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

Yahya Z A Gaafar,Anja Pecman,Christina Varveri,Luca Ferretti,Nataša Mehle,Kris De Jonghe,Marcel Westenberg,Despoina Beris,Marleen Botermans,Giovanna Muller,Denis Kutnjak,Heiko Ziebell,Nikolaos Vakirlis,Jan Kreuze,Krizbai László,Yoika Foucart

doi:10.3390/pathogens10091174

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.

Highlights

Plant viruses and virus-like diseases are ever-emerging threats to agricultural and horticultural production [1]
Twenty laboratories were invited to participate in the study and nine laboratories agreed to participate in this study, of which eight performed the study and reported their results
The number of raw reads obtained after sequencing varied where the sequencing company provided between ≈ 13 and 54 M reads for Pool 1 (≈76 M for Lab ID TPS_05 where they used a different sequencing provider), and between

Summary

Introduction

Plant viruses and virus-like diseases are ever-emerging threats to agricultural and horticultural production [1]. Conventional detection methods for plant viruses and virus-like diseases including molecular, serological, and biological indexing are primary tools used by plant virologists [2,3]. Conventional methods rely on previous information of the virus, e.g., the virus sequence, to design primers for PCR-based methods [2] or purified virions for the production of virus-specific antibodies. HTS does not require prior information as it enables the sequencing of all nucleic acids in a given sample [5]. Viral nucleic acids require an enrichment method prior to the library construction for HTS as their sequences are present in the background of their host sequences [6]. There are different enrichment methods for virus sequences derived from plant samples, e.g., ribosomal RNA (rRNA)

Methods

Results

Discussion

Conclusion