VIRTIGATION partners KU Leuven and EMWEB have developed new protocols for full-length genome sequencing of circular DNA and linear RNA plant viruses.
Article by Marianna Granatier from the VIRTIGATION project
Better profiling of DNA & RNA plant viruses
Fighting emerging viral plant viruses such as ToBRFV, ToLCNDV and TYLCV in tomatoes and cucurbits requires a deep understanding of their molecular characteristics and detection methods. To this end, VIRTIGATION partners KU Leuven and EMWEB have established a new rapid-response pipeline for improved profiling of emerging DNA and RNA plant viruses. In a recent report, KU Leuven and EMWEB researchers present several new protocols to better profile viral circular DNA and linear RNA in infected plants. Each protocol consists of three main steps:
- Enrichment of viral RNA and DNA via direct amplification or capturing the DNA or RNA sequence.
- Sequencing of the virus by Oxford Nanopore technology (sequencing technology that enables real-time analysis of native DNA or RNA and sequencing the fragment of any length achieving short to ultra-long read lengths).
- Sequencing data analysis with EMWEB’s Genome Detective online analytical tool.
Pipeline for full genome sequencing of DNA plant viruses
Thanks to the high sensitivity of this KU Leuven and EMWEB pipeline, utilising both raw and duplex Nanopore sequencing data, they could firstly produce a detailed profiling of TYLCV virus population (see figure below). However, the VIRTIGATION scientists deemed the accuracy of the pipeline based solely on raw reads as insufficient to reliably distinguish true mutations from sequencing errors without validation using Short Read Sequencing (SRS)—a technology that sequences shorter DNA fragments with higher accuracy. In contrast, duplex reads significantly improved the overall quality and accuracy of the analysis. The project researchers then developed two optimised protocols specifically tailored for begomoviruses. The full methodology for both DNA genome sequencing protocols is detailed in their report.
Pipeline for full genome sequencing of RNA plant viruses
The second pipeline enables the generation of high-quality, full-genome RNA sequences of tobamovirus variants (see figure below). KU Leuven and EMWEB researchers developed a total of four protocols for RNA sequencing, each tailored to different experimental needs. Using this pipeline, the VIRTIGATION researchers could successfully reconstruct two full-genome variants of the ToBRFV virus from a field-collected tomato sample in Belgium. The full methodology for all RNA genome sequencing protocols is further detailed in their report.
A key tool for epidemiological monitoring
The new protocols for full genome sequencing by VIRTIGATION partners KU Leuven and EMWEB constitute a new key tool for improved epidemiological monitoring of emerging plant viruses. By decoding the complete genetic material of viral DNA and RNA, researchers can more accurately identify virus strains, monitor mutations, and track the emergence of new variants. Such detailed sequencing data also supports the creation of new advanced diagnostic tools that enable earlier detection and faster response to viral outbreaks. Finally, the KU Leuven and EMWEB findings will also support value chain actors in better tracing virus spread across regions, thereby helping to inform quarantine and containment efforts.
Read here the deliverable “Protocols for full genome sequencing” produced by VIRTIGATION partners KU Leuven and EMWEB, which describes their research in more detail.
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