12S gene metabarcoding with DNA standard quantifies marine bony fish environmental DNA, identifies threshold for reproducible detection, and overcomes distortion due to amplification of non-fish DNA

Single-species PCR assays accurately measure eDNA concentration. Here we test whether multi-species PCR, that is, metabarcoding, with an internal standard can quantify eDNA of marine bony fish. Replicate amplifications with Riaz 12 S gene primers were spiked with known amounts of a non-fish vertebrate DNA standard, indexed separately, and sequenced on an Illumina MiSeq. Fish eDNA copies were calculated by comparing fish and standard reads. Relative reads were directly proportional to relative DNA copies, with average and maximum variance between replicates of about 1.3-and 2-fold, respectively. There was an apparent minimum threshold for consistent amplification of about 10 eDNA copies per PCR reaction. The internal DNA standard corrected for suppression of fish read counts due to amplification of non-fish DNA. To assess potential PCR bias among species, we compared reads obtained with Riaz 12 S primers to those with modified MiFish primers, which target a different 12 S gene segment. Our results provide evidence that Riaz 12 S gene metabarcoding with an internal DNA standard quantifies marine bony fish eDNA over a range of about 10–5000 copies per reaction, without indication of significant PCR bias among teleost species. In mid-Atlantic coastal samples, eDNA rarity was the main limitation to reproducible detection and quantification, and this was partly overcome by increasing the amount of a DNA sample amplified. Our findings support incorporating a DNA standard in 12 S metabarcoding to help quantify eDNA abundance for marine bony fish species.