DOI: 10.5061/dryad.ttdz08m93
title: Data from: Longer amplicon metabarcoding primers enhance fish taxonomic resolution in eDNA samples

description: 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). 
description: # 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 sets designed as a part of this study. #### File: Supplementary_File_S2_-_12S_marker_efficiencies.xlsx **Description:**  This datafile includes the result of an analysis of estimated primer set amplification efficiencies for the 169 Canadian freshwater fish species included in the "Supplementary_File_S1_-_12S_Alignment_for_169_Canadian_Freshwater_Species.fasta". The analysis was conducted using the *DECIPHER *software, and include the following variables: ##### Variables * Species: The latin name of the species from which the sequence originates * Reference sequence: The ID of the reference sequence for the corresponding species. * MiFish, Mmito, Mteleo, and Teleo columns: The estimated amplification efficiencies for each species, corresponding to the four primer sets evaluated as a part of the study (the specific primer set corresponds to the column title). * Data on amplification efficiencies for individual markers (MiFish, Mmito, Mteleo, and Teleo) can be found on secondary tabs. ## Code/software The .fasta file can be viewed using any software employed to visualize genetic data (e.g., GENEIOUS, or for a free software, BioEdit ([https://bioedit.software.informer.com/](https://bioedit.software.informer.com/)). The "Supplementary File S2 - 12S marker efficiencies" file can be viewed using excel, or for a free open source spreadsheet viewer, LibreOffice ([https://www.libreoffice.org/](https://www.libreoffice.org/)). Amplification efficiency data were predicted using the AmplifyDNA function of the R package DECIPHER v. 3.0.0 (Wright 2016). Parameters were annealing temperatures and primer concentrations given in Table 2, ion concentration of 0.5 M, and maximum product size 500 bp. -Wright, E.S. 2016. Using DECIPHER v2.0 to Analyze Big Biological Sequence Data in R. R J. **8**: 352–359. ## Access information Other publicly accessible locations of the data: * NA Data was derived from the following sources: * NA