Facilitating the adoption of high‐throughput sequencing technologies as a plant pest diagnostic test in laboratories: A step‐by‐step description

Abstract

High-throughput sequencing (HTS) is a powerful tool that enables the simultaneous detection and potential identification of any organisms present in a sample. The growing interest in the application of HTS technologies for routine diagnostics in plant health laboratories is triggering the development of guidelines on how to prepare laboratories for performing HTS testing. This paper describes general and technical recommendations to guide laboratories through the complex process of

Viroscope: Plant viral diagnosis from high-throughput sequencing data using biologically-informed genome assembly coverage

“…This is a fundamental issue that still needs to be addressed at 'invitro' and 'in-silico' level during virus diagnosis using HTS pipelines. In agreement with many scientific researchers in the field (Rott et al, 2017;Adams et al, 2018;Jones and Naidú, 2019;Mehetre et al, 2021;Gauthier et al, 2022;Lebas et al, 2022;Villamor et al, 2022), we endorse that plant pathogen molecular diagnostics by HTS is becoming a more cost-effective and scalable solution with new accessible sequencing technologies already available to perform precise diagnosis. The enormous benefit of HTS applied to plant health is indispensable to ensure reliable diagnosis of known and unknown pathogens and will contribute to more sustainable agriculture and safer international plant trade practices.…”

“…In addition, it has proved to be a major advance for crops, imported plants and germplasm in which disease symptoms are absent, unspecific or only triggered by multiple viruses (Massart et al, 2017;Mehetre et al, 2021). Currently, useful guidelines and reports are available to facilitate and standardize the adoption of HTS technologies in plant pest routine diagnostics (Gaafar et al, 2021;Tamisier et al, 2021;Lebas et al, 2022). Nonetheless and in face of a changing farming paradigm, more efforts on HTS data analysis and its accurate interpretation must be established.…”

“…Functional annotation may aid in increasing certainty of diagnosis in virus detection in these cases of low abundance, due to its direct implication in viral physiology and potential infectivity. Additionally, cross-contamination can occur at different steps of the laboratory process (i.e., nucleic acid extraction, library preparation, and/or sequencing) (Rott et al, 2017;Gauthier et al, 2022;Lebas et al, 2022). This is a fundamental issue that still needs to be addressed at 'invitro' and 'in-silico' level during virus diagnosis using HTS pipelines.…”

PM 7/151 (1) Considerations for the use of high throughput sequencing in plant health diagnostics1

“…It was reviewed by the Panel on Diagnostics and Quality Assurance. The writing of VALITEST deliverable D 2.2 involved many international experts and lead to the publication of two scientific articles (Lebas et al, 2022; Massart et al, submitted).…”

Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests

“…For example, each step of the bioinformatic analysis (e.g., quality control, target identification and control evaluation) can be influenced by the bioinformatics triade: the algorithms, their parameters and thresholds, and the reference database(s). The steps of the laboratory and bioinformatics components have been described in detail in the companion paper proposing guidelines for the preparation of laboratories before the use of HTS technologies in plant pest diagnostics (Lebas et al, 2022). The large number of operations and the high complexity of some of them increase the risks of creating bias during the analysis and can cause a lack of reliability of the pest detection, e.g., false positive or false negative, which could have dramatic consequences for regulated plant pests.…”