Interlaboratory comparison study on ribodepleted total RNA high-throughput sequencing for plant virus diagnostics and bioinformatic competence

Yahya Gaafar, Marcel Westenberg, Marleen Botermans, Krizbai Laszlo, Kris De Jonghe, Yoika Foucart, Lucca Ferretti, Denis Kutnjak, Anja Pecman, Natasa Mehle, Jan Kreuze, Giovanna Muller, Nikolaos Vakirlis, Despoina Beris, Christina Varveri, Heiko Ziebell

Onderzoeksoutput: Bijdrage aan tijdschriftA1: Web of Science-artikelpeer review


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
Oorspronkelijke taalEngels
Pagina's (van-tot)1-17
Aantal pagina’s17
PublicatiestatusGepubliceerd - 12-sep.-2021

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