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; second author Institute for Plant Sustainable Protection National Research Center Via Amendola 165 A 70126 Bari Italy; third and fifth authors Plant Sciences Unit Flanders Research Institute for Agriculture Fisheries and Food Gembloux Agro Bio Tech University of Liège Passage des Déportés 2 5030 Gembloux Belgium; and twenty eighth author UMR 1332 BFP INRA Université de Bordeaux CS20032 33882 Villenave d'Ornon Cedex France

Citace poskytuje Crossref.org

Multiple Infections with Viruses of the Family Tymoviridae in Czech Grapevines

Detection of single nucleotide polymorphisms in virus genomes assembled from high-throughput sequencing data: large-scale performance testing of sequence analysis strategies

Detection of Apple Hammerhead Viroid, Apple Luteovirus 1 and Citrus Concave Gum-Associated Virus in Apple Propagation Materials and Orchards in the Czech Republic and Hungary

High Diversity of Novel Viruses in the Tree Pathogen Phytophthora castaneae Revealed by High-Throughput Sequencing of Total and Small RNA