Laura Wenzel: Applications of nanopore sequencing for plant pathogen detection

Laura opened with a discussion of “Why we care about plant health”. With the aim of monitoring and promoting plant health, Laura explained how crop losses through disease cost a huge amount of money and gave the specific example of panama disease. This being a disease in bananas cause by Fusarium oxysporum. This was such a problem in the 1950s the most popular banana cultivar at the time became economically non-viable for production. There are only limited resistant genotypes, but management of the disease is generally restricted to the use of pathogen free planting stock. Plant health across species was important to Laura, from food crops to ascetic angiosperms. In order to monitor and contain plant diseases, pathogen detection in seeds, propagation material and mature plants is needed. Current methods to do this involve using procedures such as indicator plants, ELISAs and qPCR methods. The problem being that many of these approaches work in a targeted fashion, i.e. one must know what to look for. Laura and her team are aiming to use nanopore sequencing to complement or even replace some of these targeted, high skill approaches.

Using an example RNA viral infection in Campanula, a flowing plant, Laura suggested that PCR/antibody-based detection methods could be replaced by sequencing the RNA via cDNA synthesis and taxonomic classification. Campanula can be infected by a number of single stranded RNA viruses, most specifically, Arabis mosaic virus, Cucumber mosaic virus, and Phlox virus S, however there are not defined tests for all potential viral pathogens and thus you can only find what you are looking for. cDNA sequencing using the Nanopore PCS-108 kit and LSK-108 kit reveled that, after taxonomic classification, all three of the above pathogens could be identified, with Arabis mosaic virus sequences dominating the samples.  However, Laura pointed out that things got a little strange when HIV virus was also detected and was obviously a false positive. Laura then asked, “why do we get false positives?”. It transpired that a lot of this was due to the sort nature of the query sequence used in the assignment algorithm. Laura suggested that if using something like BLAST the result would be better but as yet she has had little luck. However, using longer hits or splitting the reference sequences up into sections has helped. Moving on Laura showed how she could, as has become a common theme over the course of the conference, detect full viral genomes in single reads. Therefore, again, no assembly was required.

Looking at coverage statistics of some of the sequences from the plant viruses, Laura realized that, one of the plant pathogens of interest, cucumber mosaic virus, was not a poly-A tailed virus and thus was not directly compatible with polyT based reverse transcription priming. Laura stated that random hexamer-based methods could reverse transcribe this virus, allowing correct taxonomic identification.

Laura then moved onto discuss how nanopore sequencing could be used to support current detection methods by generating more complete genomes of plant pathovars so specific genomic detection assays, such as QPCR can be designed to discriminate between closely related by phenotypically different disease-causing organisms. Here a quick example was shown where a bacterial pathogen was sequenced from a lab isolate and was assembled into a single circular contig using 50 K reads and 100 X coverage. This was then compared with 185 contigs produced by 100 x coverage of short reads demonstrating a much more complete genome could be obtained for the same level of coverage.