Application of Oxford Nanopore Technology to Plant Virus Detection

The adoption of Oxford Nanopore Technologies (ONT) sequencing as a tool in plant virology has been relatively slow despite its promise in more recent years to yield large quantities of long nucleotide sequences in real time without the need for prior amplification. The portability of the MinION and Flongle platforms combined with lowering costs and continued improvements in read accuracy make ONT an attractive method for both low- and high-scale virus diagnostics. Here, we provide a detailed step-by-step protocol using the ONT Flongle platform that we have developed for the routine application on a range of symptomatic post-entry quarantine and domestic surveillance plant samples. The aim of this methods paper is to highlight ONT’s feasibility as a valuable component to the diagnostician’s toolkit and to hopefully stimulate other laboratories towards the eventual goal of integrating high-throughput sequencing technologies as validated plant virus diagnostic methods in their own right.

A Primer on the Analysis of High-Throughput Sequencing Data for Detection of Plant Viruses

High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.

High Throughput Sequencing For Plant Virus Detection and Discovery

Over the last decade, virologists have discovered an unprecedented number of viruses using high throughput sequencing (HTS), which led to the advancement of our knowledge on the diversity of viruses in nature, particularly unraveling the virome of many agricultural crops. However, these new virus discoveries have often widened the gaps in our understanding of virus biology; the forefront of which is the actual role of a new virus in disease, if any. Yet, when used critically in etiological studies, HTS is a powerful tool to establish disease causality between the virus and its host. Conversely, with globalization, movement of plant material is increasingly more common and often a point of dispute between countries. HTS could potentially resolve these issues given its capacity to detect and discover. Although many pipelines are available for plant virus discovery, all share a common backbone. A description of the process of plant virus detection and discovery from HTS data are presented, providing a summary of the different pipelines available for scientists’ utility in their research.

Application of Next Generation Sequencing for Diagnostic Testing of Tree Fruit Viruses and Viroids

Conventional detection of viruses and virus-like diseases of plants is accomplished using a combination of molecular, serological, and biological indexing. These are the primary tools used by plant virologists to monitor and ensure trees are free of known viral pathogens. The biological indexing assay, or bioassay, is considered to be the “gold standard” as it is the only method of the three that can detect new, uncharacterized, or poorly characterized viral disease agents. Unfortunately, this method is also the most labor intensive and can take up to three years to complete. Next generation sequencing (NGS) is a technology with rapidly expanding possibilities including potential applications for the detection of plant viruses. In this study, comparisons are made between tree fruit testing by conventional and NGS methods, to demonstrate the efficacy of NGS. A comparison of 178 infected trees, many infected with several viral pathogens, demonstrated that conventional and NGS were equally capable of detecting known viruses and viroids. Comparable results were obtained for 170 of 178 of the specimens. Of the remaining eight specimens, some discrepancies were observed between viruses detected by the two methods, representing less than 5% of the specimens. NGS was further demonstrated to be equal or superior for the detection of new or poorly characterized viruses when compared with a conventional bioassay. These results validated both the effectiveness of conventional virus testing methods and the use of NGS as an additional or alternative method for plant virus detection.