Side-by-Side Comparison of Post-Entry Quarantine and High Throughput Sequencing Methods for Virus and Viroid Diagnosis

Abstract

Rapid and safe access to new plant genetic stocks is crucial for primary plant industries to remain profitable, sustainable, and internationally competitive. Imported plant species may spend several years in Post Entry Quarantine (PEQ) facilities, undergoing pathogen testing which can impact the ability of plant industries to quickly adapt to new global market opportunities by accessing new varieties. Advances in high throughput sequencing (HTS) technologies provide new opportunities for a broad range of fields, including phytosanitary diagnostics. In this study, we compare the performance of two HTS methods (RNA-Seq and sRNA-Seq) with that of existing PEQ molecular assays in detecting and identifying viruses and viroids from various plant commodities. To analyze the data, we tested several bioinformatics tools which rely on different approaches, including direct-read, de novo, and reference-guided assembly. We implemented VirusReport, a new portable, scalable, and reproducible nextflow pipeline that analyses sRNA datasets to detect and identify viruses and viroids. We raise awareness of the need to evaluate cross-sample contamination when analyzing HTS data routinely and of using methods to mitigate index cross-talk. Overall, our results suggest that sRNA analyzed using VirReport provides opportunities to improve quarantine testing at PEQ by detecting all regulated exotic viruses from imported plants in a single assay.

Implementation of GA-VirReport, a Web-Based Bioinformatics Toolkit for Post-Entry Quarantine Screening of Virus and Viroids in Plants

“…A subset of eight plants from the Gauthier et al [ 20 ] study were selected for comparative pilot implementation and testing of the GA-VirReport workflow. These plants belonging to Citrus ( n = 3), Prunus ( n = 1), Rubus ( n = 1), Miscanthus ( n = 1), Iris ( n = 1) and Ipomoea ( n = 1) genera were maintained in the PEQ glasshouses, Mickleham, Australia, and were re-sampled in March 2021 for the current study.…”

“…These plants were from a range of plant genera, namely Solanum ( n = 9), Fragaria ( n = 14), Vitis ( n = 4), Malus ( n = 3), Pyrus ( n = 6) and Prunus ( n = 69). All plants were sampled between September and November 2021 according to [ 20 ].…”

“…For Rubus and Fragaria , 50 mg of the petioles were used for nucleic acid isolation as per PEQ sampling procedures. Total RNA extractions were performed as previously described [ 20 ]. The quality and concentration of total RNA were measured using a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) following manufacturer’s protocols.…”

“…Several bioinformatics toolkits were recently developed for streamlining the detection of viruses and viroids from host plant sRNA-seq data. Some of these tools include the viral surveillance and diagnosis (VSD) toolkit [ 10 ], VirusDetect [ 18 ], VirFind [ 19 ] and VirReport [ 20 ]. In plant quarantine testing settings, the bottleneck for using some of the existing automated toolkits is the requirement for either local installation or knowledge of the Linux operating system and IT security barriers relating to the potentially sensitive nature of the data being generated.…”

“…Here, we introduce a web-based counterpart of VirReport [ 20 ] that is executable on the Galaxy Australia (GA) platform [ 23 ], which we called GA-VirReport, and a harmonized plant virus and viroid database (PVirDB). A secondary GA-VirReport-Stats pipeline generates confidence statistics for detections and includes reference-guided consensus sequence generation.…”