Validation of high throughput sequencing as virus indexing test for Musa germplasm: performance criteria evaluation and contamination monitoring using an alien control
Wei Rong1*, Johan Rollin1,2*, Marwa Hanafi1, Nicolas Roux3, Sebastien Massart1. 1Laboratory of Plant Pathology – TERRA - Gembloux Agro-Bio Tech – University of Liège, 5030 Gembloux, Belgium. 2DNAVision, 6041 Gosselies, Belgium . 3Bioversity International, Parc Scientifique Agropolis II, 34397 Montpellier, France. *These authors contributed equally to this work. https://doi.org/10.1094/PHYTOFR-03-22-0030-FI Overall, HTS used displayed a better analytical sensitivity compared to RT-PCR and a better inclusivity. Repeatability and reproducibility of virus detection were both of 100%. The diagnostic sensitivity was very high but false positive results were observed. Finally, the results also underlined the need of expert judgement in the interpretation of the results. In conclusion, the HTS test with an alien control and completed by expert evaluation has fulfilled the criteria of virus indexing protocol for Musa germplasm.
- 23 January 2023
- Yolanda Hernando Saiz
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In this study, 78 Musa accessions previously indexed for viruses by molecular tests and/or electron microscopy were tested individually or in pools using an HTS protocol based on total RNA sequencing. The external alien control, a wheat sample infected by BYDV, monitored the contamination burden and determined an adaptative detection threshold. Overall, HTS test displayed a better analytical sensitivity compared to RT-PCR and a better inclusivity, as distant isolates and new viral species were only detected by the HTS test. Repeatability and reproducibility of virus detection were both of 100% although differences in number of sequencing reads per virus were observed between replicates. The diagnostic sensitivity was very high but false positive results were observed. Finally, the results also underlined the need of expert judgement in the interpretation of the results. In conclusion, the HTS test with an alien control and completed by expert evaluation has fulfilled the criteria of virus indexing protocol for Musa germplasm
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