Longer amplicon metabarcoding primers enhance fish taxonomic resolution in eDNA samples

Many previously designed fish eDNA metabarcoding primers amplify short regions ranging from 70-170 bp. However, the capacity to differentiate related taxa is positively correlated with amplicon size. We collated an alignment of full 12S gene sequences for 169 Canadian Freshwater species, and used this to design and/or modify existing primers to develop two novel primer pairs that target Canadian freshwater fish and which produce amplicons approximately 210 and 315 bp in size. Together, these amplicons covered ~57% of the 12S gene and included all but a single variable region useful for differentiating taxa. Using an in-silico analysis of sequences from 173 Canadian fish species, we demonstrated that these primer pairs amplified more efficiently and can more readily distinguish closely related taxa relative to commonly employed shorter-amplicon primer pairs. We additionally validated their in-situ sensitivity in natural ecosystems by sampling two urban pond ecosystems in Hamilton, Ontario, that were also surveyed using conventional methods, including one pond that was drained for a complete census. Both primer pairs detected all captured species, including four rare species (1-3 individuals of ~1700).