Environmental DNA (eDNA) metabarcoding has gained growing attention as a strategy for monitoring biodiversity in ecology. However, taxa identifications produced through metabarcoding require sophisticated processing of high-throughput sequencing data from taxonomically informative DNA barcodes. Various sets of universal and taxon-specific primers have been developed, extending the usability of metabarcoding across archaea, bacteria and eukaryotes. Accordingly, a multitude of metabarcoding data analysis tools and pipelines have also been developed. Often, several developed workflows are designed to process the same amplicon sequencing data, making it somewhat puzzling to choose one among the plethora of existing pipelines. However, each pipeline has its own specific philosophy, strengths and limitations, which should be considered depending on the aims of any specific study, as well as the bioinformatics expertise of the user. In this review, we outline the input data requirements, supported operating systems and particular attributes of thirty-two amplicon processing pipelines with the goal of helping users to select a pipeline for their metabarcoding projects.

Agroécologie INRAE Institut Agro Univ Bourgogne Franche Comté Dijon France

Aquatic Ecosystem Research University of Duisburg Essen Essen Germany

Center for Applied Microbiome Science Pathogen and Microbiome Institute Northern Arizona University Flagstaff Arizona USA

Center for Forest Mycology Research Northern Research Station US Forest Service Madison Wisconsin USA

Department of Biological and Environmental Science University of Jyväskylä Jyväskylä Finland

Department of Forest Mycology and Plant Pathology Swedish University of Agricultural Sciences Uppsala Sweden

Department of Integrative Biology and Centre for Biodiversity Genomics University of Guelph Guelph Ontario Canada

Department of Marine Microbiology and Biogeochemistry NIOZ Royal Netherlands Institute for Sea Research Texel Netherlands

Department of Population Health and Pathobiology College of Veterinary Medicine and Bioinformatics Research Center North Carolina State University Raleigh North Carolina USA

Earlham Institute Norwich Research Park Norfolk UK

GenPhySE Université de Toulouse INRAE ENVT Castanet Tolosan France

Gut Microbes and Health Quadram Institute Bioscience Norfolk UK

INRAE AgroParisTech GABI Université Paris Saclay Jouy en Josas France

INRAE SIGENAE Jouy en Josas France

Institut de Biologie Intégrative et des Systèmes Université Laval Québec Québec Canada

Institute of Ecology and Earth Sciences University of Tartu Tartu Estonia

KU Leuven Department of Microbiology Immunology and Transplantation Rega Institute for Medical Research Laboratory of Molecular Bacteriology Leuven Belgium

Laboratory of Environmental Microbiology Institute of Microbiology of the Czech Academy of Sciences Praha Czech Republic

Quantitative Biology Center University of Tübingen Tübingen Germany

School of Biological Sciences University of Reading Reading UK

UK Centre for Ecology and Hydrology Oxfordshire UK

Unit of Computational Biology Research and Innovation Centre Fondazione Edmund Mach Italy

Vermont Biomedical Research Network University of Vermont Burlington Vermont USA

Zachary Gold NOAA Pacific Marine Environmental Laboratory Seattle Washington USA

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Comparative Analysis of Pasture Composition: DNA Metabarcoding Versus Quadrat-Based Botanical Surveys in Experimental Grassland Plots