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

Abstract

High-throughput sequencing (HTS) technologies have brought tremendous improvements in the ability to detect plant viruses and have a great potential for application in virus routine diagnostics. The performance criteria of an HTS test need therefore to be estimated and compared to traditional virus indexing tests before it can be used in routine diagnostic. 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 analyzed for virus detection. 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 than the classical indexing protocol, 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.

Cont-ID: Detection of samples cross-contamination in viral metagenomic data

“…The X will impact the first rule that considers the relative proportion of reads of a virus in this sample compared to the sample with the maximum read of this virus. This threshold is a refinement of the "alien threshold" described earlier (5). The default value proposed is 2.…”

“…The first datasets (batches A to D) were generated in our laboratory by total RNA sequencing protocol with ribodepletion applied to RNA extracted from banana plants (belonging to the Musa genus) (5). These data were generated to compare the test performance criteria of high throughput sequencing with classical virus testing protocols that include ImmunoCapture (IC)-(RT)-PCR and electron microscopy (36).…”

“…For all datasets, read quality control (quality trimming, reads deduplication) was performed using a standard procedure described elsewhere (5). The cleaned reads were then mapped to a custom-built database (DB) containing all complete genome sequences from previously detected viruses in the datasets.…”

Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests

“…The rapid pace of development of nucleic acids preparation protocols (e.g., library kits), sequencing platforms (e.g., length of reads, chemistry/technology) and bioinformatic triade (e.g., novel algorithms, updated database) may require updates of HTS tests already validated and used for pest detection in the laboratory. A kit might even be discontinued during the performance criteria evaluation of an HTS test as succeeded for Musa viruses (Rong et al, 2022). As recommended by EPPO in standard PM 7/98 (2019), the impact of the update should be evaluated by competent personnel who will decide whether a verification or a validation is required.…”

“…In addition, detected viral sequences may correspond to a bona fide virus infecting other organisms associated with the sample, including bacteria, fungi or arthropods (Al Rwahnih et al, 2011;Marzano & Domier, 2016) or to viral sequences integrated into the plant genome (Baizan-Edge et al, 2019;Massart et al, 2017;Rong et al, 2022). Bacterial, fungal or viral sequences attributed to a pest species might be originating from closely related species (see analytical specificity issues) that are not pathogenic but living as endophytes without causing any harm under the specific environmental conditions.…”