Selective magnetic bead-based RNA capture assisted nanopore sequencing of tobamoviruses infecting tomato

Accurate and early detection of viral infections is crucial for disease management and is often hindered by low viral titers and high host RNA background. This study developed a magnetic bead-based RNA capture method to enrich tobamovirus sequences for nanopore sequencing. We designed three 5'-biotinylated oligonucleotide probes (84-100 nt) targeting conserved regions of:  Tobamovirus viridimaculae, formerly cucumber green mottle mosaic virus (CGMMV) Tobamovirus capsici, formerly pepper mild mottle mosaic virus (PMMoV); Tobamovirus tabaci, formerly tobacco mosaic virus (TMV); and Tobamovirus tomatotessellati, formerly tomato mosaic virus (ToMV) isolates. Total RNA from Solanum lycopersicum spiked with lyophilized positive controls was hybridized with these oligo probes and captured on streptavidin-coated magnetic beads. Subsequent template-switching cDNA amplification and nanopore sequencing revealed bead-based capture enriched viral sequences 1.95- to 56.77-fold. Using these twelve baits in a single panel for the multiplex detection of these tobamoviruses increased viral sequence abundance 1.49- to 6.04-fold. Genome coverage of ToMV was significantly higher in non-captured RNA samples (mean: 92.24% ± 1.12% vs. 64.07% ± 4.95%). Host RNA background was significantly reduced for CGMMV, ToMV, and coinfections. Our study demonstrates the enrichment of viral pathogens infecting important crops, improving the sensitivity in a nanopore sequencing framework. Future directions include optimizing and validating this method on a variety of low and higher titer plant viruses.