A Validation Study of a High-Throughput Sequencing Workflow for the Detection of Grapevine Viruses and Viroids

Grapevine (Vitis vinifera L.) hosts a diverse range of viruses and viroids that differ in genome type, abundance, and distribution, creating challenges for reliable detection. High-throughput sequencing (HTS) is a powerful non-targeted tool for pathogen diagnostics, but its routine implementation requires validated workflows and objective interpretation criteria. Here, we present a validation framework for HTS-based plant virus and viroid diagnostics and demonstrate its application using an in-house grapevine pathogen detection workflow. Ten composite reference samples containing 85 virus and viroid occurrences were analyzed across a dilution series to evaluate diagnostic accuracy, detection limits, repeatability, reproducibility, and contamination risk. Results showed that sequencing quality metrics alone were insufficient for diagnostic interpretation. Additional indicators, including mapped reads and internal control depth, were necessary to assess sample suitability and define minimum performance requirements. Viral detection was evaluated using genome coverage and relative abundance (weight) metrics. A positive relationship between these parameters enabled the establishment of quantitative detection thresholds and a three-tier classification system: positive, likely positive, and likely negative. Diagnostic accuracy decreased with dilution because of reduced sensitivity for low-abundance pathogens, whereas repeatability and within-laboratory reproducibility remained high and contamination signals stayed below detection thresholds. Although the performance metrics are workflow-specific, this study provides a practical framework for laboratory validation of HTS-based plant virus and viroid diagnostics.