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, Ontari..., , # Data from: Longer amplicon metabarcoding primers enhance fish taxonomic resolution in eDNA samples

Dataset DOI: [10.5061/dryad.ttdz08m93](10.5061/dryad.ttdz08m93)

## Description of the data and file structure

These data were used to design and validate two novel long-read eDNA metabarcoding primer sets. The data include an alignment of full mitochondrial 12S genomes from 169 Canadian freshwater fish species, as well as the results of an in-silico analysis using the DECIPHER program to estimate the efficiency at which the two novel primer sets, and two previously designed primer sets, amplify the target species' DNA.

### Files and variables

#### File: Supplementary_File_S1_-_12S_Alignment_for_169_Canadian_Freshwater_Species.fasta

**Description:** 

This datafile includes full 12S mitochondrial sequences for 169 Canadian freshwater fishes. Sequences are described by their species of origin, and included in the file are the forward and reverse primers for the long-amplicon primer s...,