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

Abstract

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.

MiFish metabarcoding: a high-throughput approach for simultaneous detection of multiple fish species from environmental DNA and other samples

“…To avoid such failures in the library preparation, it is important to: (1) purify the 1st PCR products to eliminate redundant tailed primers and the primer dimers (hybridized tailed-primer molecules) using either a spin column or beads; (2) quantify the purified 1st PCR products using an automated electrophoresis system, such as BioAnalyzer or TapeStation (both Agilent Technologies, Inc., San Diego, CA, USA) (Fig. 12); (3) dilute the quantified 1st PCR products to a fixed concentration (e.g., 0.1 ng/µl) as templates for the second-round PCR (2nd PCR); and (4) size-select the 2nd PCR products at around 370 bp using gel electrophoresis to remove extra bands at around 440 bp (presumed to be a product derived from the 16S rRNA gene of a microorganism; see also Gold et al 2020). Note that those extra bands are more prominent in waters that are susceptible to anthropogenic effects, such as estuaries and bay areas near the big cities, than in other areas.…”

“…38) over a 5-year period between 2015 and 2020 after publication of . For taxonomic coverage, see text initiative among related universities and research institutes in the California Current region has built its own reference database for enhanced MiFish eDNA metabarcoding, assembling reference sequences from 712 species among the 864 known species from the region (Gold et al 2020).…”

“…Andruszkiewicz et al (2017b) found that there were differences in vertebrate community composition identified by eDNA metabarcoding between the surface and subsurface waters, and between neritic (< 200 m depth) and deep-water stations, concluding that eDNA metabarcoding offers enormous potential for identifying spatial structures of vertebrate communities. This paper was followed by a series of subsequent eDNA metabarcoding studies using MiFish primers off California (Andruszkiewicz et al 2017a;Truelove et al 2019;Closek et al 2019;Djurhuus et al 2020;Gold 2020).…”

“…Kacev et al (2018) concluded that continued refinement of this new method (in particular that of reference libraries) will increase the utility of this high-throughput genetic approach for routine monitoring and assessment. Actually, subsequent efforts to assemble the MiFish sequences from fishes of the California Current region resulted in 717 reference sequences, which cover 84.7% of the total fauna, with 864 fish species currently included (Gold et al 2020).…”

“…The second initiative involves several universities and research institutes in the California Current region in a joint initiative to consider using MiFish eDNA metabarcoding to supplement traditional capturebased sampling to improve the efficiency of biodiversity monitoring in that region (Kacev et al 2018). As part of this effort, the MiFish eDNA metabarcoding library preparation protocol has been published online (Pitz et al 2020), and the California Current specific reference database (FishCARD) has been assembled for more accurate taxonomic assignment in MiFish eDNA metabarcoding (Gold et al 2020). Thus, these two global initiatives in different geographic regions are expected to use MiFish eDNA metabarcoding in their future studies.…”