Dissertation
Combining target enrichment and third-generation sequencing to identify multiple pathogenic viruses in plants
University of the Sunshine Coast, Queensland
Doctor of Philosophy, University of the Sunshine Coast, Queensland
2026
DOI:
https://doi.org/10.25907/01043
Abstract
Modern diagnostic techniques for detection of plant viral pathogens typically require individual assays to be conducted for each target pathogen. With increasing movement of high-risk plant material across Australian borders, this process is becoming increasingly time consuming, costly, and ultimately unsustainable as existing pathogens mutate and new pathogens emerge, requiring the development and validation of more and more new tests. The emergence of high throughput sequencing (HTS) means more plant material can be analysed for pathogens at any one time. This has led to faster detection and identification of novel and existing pathogens on infected plant tissue. However, technical challenges arise when targeting plant viruses. Sequence variability, mutation rates and unreliable reference databases can all lead to virus misidentification. Although HTS pipelines are becoming increasingly accessible for the purpose of plant pathogen diagnostics, limitations exist in their capacity to detect low-titre viruses. In a biosecurity regulatory environment, a failure to correctly identify high-risk plant viruses can have catastrophic consequences. This research focuses on using hybrid-capture probes combined with Nanopore sequencing to develop a multi-purpose sequencing method that can cover a large virus scope yet is both sensitive and cost-effective. Testing on synthetic DNA in a plant background revealed that successful capture was achieved when using probes with sequence divergence of 30% and spacing between probes. Cucumber green mottle mosaic virus (CGMMv) was used as a model virus to test sample preparation and found that polyA RACE synthesis and ribo-depletion was the most beneficial sample preparation method to overcome nucleic acid quantity limitations when converting RNA to cDNA, prior to hybridisation. A panel was designed to target a range of viruses with increased probe spacing compared to commercial panels. This workflow was tested on a diverse panel of 11 different viruses with significance to Australian biosecurity, including tomato brown rugose fruit virus (ToBRFv), tomato mottle mosaic virus (ToMMv), prunus necrotic ringspot virus (PepMv), tomato yellow leaf curl virus (TYLCv), sweet potato leaf curl virus (SPLCv), pepino mosaic virus (PepMv), sweet potato feathery mottle virus (SPFMv), strawberry latent ringspot virus (SLRSv), citrus tristeza virus (CTv), bean yellow mosaic virus (BYMv), and CGMMv. These viruses present a major risk to Australian agricultural industries, particularly the vegetable industry which relies heavily on seed imports, and where disease detection can be difficult. The panel included tests for strain differentiation, lowered probe stringencies, DNA and RNA viruses, and plant and seed background tissue. The workflow and accompanying panel demonstrated successful hybridisation to viruses when probe identities were >70% when designing against a reference genome at 220 bp spacing. Of these successfully hybridised samples, all demonstrated increases of target to non-target reads. Ranges varied in each sample however ssDNA viruses saw the biggest increase in reads compared to unhybridised samples. This method shows benefit to virus coverage and depth to a wide range of viruses capable of infecting both plant and seed. Further research is recommended to reduce sample preparation time and cost, however the workflow demonstrated benefit to increasing read coverage and depth of viruses using Nanopore sequencing.
Details
- Title
- Combining target enrichment and third-generation sequencing to identify multiple pathogenic viruses in plants
- Authors
- Thomas Farrall - University of the Sunshine Coast, Queensland, School of Science, Technology and Engineering
- Contributors
- Mark Brown (Principal Supervisor) - University of the Sunshine Coast, Queensland, Forest Industries Research CentreHelen Nahrung (Co-Supervisor) - University of the Sunshine Coast, Queensland, Forest Industries Research CentreJeremy Brawner (Consultant Supervisor) - University of the Sunshine Coast, Queensland, Forest Research InstituteAdrian Dinsdale (Consultant Supervisor)
- Awarding institution
- University of the Sunshine Coast, Queensland
- Degree awarded
- Doctor of Philosophy
- Publisher
- University of the Sunshine Coast, Queensland
- DOI
- 10.25907/01043
- Grant note
- Australian Department of Agriculture, Fisheries and Forestry (DAFF), under the modern technologies and diagnostic tools (MTDT) project.
- Organisation Unit
- School of Science, Technology and Engineering; Forest Research Institute
- Language
- English
- Record Identifier
- 991230028602621
- Output Type
- Dissertation
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