The Development of Wet Laboratory Methods for Improved Downstream Analysis of High Throughput Sequencing Data for Virus and Viroid Diagnostics of Vitis vinifera in Australian Post-Entry Quarantine

High throughput sequencing (HTS) methods are an essential tool in Australia’s Post-Entry Quarantine (PEQ) facility to facilitate the rapid, reliable and sensitive diagnostics of plant viruses and viroids. Small RNA sequencing (sRNAseq) for the detection of viruses and viroids has been implemented for several plant commodities in PEQ, including clonal grasses, Prunus spp., Rubus spp., and Fragaria spp., with the potential to expand to further plant genera. Currently, imported grapevine ( Vitis spp.) require many PCR assays (22) which are both laborious and time consuming. In this study we compare different wet laboratory processes for Vitis vinifera ribonucleic acid (RNA) extraction, including different tissue types, tissue weights and elution volumes and their effects on detecting viruses and viroids by Illumina sRNA sequencing. Overall, our results suggest that increased RNA concentration of V. vinifera can be achieved by using cambium tissue, reducing elution volume and decreasing the tissue weight. Furthermore, when comparing sRNAseq to current PCR methods in post-entry quarantine, sRNAseq outperformed the prescribed PCRs.