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

Area of Science:

• Environmental DNA (eDNA) research
• Aquatic ecology
• Molecular biology techniques

Background:

• Environmental DNA (eDNA) analysis is crucial for fish monitoring, utilizing techniques like quantitative PCR (qPCR), digital droplet PCR (ddPCR), and metabarcoding.
• Standardization of eDNA workflows, particularly DNA extraction and sequence analysis, is lacking, hindering sensitivity and cross-study comparisons.
• DNA extraction significantly influences eDNA detection efficiency, making it a critical step for optimization.

Purpose of the Study:

• To evaluate and compare two commercial DNA extraction kits (Qiagen and Macherey-Nagel) for eDNA-based fish detection.
• To assess the performance of these kits across various analytical methods, including qPCR, ddPCR, and metabarcoding with different bioinformatics pipelines.
• To provide a framework for optimizing and standardizing eDNA methods for reliable fish monitoring.

Main Methods:

• Two commercial DNA extraction kits (Qiagen, Macherey-Nagel) were tested using environmental samples containing brook trout DNA.
• Detection of brook trout eDNA was performed using qPCR, ddPCR, and metabarcoding across three distinct bioinformatics pipelines.
• Kit performance was evaluated based on detection success rates, DNA concentration, and cost-effectiveness.

Main Results:

• Both kits successfully detected brook trout eDNA in all samples using metabarcoding (100% success).
• ddPCR showed higher detection rates (90% vs. 70%) and DNA concentrations with the Qiagen kit compared to Macherey-Nagel.
• qPCR detection rates were equal (70%) for both kits, with no significant difference in DNA concentration; however, metabarcoding yielded more reads with Qiagen using the MetaWorks pipeline.

Conclusions:

• The Qiagen DNA extraction kit is recommended for eDNA fish monitoring due to its superior or equal performance across multiple detection methods and lower cost.
• Standardizing DNA extraction methods is essential for improving the sensitivity and comparability of eDNA surveys.
• The study provides a replicable framework for evaluating new eDNA extraction kits to advance the field of environmental DNA monitoring.