How <scp>eDNA</scp> data filtration, sequence coverage, and primer selection influence assessment of fish communities in northern temperate lakes

For nearly 15 years now, environmental DNA has demonstrated its effectiveness in monitoring biodiversity. Methodological and technical improvements have significantly enhanced the field. However, effect of factors such as sequence coverage, bioinformatic filtration, primer choice been less explored or need to be optimized according specific survey objectives study site characteristics. We evaluated these help optimize fish biodiversity North American temperate lakes. sampled water for community eDNA analysis 12 lakes from southwestern Québec, Canada. The were selected encompass a wide range surface areas species richness. total 520 sites (25–50 per lake) analyzed three mitochondrial regions (12S rRNA; 16S cytb) using NovaSeq sequencing. Our results, based on rarefied count matrices (from sequencing depth 100,000 minimum 1000 reads sample), showed that keeping only each sample if they represented at least one thousandth (species read proportion threshold = 0.001) sample's was adequate remove false positives had limited negative impact true with low counts. found negligible accuracy assessment given lake. With same complete local reference database set, single set produced similar richness medians than combination two sets. Overall, 12S detected more provided consistent profiles cytb. Based our observations, we suggest MiFish-U applying 0.001 monitor north-temperate lentic freshwater communities.