Dataset for: How eDNA data filtration, sequence coverage, and primer selection influence assessment of fish communities in northern temperate lakes

<p><span>For nearly 15 years now, environmental DNA</span><span> has demonstrated</span><span> its effectiveness in monitoring biodiversity. Methodological and technical improvements have significantly enhanced the field. However, the effect of factors such as sequence coverage, bioinformatic filtration and primer choice have been less explored or need to be optimized according </span><span>to </span><span>specific survey objectives and </span><span>study </span><span>site characteristics. We evaluated these factors </span><span>to </span><span>help optimize monitoring fish biodiversity in North American temperate lakes. We sampled water for fish community eDNA analysis in 12 lakes from southwestern Québec, Canada. The lakes were selected to encompass a wide range of surface areas and species richness. We sampled water from a total of </span><span>520</span><span> sites (25 to 50 per lake) and analyzed three mitochondrial DNA regions (12S rRNA; 16S rRNA; and cytb) using NovaSeq</span><span> sequencing. Our results, based on rarefied count matrices (from a sequencing depth of 100,000 to a minimum </span><span>depth </span><span>of 1,000 reads per sample), </span><span>showed</span><span> that </span><span>keeping only</span><span> species </span><span>in each sample if they</span><span> represented </span><span>at least one thousandth (species </span><span>minimum </span><span>read proportion threshold =</span><span> 0.001</span><span>)</span><span> of the </span><span>sample's</span><span> reads was adequate to remove false positives </span><span>and had a limited negative</span><span> impact on true positives</span><span> with low read counts. The</span><span> sequencing depth </span><span>was found to have</span><span> a negligible impact </span><span>on the accuracy</span><span> of fish </span><span>community assessment in a given lake. With the same sequencing depth and a complete local reference database for each primer set, </span><span>a single primer set </span><span>produced</span><span> similar species richness medians than the combination of two or three primer sets. Overall, 12S and 16S detected more species and provided more consistent community profiles than cytb. </span><span>Based on our observations, we suggest using the 12S MiFish-U primer set and applying a minimum proportion of 0.001 reads per species and site to monitor north-temperate lentic freshwater fish communities.</span></p>