Data from: Longer amplicon metabarcoding primers enhance fish taxonomic resolution in eDNA samples
Data files
Oct 22, 2025 version files 241.68 KB
Abstract
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).
Dataset DOI: 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/).
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/).
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:
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Data was derived from the following sources:
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