erythropoietin (EPO) is one target of interest. Since 2010, several protocols have been proposed to identify EPO gene doping by focusing on the presence in blood of a transgene that differ in size from the endogenous gene sequence, normally found in the human DNA. In this work, our aim was to validate an easily applicable method for EPO gene doping detection in dried blood spots (DBS). We evaluated the detection of EPO transgene in 20-μl DBS after the spike of a plasmid carrying the EPO transgene in whole blood. Three different DBS were compared: Nucleic-Card™, Whatman ® 903, and the volumetric 20-μl VAMS™. Detection was performed with real-time polymerase chain reaction (PCR) and validated with two Taqman assays (one commercial and one custom) specific for the EPO transgene. The initial testing procedure could be done using one assay (custom) and the confirmation using the second one (commercial Taqman) with a final check of the size of the PCR product. Starting from 20-μl dried blood, 1000 copies of EPO transgene could efficiently be detected with the three types of DBS, VAMS showing a slightly better sensitivity. No loss of sensitivity was observed after 1-month storage of DBS at room temperature. This method could be applied to DBS collected during doping controls and allows reanalysis.

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Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing

Cheung

Wong

Lin

et al. 2022

Drug Testing and Analysis

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The concern about gene doping has remained high in horseracing and other equestrian competitions. Our laboratory has previously developed a duplex quantitative polymerase chain reaction (qPCR) assay capable of detecting in equine blood the human erythropoietin (hEPO) transgene and equine tubulin α 4a (TUBA4A) gene as an internal control the latter providing quality control over DNA extraction and qPCR. This study aimed to optimize the method for routine testing of regulatory samples. The use of an automated DNA extraction system has increased the sample throughput, consistency of DNA extraction, and recovery of reference materials. The use of reduced concentration of primers and hydrolysis probe for internal control minimized their competition with transgene amplification and improved the assay sensitivity. Spike‐in of an exogenous internal control at low concentration for plasma analysis has also been validated. Using the new workflow, four duplex qPCR assays have been developed for the detection of transgenes, namely, hEPO, human growth hormone (hGH), insulin‐like growth factor 1 (hIGF‐1), and equine EPO (eEPO). The estimated limits of detection (LODs) of each transgene were 2000 copies/mL of blood and 200 copies/mL of plasma. This method could detect the presence of transgene in blood and plasma collected from a horse administered intramuscularly (IM) with recombinant adeno‐associated virus (rAAV) carrying the hEPO transgene. A longer detection time was observed in blood than in plasma. The methods have been applied to the screening of over a thousand official racehorse samples since June 2020 for the presence of these transgenes.

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Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control

Cited by 17 publications

References 32 publications

Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports

Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports

EPO transgene detection in dried blood spots for antidoping application

Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing

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