Validation of an In-House High-Throughput Total RNA Sequencing Test for the Detection of Plant Viruses and Viroids

High-throughput sequencing is becoming the method of choice for plant diagnostics. It allows the detection of known and novel viruses and viroids, even in co-infection, without preliminary knowledge of the target. However, this method has its own limitations when compared to real-time PCR or ELISA. Laboratories that implement this type of technologies in-house must ensure that the performance criteria meet the requirements associated with their diagnostic activity. In this study, we present a workflow for in-house plant viruses and viroid detection, based on total RNA extraction, ribodepletion, Illumina sequencing and bioinformatics analyses. Performance criteria such as analytical sensitivity, analytical specificity, selectivity, repeatability, reproducibility and robustness were evaluated on the tomato brown rugose fruit virus (RNA genome), the tomato leaf curl New Delhi virus (DNA genome), and the pepper chat fruit viroid (RNA genome). The performance levels obtained meet the requirements for virus and viroid detection in symptomatic plant samples.