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      Virus detection by high-throughput sequencing of small RNAs: large scale performance testing of sequence analysis strategies

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          Abstract

          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.

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          Author and article information

          Journal
          Phytopathology
          Phytopathology
          Scientific Societies
          0031-949X
          August 02 2018
          August 02 2018
          Affiliations
          [1 ]Université de Liège, Terra-Gembloux Agro-Bio Tech, Plant Pathology Laboratory, Passage des Déportés, 2, Gembloux, Belgium, 5030, ;
          [2 ]National Research Council, Institute for Sustainable Plant Protection, Sezione di Bari, Puglia, Italy;
          [3 ]Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Plant Sciences, Burgemeester Van Gansberghelaan 96, Merelbeke, Belgium, 9820, ;
          [4 ]National Agri-Food Innovation Campus, Sand Hutton, York, YO41 1LZ, United Kingdom of Great Britain and Northern Ireland;
          [5 ]Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Merelbeke, Belgium;
          [6 ]Biologicke Centrum Akademie ved Ceske republiky, 90801, Dep. Institute of Plant molecular biology, Unit. Plant Virology, Ceske Budejovice, Czech Republic;
          [7 ]Crop Research Institute, Division of Plant Health, Prague, Czech Republic;
          [8 ]International Potato Center, Germplasm Enhancement and Crop improvement, Avenida La Molina 1895, Lima, Peru, Lima 12,
          [9 ]Swedish University of Agricultural Sciences, Plant Biology and Forest Genetics, Uppsala, Sweden;
          [10 ]Nacionalni institut za biologijo, 54766, Department of Biotechnology and Systems Biology, Ljubljana, Slovenia;
          [11 ]Thessaloniki, Greece;
          [12 ]University of Liège, TERRA-Gembloux Agro-Bio Tech, Plant Pathology Laboratory, Gembloux, Belgium;
          [13 ]Aristotle University of Thessaloniki, School of Agriculture, Aristotle University of Thessaloniki, School of Agriculture, Plant pathology lab, P.O.B. 269, 54124 Thessaloniki Greece, Thessaloniki, Greece, Greece, 54124, , ;
          [14 ]Stellenbosch University, Stellenbosch, Stellenbosch, South Africa, 7600;
          [15 ]Walloon Agricultural Research Centre (CRA-W), Life Sciences Department, Gembloux, Belgium;
          [16 ]Instituto Valenciano de Investigaciones Agrarias (IVIA), Departamento Protección Vegetal y Biotecnología, Ctra Moncada Naquera km 5, Moncada, Valencia, Spain, 46113;
          [17 ]University of Basel, Hebelstarsse 1, Basel, Switzerland, 4058;
          [18 ]Agroscope, Virology and Phytoplasmology, Route de Duillier 50, Nyon, Switzerland, 1260;
          [19 ]Instituto Valenciano de Investigaciones Agrarias (IVIA), Plant Protection and Biotechnology, Ctra Moncada-Naquera km 5, Moncada, Valencia, Spain, 46113, ;
          [20 ]Univerzita Palackeho Prirodovedecka Fakulta, 98735, Dept. of Cell Biology and Genetics, Slechtitelu 27, Olomouc, Czech Republic, 77146;
          [21 ]Swiss tropical and public health institute, Epidemiology and Public Health, Socinstrasse 57, Basel, Switzerland, 4055;
          [22 ]Agricultural Research Organization, The Volcani Center, Department of plant pathology and weed research, Bet Dagan, Israel;
          [23 ]University of Basel, Department of Environmental Sciences, Botany, Basel, Switzerland;
          [24 ]Luonnonvarakeskus, 419837, Dep. Management and production of renewable resources, Helsinki, Uusimaa, Finland;
          [25 ]National Agricultural Research and Innovation Center, Diagnostic Group, Department of Genomics, Agricultural Biotechnology and Research Institute, Gödöllő, Hungary;
          [26 ]INRA, Domaine Saint Maurice, 67, allée des chênes, CS 60094,, Unité de Pathologie Végétale, F84143 Montfavet Cedex, France, France;
          [27 ]Dutch National Plant Protection Organization (NPPO-NL), Wageningen, Netherlands;
          [28 ]Universite de Liege Gembloux Agro-Bio Tech, 82209, AGROBIOCHEM-Terra Research Center-Statistiques, Informatiques et Mathématiques appliqués à la bioingénierie, Gembloux, Belgium;
          [29 ]INRA, UMR 1332 BFP, CS20032, Villenave d'Ornon Cedex, France, 33882;
          Article
          10.1094/PHYTO-02-18-0067-R
          30070618
          © 2018

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