Recent developments in high-throughput sequencing (HTS) technologies and bioinformatics have drastically changed research in virology, especially for virus discovery. Indeed, proper monitoring of the viral population requires information on the different isolates circulating in the studied area. For this purpose, HTS has greatly facilitated the sequencing of new genomes of detected viruses and their comparison. However, bioinformatics analyses allowing reconstruction of genome sequences and detection of single nucleotide polymorphisms (SNPs) can potentially create bias and has not been widely addressed so far. Therefore, more knowledge is required on the limitations of predicting SNPs based on HTS-generated sequence samples. To address this issue, we compared the ability of 14 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 21 variants of pepino mosaic virus (PepMV) in three samples through large-scale performance testing (PT) using three artificially designed datasets. To evaluate the impact of bioinformatics analyses, they were divided into three key steps: reads pre-processing, virus-isolate identification, and variant calling. Each step was evaluated independently through an original, PT design including discussion and validation between participants at each step. Overall, this work underlines key parameters influencing SNPs detection and proposes recommendations for reliable variant calling for plant viruses. The identification of the closest reference, mapping parameters and manual validation of the detection were recognized as the most impactful analysis steps for the success of the SNPs detections. Strategies to improve the prediction of SNPs are also discussed.

Biology Centre CAS Ceske Budejovice Czech Republic

Citrus Research International Matieland South Africa

Crop Research Institute Praha Czech Republic

Department of Biosystems Science and Engineering ETH Zurich Basel 4058 Switzerland

Department of Cell Biology and Genetics Faculty of Science Palacký University Olomouc Olomouc Czech Republic

Department of Chemistry and Biotechnology Tallinn University of Technology Tallinn Estonia

Department of Genetics Stellenbosch University Matieland South Africa

Department of Plant Protection Faculty of Agriculture Eskişehir Osmangazi University Eskişehir Turkey

Fisheries and Food Plant Sciences Unit Flanders Research Institute for Agriculture Merelbeke Belgium

GAFL Institut National de la Recherche pour l'Agriculture l'Alimentation et l'Environnement Montfavet France

Laboratory of Plant Pathology TERRA Gembloux Agro Bio Tech University of Liège Gembloux Belgium

Laboratory of Statistics Computer Science and Modelling Applied to Bioengineering TERRA Gembloux Agro Bio Tech Teaching and Research Centre University of Liège Gembloux Belgium

Mendeleum Institute of Genetics Faculty of Horticulture Mendel University in Brno Lednice Czech Republic

Natural Resources Institute Finland Helsinki Finland

Pathologie Végétale Institut National de la Recherche pour l'Agriculture l'Alimentation et l'Environnement Montfavet France

Plant Production and Technologies Department Ayhan Şahenk Faculty of Agricultural Science and Technologies Niğde Ömer Halisdemir University Niğde Turkey

Plant Protection Department Agricultural Faculty Hatay Mustafa Kemal University Hatay Turkey

Plant Protection Department Agroscope Nyon Switzerland

Plant Protection Department Faculty of Agriculture University of Maragheh Maragheh Iran

Swiss Institute of Bioinformatics Basel Switzerland

Wageningen University and Research Wageningen The Netherlands

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