Assessing the efficiency of COI primers in eDNA metabarcoding for marine metazoan biodiversity.

Using environmental DNA (eDNA) metabarcoding with mitochondrial COI gene markers for biodiversity assessments has been gaining popularity. This approach is particularly advantageous in marine ecosystems due to the significant challenges posed by traditional sampling methods. However, limitations like primer specificity and primer-template bias during the PCR amplification can affect the accuracy of biodiversity assessments, though these issues have not been quantitatively evaluated to date. Here, we assessed the performance of four widely used COI primer sets for eDNA metabarcoding using in silico PCR. We analyzed 4267 COI gene sequences obtained from the NCBI RefSeq database. Our findings revealed marked differences in amplification efficiencies and taxonomic coverage across the four primer sets. Importantly, the primer set pair3 (mlCOIintF-XT/jgHCO2198) demonstrated superior effectiveness for most marine metazoans. The percentages of completely matched sequences for both forward primer (F2) and reverse primer (R1) significantly exceeded those of other primer sets, indicating their optimal performance. The percentage of sequences that could be amplified across the major phyla ranged from 81.6 % to 99.4 %, with higher values in Arthropoda, Annelida, Mollusca, Echinodermata and Nematoda. However, the performance of these primer sets was less effective in Acanthocephala, Brachiopoda, Cnidaria, Ctenophora, Platyhelminthes and Porifera, which were likely to be underestimated or overlooked. By assessing primer mismatches in this study, researchers can enhance primer selection in the eDNA metabarcoding, thereby improving species detection in marine metazoan communities. Moreover, standardizing primer selection can enhance comparability and reproducibility across various research studies.