Reference DNA pools
To compare the amplification performance of all 22 markers, we constructed two pools with equal concentrations of extracted DNA from each of 98 marine and freshwater teleost fish and five representatives of elasmobranch, crustacean, and cephalopod species, in total spanning 88 genera and 60 families (full reference, FR pool; Table 2). Samples were obtained primarily from museums, but also from fish markets when commercially important species were otherwise unavailable. Although we used internal (not surface) tissue for DNA extractions, samples obtained from seafood markets could have come into contact with other fish products, potentially carrying trace contamination that might erroneously be considered false positives if detected by metabarcoding. To better avoid these potential sources of trace contamination, we constructed a second, more restricted reference pool including only the 73 DNA extracts from vouchered museum specimens (vouchered reference, VR).