Development and optimization of a diagnostic system based on Illumina sequencing of genus wide PCR amplicons for the detection of viruses of grapevines

Abstract

In this study we present a poly-specific PCR-based, high-throughput sequencing (HTS) diagnostic system together with an appropriate data analysis pipeline for the diagnosis of grapevine viruses. Poly-specific and virus-specific primers were established to be capable of detecting and identifying 37 grapevine infecting viruses from 11 genera. An analysis pipeline using CLC Genomics workbench was developed by utilising various defined artificial samples which were assembled and sequenced on the Illumina MiSeq platform. A threshold for percentage mapped reads of 0.4% during reference mapping was established to discriminate between presence or absence of viruses associated with reads. Various criteria for the evaluation of de novo assembled contigs and BLAST results were identified based on virus hits, E-value, percentage query overlap and percentage amplicon overlap. Various RT-PCR systems were used to screen 62 grapevine samples (field collected and candidate nuclear vines) for their virus populations. Seven samples were selected for Illumina MiSeq sequencing, and the data was analysed as per the optimized pipeline. The threshold established for reference mapping and the criteria for BLAST analysis was successfully implemented, proving the applicability of this PCR and HTS-based system in grapevine diagnostics. This system was compared to the standard ELISA system routinely utilised during certification. In our study, when samples evaluated by RT-PCR were tested using ELISA for the presence of GLRaV-1, -2 and -3, a false-negative rate in ELISA of 14.3% was observed, confirming that RT-PCR is the more sensitive test of the two. The capability of RTPCR to readily detect viruses present in low concentrations in woody plants, the availability of primers for virus identification, the ease and rapidity of the technique, together with constant improvement of HTS platforms especially in the area of cost makes this an extremely useful method for grapevine virus diagnostics.