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

Abstract

High-throughput sequencing (HTS) of host plant small RNA (sRNA) is a popular approach for plant virus and viroid detection. The major bottlenecks for implementing this approach in routine virus screening of plants in quarantine include lack of computational resources and/or expertise in command-line environments and limited availability of curated plant virus and viroid databases. We developed: (1) virus and viroid report web-based bioinformatics workflows on Galaxy Australia called GA-VirReport and GA-VirReport-Stats for detecting viruses and viroids from host plant sRNA extracts and (2) a curated higher plant virus and viroid database (PVirDB). We implemented sRNA sequencing with unique dual indexing on a set of plants with known viruses. Sequencing data were analyzed using GA-VirReport and PVirDB to validate these resources. We detected all known viruses in this pilot study with no cross-sample contamination. We then conducted a large-scale diagnosis of 105 imported plants processed at the post-entry quarantine facility (PEQ), Australia. We detected various pathogens in 14 imported plants and discovered that de novo assembly using 21–22 nt sRNA fraction and the megablast algorithm yielded better sensitivity and specificity. This study reports the successful, large-scale implementation of HTS and a user-friendly bioinformatics workflow for virus and viroid screening of imported plants at the PEQ.

Keywords

• high-throughput sequencing
• small RNA
• plant virus and viroid detection
• post-entry quarantine
• VirReport