Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported but little attention has been paid thus far to their sensitivity and reliability for diagnostic purposes. Therefore, we compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large-scale performance test using 10 datasets of 21- to 24-nucleotide small RNA (sRNA) sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false-positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty in detecting viral agents when they are novel or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases, and (iv) the significant level of scientific expertise needed when interpreting pipeline results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.

1st and eleventh authors Integrated and Urban Plant Pathology Laboratory TERRA Gembloux Agro Bio Tech University of Liège Passage des Déportés 2 5030 Gembloux Belgium

eighth author Crop Protection Division International Potato Center Aveninda La Molina 1895 La Molina Lima Peru

fifteenth and eighteenth authors Center of Plant Protection and Biotechnology Instituto Valenciano de Investigaciones Agrarias Ctra Moncada Náquera km 4 5 46113 Moncada Valencia Spain

fourteenth author Life Sciences Department Walloon Agricultural Research Center Rue de Liroux 4 Gembloux Belgium

fourth author Fera Science Ltd National Agri Food Innovation Campus Sand Hutton York YO41 1LZ United Kingdom

ineteenth author Department of Cell Biology and Genetics Faculty of Science Palacky University in Olomouc Slechtitelu 27 783 71 Olomouc Czech Republic

inth author Department of Biotechnology and Systems Biology National Institute of Biology Večna pot 111 1000 Ljubljana Slovenia

second author Institute for Plant Sustainable Protection National Research Center Via Amendola 165 A 70126 Bari Italy

seventeenth author Virology Phytoplasmology Laboratory Agroscope 1260 Nyon Switzerland

seventh author Crop Research Institute Drnovska 507 161 06 Prague Czech Republic

sixteenth and twenty second authors University of Basel Department of Environmental Sciences Botany 4056 Basel Switzerland

sixteenth author Institut national de la recherche agronomique 34398 Montpellier France

sixth author Biology Centre CAS Branisovska 31 370 05 Ceske Budejovice Czech Republic

tenth and twelfth authors Laboratory of Plant Pathology Faculty of Agriculture Forestry and Natural Environment School of Agriculture Aristotle University of Thessaloniki 54124 Thessaloniki Greece

third and fifth authors Plant Sciences Unit Flanders Research Institute for Agriculture Fisheries and Food Burg Van Gansberghelaan 96 9820 Merelbeke Belgium

thirteenth author Agricultural Research Council Infruitec Nietvoorbij Institute for Deciduous Fruit Vines and Wine Nietvoorbij Farm R44 7600 Stellenbosch South Africa

twentieth author Department of Medical Parasitology and Infection Biology Swiss Tropical and Public Health Institute University of Basel Socinstrasse 57 4051 Basel Switzerland

twenty eighth author UMR 1332 BFP INRA Université de Bordeaux CS20032 33882 Villenave d'Ornon Cedex France

twenty fifth author Pathologie Végétale INRA F 84140 Montfavet France

twenty first author Department of Plant Pathology and Weed Research Agricultural Research Organization The Volcani Center 50250 Bet Dagan Israel

twenty fourth author Diagnostic Group Department of Genomics Agricultural Biotechnology and Research Institute National Agricultural Research and Innovation Center Szent Gyorgyi A street 4 H 2100 Gödöllő Hungary

twenty seventh author Applied Statistics Computer Science and Mathematics Gembloux Agro Bio Tech University of Liège Passage des Déportés 2 5030 Gembloux Belgium

twenty sixth author Dutch National Plant Protection Organization P O Box 9102 6700 HC Wageningen The Netherlands

twenty third author Natural Resources Institute Finland Latokartanonkaari 9 00790 Helsinki Finland

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