Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring.

AZTI Marine Research Basque Research and Technology Alliance Pasaia Gipuzkoa Spain; IKERBASQUE Basque Foundation for Science Bilbao Bizkaia Spain

Bielefeld University Animal Ecology 33615 Bielefeld Germany

Biotechnical Faculty University of Ljubljana Jamnikarjeva 101 1000 Ljubljana Slovenia; Faculty of Mathematics Natural Sciences and Information Technologies University of Primorska Glagoljaška 8 6000 Koper Slovenia

Center for Microbiome Innovation University of California San Diego La Jolla CA USA

Centre of Molecular and Environmental Biology University of Minho Campus de Gualtar 4710 057 Braga Portugal

Coastal and Freshwater Group Cawthron Institute Private Bag 2 Nelson 7042 New Zealand

Coastal and Freshwater Group Cawthron Institute Private Bag 2 Nelson 7042 New Zealand; Institute of Marine Science University of Auckland Warkworth 0941 New Zealand

Czech University of Life Sciences Dept of Microbiology Nutrition and Dietetics Prague Czech Republic

Department of Genetics and Evolution University of Geneva Geneva Switzerland; ID Gene Ecodiagnostics 1202 Geneva Switzerland

Department of Genetics and Evolution University of Geneva Geneva Switzerland; Institute of Oceanology Polish Academy of Sciences 81 712 Sopot Poland; ID Gene Ecodiagnostics 1202 Geneva Switzerland

Department of Genetics and Evolution University of Geneva Geneva Switzerland; NORCE Climate NORCE Norwegian Research Centre AS Bjerknes Centre for Climate Research Jahnebakken 5 5007 Bergen Norway

Department of Life and Environmental Sciences Polytechnic University of Marche Via Brecce Bianche Ancona 60131 Italy

Department of Materials Environmental Sciences and Urban Planning Polytechnic University of Marche Via Brecce Bianche Ancona 60131 Italy

Department of Pure and Applied Sciences Urbino University Urbino Italy

ID Gene Ecodiagnostics 1202 Geneva Switzerland

INRAE CARRTEL 74200 Thonon les Bains France

Institute of Technology University of Tartu Tartu 50411 Estonia

Institute of Technology University of Tartu Tartu 50411 Estonia; Chair of Hydrobiology and Fishery Institute of Agricultural and Environmental Sciences Estonian University of Life Sciences Tartu Estonia; Chair of Aquaculture Institute of Veterinary Medicine and Animal Sciences Estonian University of Life Sciences Estonia

National Museum of Natural History Luxembourg 25 Rue Münster L 2160 Luxembourg Luxembourg

NatureMetrics Ltd CABI Site Bakeham Lane Egham TW20 9TY UK

Norwegian Research Center AS NORCE Environment Marine Ecology Group Mekjarvik 12 4070 Randaberg Norway

Swiss Centre for Applied Ecotoxicology EPFL ENAC IIE GE 1015 Lausanne Switzerland

Technische Universität Kaiserslautern Ecology Group D 67663 Kaiserslautern Germany

Univ Savoie Mont Blanc INRAE CARRTEL 74200 Thonon les Bains France

University of Duisburg Essen Faculty of Biology Aquatic Ecosystem Research Germany

University of Salzburg Dept of Biosciences 5020 Salzburg Austria

Water Research Institute National Research Council of Italy Monterotondo Rome Italy

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