Optimization of environmental DNA-based methods: A case study for detecting brook trout (Salvelinus fontinalis)

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.