Abstract
Properly designed (randomized and/or balanced) experiments are standard in ecological research. Molecular methods are increasingly used in ecology, but studies generally do not report the detailed design of sample processing in the laboratory. This may strongly influence the interpretability of results if the laboratory procedures do not account for the confounding effects of unexpected laboratory events. We demonstrate this with a simple experiment where unexpected differences in laboratory processing of samples would have biased results if randomization in DNA extraction and PCR steps do not provide safeguards. We emphasize the need for proper experimental design and reporting of the laboratory phase of molecular ecology research to ensure the reliability and interpretability of results.
Highlights
Ecological studies regularly ensure that the experimental setup is randomized and/or balanced
We demonstrate this with a simple experiment where unexpected differences in laboratory processing of samples would have biased results if randomization in DNA extraction and PCR steps do not provide safeguards
We emphasize the need for proper experimental design and reporting of the laboratory phase of molecular ecology research to ensure the reliability and interpretability of results
Summary
Ecological studies regularly ensure that the experimental setup is randomized and/or balanced. The importance of randomized experimental setups (Fisher, 1936) along with balanced designs (Student & Student 1938) is well known Such designs are enforced today in manipulative or observational ecological research (Hurlbert, 1984; Smith, Anderson, & Pawley, 2017). As molecular ecology studies mostly work with high observation numbers (thousands of SNPs over genomes, thousands of operational taxonomic units—OTUs—in hundreds of samples, etc.), even small chance events may result in statistically significant results (Carver, 1993). We demonstrate this with taxonomically informative marker gene fragments amplified from environmental DNA (eDNA metabarcoding). We evaluated several sources of variation: (1) expected laboratory biases (DNA extraction kit, Deiner, Walser, Mächler, & Altermatt, 2015; Barlow et al, 2016), (2) unexpected laboratory biases (e.g., a sudden change in laboratory personnel), and (3) an ecologically interesting predictor (either the age of the sediment or the effects of a power plant)
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