Development and Validation of a Bioinformatic Workflow for the Rapid Detection of Viruses in Biosecurity

Abstract

The field of biosecurity has greatly benefited from the widespread adoption of high-throughput sequencing technologies, for its ability to deeply query plant and animal samples for pathogens for which no tests exist. However, the bioinformatics analysis tools designed for rapid analysis of these sequencing datasets are not developed with this application in mind, limiting the ability of diagnosticians to standardise their workflows using published tool kits. We sought to assess previously published bioinformatic tools for their ability to identify plant- and animal-infecting viruses while distinguishing from the host genetic material. We discovered that many of the current generation of virus-detection pipelines are not adequate for this task, being outperformed by more generic classification tools. We created synthetic MinION and HiSeq libraries simulating plant and animal infections of economically important viruses and assessed a series of tools for their suitability for rapid and accurate detection of infection, and further tested the top performing tools against the VIROMOCK Challenge dataset to ensure that our findings were reproducible when compared with international standards. Our work demonstrated that several methods provide sensitive and specific detection of agriculturally important viruses in a timely manner and provides a key piece of ground truthing for method development in this space.

Enhancing biosecurity against virus disease threats to Australian grain crops: current situation and future prospects

“…However, Illumina produces shorter read lengths and involves a longer turn around time before results become available than thirdgeneration platforms such as Oxford Nanopore Technology (ONT) (Pecman et al, 2022). Also, ONT provides a convenient means of identifying plant viruses (Bronzato-Badial et al, 2018;Ben Chehida et al, 2021;Liefting et al, 2021;Maina et al, 2022;Pecman et al, 2022;Waite et al, 2022). Its ONT direct RNA sequencing approach involves sequencing RNA without the need for prior amplification and it directly reads RNA without the need for reverse transcription (Phannareth et al, 2020).…”

“…Under these circumstances, preventing incursions of damaging viruses and their virus vectors, and eradicating or containing any that establish successfully, is becoming an increasingly difficult challenge for biosecurity authorities located within different world regions and countries. Fortunately, rapid technological advances that improve the effectiveness of virus diagnosis in preborder, border and postborder situations are providing new opportunities to detect viruses earlier and more reliably (Jones, 2014;Martin et al, 2016;Kreuze, et al, 2023;Waite et al, 2022;Adams and Fox, 2016;Barrero et al, 2017;Pecman et al, 2017;Bronzato-Badial et al, 2018;FAO, 2019;Piper et al, 2019;Liefting et al, 2021;Maina et al, 2021;Whattam et al, 2021;Gauthier et al, 2022;Lelwala et al, 2022;Mackie et al, 2022;AlcaláBriseño et al, 2023).…”

Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (Olea europaea) Twigs

“…Nanopore sequencing has been used for phytodiagnostics of different pathogenic species and following different approaches. These include the detection of different plant viruses in potato [14], tomato [15], cassava [10], wheat [16], yam [17] and various other plants [15,18,19] either following standard MinION protocols or elaborated cost-effective adaptations [18,20]. Several relevant studies provide reports on the identification of Xylella fastidiosa subspecies and sequence types from naturally infected plant material [21], tomato phytopathogenic bacteria identification [22], and mango dieback disease-associated fungal species [23].…”

State-of-the-Art Plant Virus Research in Australasia

“…Paper five in the series [ 5 ] was about preventing exotic viruses from gaining new geographical territory. The paper presented a bioinformatic workflow for detecting viruses for which no current tests exist.…”