Validation of High-Throughput Sequencing as Virus Indexing Test for Musa Germplasm: Performance Criteria Evaluation and Contamination Monitoring Using an Alien Control

High-throughput sequencing (HTS) technologies have brought tremendous improvements in the ability to detect plant viruses and have great potential for application in virus routine diagnostics. The performance criteria of an HTS test
need therefore to be estimated and compared with traditional virus indexing tests
before it can be used in routine diagnostics.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 analytical sensitivity of HTS and RT-PCR was also compared by
independent testing on serial dilutions of RNA extracts.In total,136 libraries were
sequenced in five batches, and the sequences were analyzed for virus detection. The external alien control, a wheat sample infected by barley yellow dwarf
virus, monitored the contamination burden and determined an adaptative detection threshold. Overall, the HTS test displayed a better analytical sensitivity than
the RT-PCR and a better inclusivity than the classical indexing protocol,as distant
isolates and new viral species were only detected by the HTS test. The repeatability and reproducibility of virus detection were both 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 for expert judgement in the interpretation of the results.In conclusion,the HTS test with an alien control and completed
by expert evaluation fulfilled the criteria of the virus indexing protocol for Musa
germplasm.