DNA metabarcoding is an important tool for molecular ecology. However, its effectiveness hinges on the quality of reference sequence databases and classification parameters employed. Here we evaluate the performance of MiFish 12S taxonomic assignments using a case study of California Current Large Marine Ecosystem fishes to determine best practices for metabarcoding. Specifically, we use a taxonomy cross-validation by identity framework to compare classification performance between a global database comprised of all available sequences and a curated database that only includes sequences of fishes from the California Current Large Marine Ecosystem. We demonstrate that the curated, regional database provides higher assignment accuracy than the comprehensive global database. We also document a tradeoff between accuracy and misclassification across a range of taxonomic cutoff scores, highlighting the importance of parameter selection for taxonomic classification. Furthermore, we compared assignment accuracy with and without the inclusion of additionally generated reference sequences. To this end, we sequenced tissue from 605 species using the MiFish 12S primers, adding 253 species to GenBank’s existing 550 California Current Large Marine Ecosystem fish sequences. We then compared species and reads identified from seawater environmental DNA samples using global databases with and without our generated references, and the regional database. The addition of new references allowed for the identification of 16 native taxa and 17.0% of total reads from eDNA samples, including species with vast ecological and economic value. Together these results demonstrate the importance of comprehensive and curated reference databases for effective metabarcoding and the need for locus-specific validation efforts.

DNA metabarcoding is an important tool for molecular ecology. However, metabarcoding effectiveness hinges on the quality of reference databases for taxa and loci of interest. This limitation is true for metabarcoding of marine fishes in the California Current Large Marine Ecosystem where there is a paucity of reference 12S barcodes. Here we present FishCARD, a California Current-specific fish 12S-specific reference barcode database. We barcoded 612 species using the MiFish metabarcoding primers; an addition of 258 species to the 459 California Current fish species with existing 12S barcodes from GenBank. The resulting FishCARD database covers 82.7% of California Current fishes, and it includes virtually all fishes sampled by large marine monitoring programs such as the Partnership for Interdisciplinary Studies of Coastal Oceans and California Cooperative Oceanic Fisheries Investigation. To demonstrate the importance of complete reference databases for eDNA metabarcoding, we compared species and reads identified from three 1L seawater samples collected off Santa Cruz Island, CA using GenBank sequences with and without our generated barcodes, as well as the FishCARD database curated here. The inclusion of our generated barcodes allowed the additional identification of 15 native taxa and 21.8% of total reads from eDNA samples. However, we found that half of all amplicon sequence variants (ASVs) generated by MiFish 12S primers were of non-vertebrate 16S origin, demonstrating a clear limitation of a widely employed fish metabarcoding primers. Despite these limitations, FishCARD provides an important genetic resource to enhance the effectiveness of marine metabarcoding efforts in the California Current Large Marine Ecosystem.