Interlaboratory Comparison Study on Ribodepleted Total RNA High-Throughput Sequencing for Plant Virus Diagnostics and Bioinformatic Competence

Abstract

High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and validated) to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data

Side-by-Side Comparison of Post-Entry Quarantine and High Throughput Sequencing Methods for Virus and Viroid Diagnosis

“…Academic and governmental research groups both acknowledge the need for extant protocols to independently confirm the biological significance of detected viruses via HTS. Several international efforts are underway to critically catalogue methods available [ 17 , 21 , 22 , 23 ] and provide researchers and diagnostics laboratories with open-source reference datasets to compare and validate their detection pipelines [ 16 , 24 ]. There is also an increase in comparative studies reporting the performance of different approaches [ 14 , 15 , 16 , 21 , 25 , 26 ].…”

“…Several international efforts are underway to critically catalogue methods available [ 17 , 21 , 22 , 23 ] and provide researchers and diagnostics laboratories with open-source reference datasets to compare and validate their detection pipelines [ 16 , 24 ]. There is also an increase in comparative studies reporting the performance of different approaches [ 14 , 15 , 16 , 21 , 25 , 26 ]. Together, this will help improve and accelerate harmonization, comparability, and reproducibility of HTS methods.…”

Symptomless infection by strawberry virus 1 (StrV‐1) leads to losses in strawberry yields

“…However, it cannot be excluded that recipient plants were infected by pathogens other than those tested for, though we believe that the likelihood is minimal. To address this point, HTS may be employed; however, the method is still not appropriate for routine practice primarily due to the number of different protocols used, the need for a significant level of virology expertise to correctly interpret the results, and the amount of time needed for analyses, especially when new viruses are detected (Bester et al, 2021; Gaafar et al, 2021). For these reasons we decided to omit an HTS analysis in starting material and performed testing of all pathogens in our accredited laboratory to be able to proceed within the set time framework of the experiment, even with a risk that an undiscovered pathogen may be present in some plants.…”

Targeted Whole Genome Sequencing (TWG-Seq) of Cucumber Green Mottle Mosaic Virus Using Tiled Amplicon Multiplex PCR and Nanopore Sequencing

“…Enrichment methods are frequently employed to raise confidence in sequencing outcomes [ 11 , 17 , 18 , 19 ]. Indirect methods that can increase the relative virus titre of a metagenomic sample involve rRNA host depletion, and dsRNA and siRNA extractions [ 20 ].…”