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Monica Kehoe - Nanopore sequencing and analysis of plant pathogenic viruses: more than just rapid diagnostics?

London Calling 2019

The use of the portable MinION sequencer in plant pathology is rapidly increasing. Many studies have shown that the accuracy, portability and reduced time to result using the MinION are actively changing the way we do diagnostics and new diagnostic development for pests and diseases in agriculture. The first advantage of the MinION is clearly the ability to obtain rapid preliminary IDs of unknown pests and disease in the field. This was demonstrated recently by the Cassava Virus Action Project, taking just 4 hours to identify the virus present in symptomatic cassava plants in the field. The other advantage that can be overlooked is the opportunity to reduce the turn-around time to diagnosis for unknown pests and pathogens in a laboratory setting, as well as avoiding the need for expensive specialised equipment. While much has been made of the advances in in-field diagnostics, we wanted to answer the question of how does the data stack up in a laboratory setting when compared with other technologies? The small start-up costs and easy access to MinION sequencing, compared to other technologies makes it very attractive to plant virologists. Our team took a set of plant RNA samples from field pea with a known viral composition of a Potyvirus (Pea seed-borne mosaic virus - polyadenylated) and a Polerovirus (Turnip yellows virus – non polyadenylated), which we already had an Illumina data set for, and sequenced the samples using a cDNA and direct RNA kit on a MinION.  We compared downstream analyses performed with both the Illumina and MinION data. The results of our research suggest that not only is MinION suitable for rapid diagnostics in the laboratory and the field, but it is also useful in a wider research capacity.

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Monica Kehoe (Western Australian Department of Primary Industries and Regional Development) highlighted the importance of plant disease management: plant viruses cost $60bn/year, and can have a devastating effect on food security; correct identification of a virus and its vector, with suitable tools, is crucial in effectively managing their spread. She described how the sequencing of plant virus genomes with new sequencing technology is enabling rapid identification, both in the field and in the lab, with great significance to food security - but is also significant in allowing for in-house, whole genome sequencing approaches for broader research and monitoring of the viruses.

In the field, Monica described the work of The Cassava Virus Action Project, or "Tree Lab" 2018, who performed in-field extraction and sequencing of Cassava - an economically important crop and major food staple for millions of people - in Tanzania, Kenya and Uganda. Monica describes how on-site identification of viruses infecting Cassava was achieved, enabling rapid diagnosis of plant viruses in the field. This resulted in integrated pest management solutions, in which suitable plant varieties, resistant to the detected viruses, could be planted to increase yields.

Monica then introduced the work of DPIRD diagnostics in diagnosing plant viruses, using a broad range of biological, serological and molecular techniques and sequencing methods. Their work is broader than just rapid identification; they also perform whole genome sequencing of plant viruses, in-house. Monica described how, until recently, it was not possible to sequence their samples in their facility: receiving data from first-generation sequencing took a couple of days. For next-generation sequencing, the lab would send their total RNA samples to a third party short-read sequencing technology provider, who also performed library prep and QC. However, this required waiting until sufficient samples were ready to be sequenced at once; Monica noted that they then have to wait for up to three months for the data, also quoting one project which required 18 months to obtain the required sequencing data. Furthermore, the service is expensive.

Recently, the team have started using Oxford Nanopore technology to sequence in their own facility. As plant viruses can be either DNA or RNA, Monica discussed how they required "a protocol to cover all possibilities" for unidentified viruses. They settled on RNA plant virus sequencing, and developed a protocol using reverse transcription with random priming to prep samples, in case of the absence of polyA-tails, to avoid missing any viruses present. Libraries were sequenced on the MinION with MinIT, basecalled with Guppy and the data analysed with Poretools, Pomoxis, Geneious and BLAST. Their first sample was identified as pepper mild mottle virus (PMMoV), a Tobamovirus that often infects capsicums, with 99.9% concordance between long and short-read sequencing results. In their second study, results indicated a co-infection of three viruses: a Potyvirus (pea seed-borne mosaic virus) and two Poleoviruses (phasey bean mild yellows virus & turnip yellows virus); Monica noted that the mapped results were less ambiguous than those when short-read sequencing was used. De novo assemblies of all four viruses were generated: Monica noted that this approach was chosen over mapping to a reference in order to avoid biasing their answer - "we want to say 'what's in here?', not 'we think this is...'". She described how the use of nanopore long reads in assembly enabled full-length genome contigs, which was not always possible when using short reads.

Monica finished her talk by discussing the future plans of the team, including generating protocols for co-infections, developing a direct RNA method of analysis for their unknown viral samples, and also for sequencing unknown DNA and RNA samples in a single tube. She also described how the team have designed their own direct RNA adapters specific to plant families of interest. Furthermore, they plan to reduce costs further still with the use of the Flongle device.

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