NanoViromics: long-read sequencing of dsRNA for plant virus and viroid rapid detection

Abstract

There is a global need for identifying viral pathogens, as well as for providing certified clean plant materials, in order to limit the spread of viral diseases. A key component of management programs for viral-like diseases is having a diagnostic tool that is quick, reliable, inexpensive, and easy to use. We have developed and validated a dsRNA-based nanopore sequencing protocol as a reliable method for detecting viruses and viroids in grapevines. We compared our method, which we term direct-cDNA sequencing from dsRNA (dsRNAcD), to direct RNA sequencing from rRNA-depleted total RNA (rdTotalRNA), and found that it provided more viral reads from infected samples. Indeed, dsRNAcD was able to detect all of the viruses and viroids detected using Illumina MiSeq sequencing (dsRNA-MiSeq). Furthermore, dsRNAcD sequencing was also able to detect low-abundance viruses that rdTotalRNA sequencing failed to detect. Additionally, rdTotalRNA sequencing resulted in a false-positive viroid identification due to the misannotation of a host-driven read. Two taxonomic classification workflows, DIAMOND & MEGAN (DIA & MEG) and Centrifuge & Recentrifuge (Cent & Rec), were also evaluated for quick and accurate read classification. Although the results from both workflows were similar, we identified pros and cons for both workflows. Our study shows that dsRNAcD sequencing and the proposed data analysis workflows are suitable for consistent detection of viruses and viroids, particularly in grapevines where mixed viral infections are common.

Non-Targeted RNA Sequencing: Towards the Development of Universal Clinical Diagnosis Methods for Human and Veterinary Infectious Diseases

“…While currently dedicated mostly to the analysis of epigenetic modifications of native RNA molecules, the technology is rapidly evolving with advancements in software and methods [ 8 , 9 , 10 , 11 , 12 , 13 ]. This has already led to applications as diverse as viral/viroid detection in grapevines [ 14 ] and to the definition of the soil transcriptome [ 15 ]. Moreover, direct RNA sequencing holds promise for rapid viability assessment in various samples.…”

First detection and genomic characterisation of Spinach latent virus in tomato in Canada

“…resulting cDNA was sequenced on an R9.4.1 flow cell using a direct cDNA Nanopore Sequencing Kit and MinION sequencer (Javaran et al, 2023). A total of 26,530 reads (BioSample: SAMN37658768) were generated from the diseased tomato leaf.…”