The utility of eDNA for fish species and community monitoring is well-established using targeted amplification (i.e., qPCR and ddPCR) and passive sequencing approaches (i.e., metabarcoding). However, the lack of optimized and standardized methods reduces the sensitivity of this approach and precludes the reliable comparison of findings across studies, respectively. DNA extraction is a prime target for optimization efforts because the extraction method is highly variable across eDNA studies despite being the most influential factor in detection efficiency across the entire post-collection workflow. Sequence analysis is arguably the least standardized step in the workflow, with new bioinformatics pipelines frequently emerging in the literature and being implemented with innumerable unique combinations of parameter values. The current study aimed to support the optimization and standardization of eDNA methods for fish detection by assessing two commercial DNA extraction kits manufactured by Qiagen and Macherey-Nagel on cost, time, and performance specifications and comparing the success of brook trout detection by metabarcoding across three bioinformatics pipelines, qPCR, and ddPCR. Our protocols were effective in detecting brook trout in all 20 samples analyzed. Brook trout eDNA was detected by ddPCR in nine (90%) Qiagen extracts but only seven (70%) Macherey-Nagel extracts. In comparison, detection success was equal across the two extraction kits using qPCR (70%) and metabarcoding (100%). The metabarcoding pipelines performed equally well in detecting brook trout with no significant differences in read numbers associated with the target species. Under our experimental conditions, the Qiagen kit was selected as the preferred kit due to its overall good performance and considerably lower cost despite a slightly longer extraction time.
