FishCARD: Fish 12S California Current Specific Reference Database for Enhanced Metabarcoding 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.