Monica Kehoe from the Department of Primary Industries and Regional Development in Australia presented at the 2019 London Calling meeting. The session’s title was “Nanopore sequencing and analysis of plant pathogenic viruses—more than just rapid diagnostics?” Kehoe is based in Western Australia and using Nanopore sequencing to sequence RNA plant viruses. Their focus is to characterize plant disease: diagnosis of the virus is needed for control strategies. Rapid turnaround is important too. Kehoe wants to use Nanopore sequencing in-house for routine whole genome sequencing of plant viruses up to ~18kb. They noted they already use Nanopore sequencing in the field. Kehoe explained that biological testing can take weeks. Serological testing with ELISA, lateral flow, or dipstick can also be challenging. Molecular techniques include PCR, qPCR, and LAMP but do not provide sequence information. Sanger sequencing has been used, but it is tedious. In the past, they would wait until they had enough samples for an Illumina sequencing lane. Recently they have focused on Nanopore sequencing using the MinION Mk1B and FLO-MIN106. The viruses they are interested in may not have polyA tails. Thus, they use RiboMinus Plant kits with Superscript IV, SQK-LSK, and PBC-011 kits. In one example shared, Kehoe explained how they sequenced a PMMoV virus and found some differences. de novo assembly was also performed using the nanopore sequencing results. In cases of multiple virus infections, Nanopore sequencing results matched those from Illumina at >98%. Not all plant viruses have a polyA tail, emphasized Kehoe. Therefore, they had to test methods since the direct RNA sequencing kit did not work for these samples. Kehoe noted that they want to try sequencing with Flongle flow cells. Learning how they compared Illumina and Nanopore sequencing results for these viruses was interesting and could help with some of our direct RNA sequencing challenges.
