title: Metabarcoding of aquatic invertebrates
design: Aquatic invertebrate specimens were collected in Canada, Finland, France, and Germany. A small piece of tissue was subsampled from each specimen (Arthropoda: a leg or a section of a leg; Annelida: a small section of the body; Mollusca: a piece of the mantle) and the DNA was extracted in 96-well plates with a membranebased protocol (Ivanova et al. 2006), except for phylum Mollusca (CTAB-based protocol, Wallinger et al. 2017). The 658 bp barcode region of COI was amplified using the primer cocktail of C_LepFolF/C_LepFolR (HernndezTriana et al., 2014), except for Mollusca (C_GasF1_t1/GasR1_t1 (S. Prosser, unpublished data)) with initial denaturation at 94C for 2 min followed by 5 cycles of denaturation for 40 s at 94C, annealing for 40 s at 45C and extension for 1 min at 72C; then 35 cycles of denaturation for 40 s at 94C with annealing for 40 s at 51C and extension for 1 min at 72C; and a final extension for 5 min at 72C (Hebert et al. 2018; Ivanova et al. 2006). All products were sequenced in the forward and reverse directions following standard procedures on an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, California, USA).