Abstract
DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding‐based biodiversity studies is gaining popularity as a noninvasive, time‐efficient, and cost‐effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under‐surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA‐related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species‐level assignment) and universal (broad taxonomic group with genus/family‐level assignment) approaches obtained from replicates treated with the optimal and a low‐performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false‐negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol‐chloroform‐isoamyl for successful implementation of eDNA multi‐marker metabarcoding surveys.
Highlights
Environmental DNA metabarcoding is defined as the simultaneous identification of a multitude of species through ‐generation sequencing from environmental samples. eDNA surveys are gaining attention as a novel, noninvasive, time‐efficient, and cost‐effective monitoring method (Thomsen & Willerslev, 2015)
Our results demonstrate that protocol choice in both the capture and extraction step in eDNA experiments significantly affects the DNA yield obtained from marine samples
For the first time, that targeted metabarcoding assays amplifying low‐abundance taxonomic groups are far more sensitive to protocol choice and the resulting reduced DNA yields compared to broad‐ scale metabarcoding assays
Summary
Environmental DNA (eDNA) metabarcoding is defined as the simultaneous identification of a multitude of species through ‐generation sequencing from environmental samples (soil, sediment, water). eDNA surveys are gaining attention as a novel, noninvasive, time‐efficient, and cost‐effective monitoring method (Thomsen & Willerslev, 2015). Environmental DNA (eDNA) metabarcoding is defined as the simultaneous identification of a multitude of species through ‐generation sequencing from environmental samples (soil, sediment, water). EDNA surveys are gaining attention as a novel, noninvasive, time‐efficient, and cost‐effective monitoring method (Thomsen & Willerslev, 2015). Aquatic eDNA has shown great promise as an alternative monitoring method in aquatic environments (Hunter et al, 2015) and in assessing their eukaryotic biodiversity (Stat et al, 2017; Thomsen et al, 2012). A meta‐analysis of the current literature shows a broadening spectrum of protocols being used to capture and extract eDNA from aquatic samples (Figure 1a; Supplement 1). The volume of water is concentrated in a capture step using either precipitation (Dejean et al, 2012), centrifugation (Klymus, Richter, Chapman, & Paukert, 2015), or filtration (Port et al, 2016). DNA is extracted by one of a wide range of commercial kits and modified extraction protocols
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