We incorporated a prescreening step in our method based on the advice of Dr. Oliver de Hon of the Dutch Doping Authorities. He emphasised the importance of a high throughput assay that could screen thousands of athletes simultaneously. Therefore, we developed a colorimetric assay based on the extent of gold nanoparticle aggregation (Baetsen-Young et al., 2018). When target doping DNA is absent, the nanoparticle completely aggregates, resulting in a purple color. When target doping DNA is present, it forms a secondary structure with a targeting DNA probe, which stabilizes the nanoparticle from aggregating, resulting in a red color.

3.Targeted library preparation

Positive prescreened samples proceed to our novel rapid targeted next generation sequencing. Targeted library preparation relies on our innovative fusion protein consisting of a Tn5 transposase and a dxCas9. The fusion of these two proteins resulted in a target specific transposition. As a proof of concept, we showed that our fusion protein is target specific with an in vitro targeted integration assay, that was verified by visualising the amplified integration products with gel electrophoresis. Once we established the functionality and optimal conditions, we implemented the fusion protein in the established rapid next generation sequencing library preparation protocol from Oxford Nanopore Technologies (ONT). We replaced the original transposase, responsible for random integration of the sequencing adaptors, by our novel fusion protein. In our case, a specifically designed sgRNA will guide the fusion protein to the exon-exon junction target site and prepare only gene doping DNA for sequencing. Additionally, we developed a sgRNA model which identified the optimal exon-exon target site by searching for the least sgRNA’s required to cover all possible variation of the target sites due to synonymous mutations of EPO coding sequence. A sgRNA array of the resulting 12 sgRNAs can be used in a single library preparation, utilizing the multiplexing capability of dxCas9 (Cong et al., 2013). As a result, we could simultaneously test for the gene doping variants, improving the efficiency of our method.

Further, we implemented multiplexing with barcodes to improve method efficiency, reduce cost, and expand the throughput. We created a barcoding webtool to generate unique barcodes, which are integrated into the adapter sequences that are ligated to the target sequence. This allows us to sequence samples from multiple different athletes in the same run and trace the output sequence back to corresponding barcode (Bayliss et al., 2017).

4. Targeted sequencing

After library preparation, we sequence the samples using a MinION, portable real-time sequencing device, from ONT. Only targeted sequences with adapters are translocated through the pores of ONT’s via the nanopore motor protein. The remaining untagged DNA will not be sequenced. By making a simple enzyme substitution, we transformed ONT’s established next generation sequencing platform into a targeted next generation sequencing platform.

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We processed the data obtained from ONT MinION sequencing runs with our tailor-made data analysis software tool. The software consists of an algorithm that aligns all the files generated by our sequencing run sequences with our pre-existing database, containing expected gene doping sequences. Based on the alignment score, the sequences are classified into gene doping DNA or non-gene doping DNA, eliminating any false positives that might have been sequenced. The software strengthens the robustness and reliability of our method, allowing us to determine whether gene doping DNA was present in the athlete’s blood sample. To go a step further, our algorithm has the capacity of expanding the database as it detects new gene doping sequences, thereby simultaneously evolving with gene doping.

Impact

We believe that by establishing and integrating a novel detection method into valid testing systems, we will discourage athletes from using high-risk gene doping technologies. Not only has the Dutch Doping Authority, the Delft Sports Engineering Institute and Dutch Trotting and Flat Racing Association (NDR) shown interest in our technology, but so has various other non gene doping related stakeholders. Sanquin, the Dutch blood bank, and the Dutch Research Department for Food Safety (RIKILT) at Wageningen University also emphasized the potential and value of our targeted sequencing technology. These stakeholders’ interests highlight the possible applications of our targeted sequencing method in other fields, such as cancer detection, non-invasive prenatal screening, food safety regulations, and even strain identification.

References

1. Baetsen-Young, A.M., Vasher M., Matta L.L., Colgan P., Alocilja E. C., Day B. Direct Colorimetric Detection of Unamplified Pathogen DNA by Dextrin-capped Gold Nanoparticles. Biosensors and Bioelectronics 101 (2018): 29-36. doi:10.1016/j.bios.2017.10.011.
2. Bayliss, S. C., Hunt, V. L., Yokoyama, M., Thorpe, H. A., Feil, E. J. (2017). The use of Oxford Nanopore native barcoding for complete genome assembly. GigaScience, 6(3), 1–6. http://doi.org/10.1093/gigascience/gix001
3. Beiter T, Zimmermann M, Fragasso A, Hudemann J, Niess AM, et al. Direct and long-term detection of gene doping in conventional blood samples. Gene Therapy. 2011, 18: 225–231.
4. Cong L.,, Ran F. A., Cox D., Lin S., Barretto R., Habib, N., Hsu P.D., Wu X., Jiang W, Marraffini L.A., Zhang F. Multiplex Genome Engineering Using CRISPR/Cas Systems. SCIENCE. 15 FEB 2013 : 819-823
5. Ni W., Le Guiner C., Gernoux G., Penaud-Budloo M., Moullier P., Snyder R.O. Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping. Gene Therapy. 2011, 18, 709–718. doi: 10.1038/gt.2011.19