Assessment of ONT sequencing (MinION) for plant virus detection and comparison with Illumina-based sequencing

General plant health can be improved by the use of virus-tested starting material, as many viruses are spread through vegetative propagation. HTS-based untargeted detection replacing specific PCR- or ELISA-based methods, or even biological indexing, is extremely promising for this purpose: the complete virome can be assessed without the need for prior knowledge. Illumina sequencing has already proven to be a reliable and sensitive method for virus detection, yet, also the ONT technology has gained interest as a promising tool over the last years. Nanopore sequencing generates long reads, can be performed in every lab, decreases the costs, and improves accuracy. For MinION sequencing a protocol based on Liefting et al. (2021) and similar to our standardized Illumina sequencing protocol, was used. In short, total RNA extraction was followed by rRNA depletion and random primed double stranded cDNA synthesis. Barcodes and adapters were added according to the ONT Direct cDNA Sequencing Kit protocol and samples were sequenced for 72 hours on a Flow Cell. Reads were classified by comparing against the Genbank non-redundant Nucleotide database with Kraken2 and mapped to virus reference genomes with Minimap2. Five plant samples previously screened with Illumina sequencing, infected with multiple viruses/viroids from different genera, were selected for this study. Overall, most of the viruses could be detected and were identified correctly with this protocol. Viruses detected below 50 rpm with Illumina sequencing could only be detected with poor genome coverage, while some failed to be detected. It can be concluded that correct virus detection and identification is possible with this MinION protocol but sensitivity is still lower.