Towards robust and repeatable sampling methods in <scp>eDNA</scp>‐based studies

Dickie, Ian A.; Boyer, Stéphane; Buckley, Hannah; Duncan, Richard P.; Gardner, Paul P.; Hogg, Ian D.; Holdaway, Robert J.; Lear, Gavin; Makiola, Andreas; Morales, Sergio E.; Powell, Jeff R.; Weaver, Louise

doi:10.1111/1755-0998.12907

Cited by 140 publications

(156 citation statements)

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“…It may be appropriate to optimize DNA extraction and PCR methods on a case‐by‐case basis in the form of pilot studies. Together with other recently published examinations of sampling, laboratory, and analysis methods (Alberdi et al., ; Dickie et al., ; Murray et al., ), our results provide a guide for future DNA metabarcoding studies of soil biodiversity.…”

Section: Discussionsupporting

confidence: 74%

“…DNA metabarcoding analyses involve a series of methodological decisions that are likely to affect measures of biodiversity (Alberdi, Aizpurua, Gilbert, & Bohmann, 2017;Dickie et al, 2018;Murray, Coghlan, & Bunce, 2015). The effects of DNA extraction methods on DNA yield and detected microbial community composition have been examined for substrates including oral and rumen samples (Henderson et al, 2013;Vesty, Biswas, Taylor, Gear, & Douglas, 2017), permafrost (Vishnivetskaya et al, 2014), and soils (Delmont et al, 2011;Kang & Mills, 2006;Wagner, Praeg, Reitschuler, & Illmer, 2015).…”

Section: Introductionmentioning

confidence: 99%

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Impacts of DNA extraction and PCR on DNA metabarcoding estimates of soil biodiversity

et al. 2018

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Soils are ubiquitous and important components of terrestrial ecosystems, richly populated with diverse organisms with important ecological roles. DNA metabarcoding is a promising tool for efficient and taxonomically comprehensive analyses of soil biodiversity, but the outcomes of these analyses are likely affected by basic methodological factors such as sampling and laboratory protocols. We investigated the impacts of DNA extraction methodology and multiple PCRs on DNA metabarcoding biodiversity estimates for multiple taxonomic groups in soil. We applied four DNA extraction methods with different size constraints in parallel to fixed volumes of soil from a pair of forest sites, followed by sets of ten individually indexed PCRs and Illumina sequencing of prokaryote (16S) and eukaryote (18S, fungal 26S, and metazoan cytochrome c oxidase subunit I) barcodes. Methods utilizing larger soil volumes resulted in higher biodiversity estimates for arthropods but not for prokaryotes or microeukaryotes, and improved spatial discrimination of metazoan communities but not prokaryotes. Each DNA extraction method resulted in a biased community composition, and these biases were consistent across sites and amplicons. Some 36%–41% of operational taxonomic units (OTUs) were shared between all four DNA extraction methods and OTUs from many phyla had contrasting abundances from different methods. From 0.2% to 19% of OTUs, accounting for 9.4% to 97% of sequence reads, were amplified in all 10 PCRs from each DNA extraction method and sample. OTUs detected in only a single PCR accounted for 26% to 41% of OTUs but only 0.3% to 2.8% of the sequence reads from each extraction method and sample. The inferred species richness increased strongly with the number of PCRs carried out. We conclude that a consistent DNA extraction method should be used for comparisons between samples, confirm that larger samples of soil (≥15 g) should be used for analyses of metazoan biodiversity than for microbes, and that the number of PCR replicates undertaken strongly affects species richness estimates.

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Section: Discussionsupporting

confidence: 74%

Section: Introductionmentioning

confidence: 99%

Impacts of DNA extraction and PCR on DNA metabarcoding estimates of soil biodiversity

et al. 2018

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“…Taken together, these data indicate that, like many other considerations regarding eDNA experimental design (Dickie et al, ), substrate selection is a major, but largely unrecognized, factor. After exploring this multi‐substrate metabarcoding data, we advocate that a priori assumptions about the “best” substrate and level of replication required are difficult to generalize and best approached through pilot studies.…”

Section: Discussionsupporting

confidence: 52%

Environmental DNA metabarcoding studies are critically affected by substrate selection

Bunce

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Simpson

et al. 2018

Molecular Ecology Resources

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Effective biomonitoring is critical for driving management outcomes that ensure long‐term sustainability of the marine environment. In recent years, environmental DNA (eDNA), coupled with metabarcoding methodologies, has emerged as a promising tool for generating biotic surveys of marine ecosystems, including those under anthropogenic pressure. However, more empirical data are needed on how to best implement eDNA field sampling approaches to maximize their utility for each specific application. The effect of the substrate chosen for eDNA sampling on the diversity of marine taxa detected by DNA metabarcoding has not yet been systematically analysed, despite aquatic systems being those most commonly targeted for eDNA studies. We investigated the effect of four commonly used eDNA substrates to explore taxonomic diversity: (a) surface water, (b) marine sediment, (c) settlement plates and (d) planktonic tows. With a focus on coastal ports, 332 eDNA samples from Australia (Indian and Southern oceans) and Kazakhstan (Caspian Sea) were collected and analysed by multi‐assay DNA metabarcoding. Across study locations, between 30% and 52% of eukaryotic families detected were unique to a particular substrate and <6% of families were found in all four substrates. Taxonomic composition varied significantly depending on the substrate sampled implying that the suitability (and bias) of an eDNA substrate will depend on the focal taxa. These findings demonstrate that single substrate eDNA metabarcoding likely underestimates the total eukaryotic diversity. Future eDNA experimental design should consider incorporating multiple substrates or select substrate(s) best suited to the specific detection of target taxa.

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“…This is a major barrier impeding the transition of singular results into widely accepted facts. Research based on high-throughput sequencing of eDNA faces problems in this regard (Dickie et al, 2018), due not only to its recent history and rapidly developing methodological approaches, but the plethora of distortive aspects mentioned in the main text.…”

Section: Box 5 Reproducibility Replicability and Comparability Of Sementioning

confidence: 99%

Promises and pitfalls of using high‐throughput sequencing for diet analysis

Alberdi

Aizpurua

Bohmann

et al. 2018

Molecular Ecology Resources

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The application of high‐throughput sequencing‐based approaches to DNA extracted from environmental samples such as gut contents and faeces has become a popular tool for studying dietary habits of animals. Due to the high resolution and prey detection capacity they provide, both metabarcoding and shotgun sequencing are increasingly used to address ecological questions grounded in dietary relationships. Despite their great promise in this context, recent research has unveiled how a wealth of biological (related to the study system) and technical (related to the methodology) factors can distort the signal of taxonomic composition and diversity. Here, we review these studies in the light of high‐throughput sequencing‐based assessment of trophic interactions. We address how the study design can account for distortion factors, and how acknowledging limitations and biases inherent to sequencing‐based diet analyses are essential for obtaining reliable results, thus drawing appropriate conclusions. Furthermore, we suggest strategies to minimize the effect of distortion factors, measures to increase reproducibility, replicability and comparability of studies, and options to scale up DNA sequencing‐based diet analyses. In doing so, we aim to aid end‐users in designing reliable diet studies by informing them about the complexity and limitations of DNA sequencing‐based diet analyses, and encourage researchers to create and improve tools that will eventually drive this field to its maturity.

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Towards robust and repeatable sampling methods in eDNA‐based studies

Cited by 140 publications

References 58 publications

Impacts of DNA extraction and PCR on DNA metabarcoding estimates of soil biodiversity

Impacts of DNA extraction and PCR on DNA metabarcoding estimates of soil biodiversity

Environmental DNA metabarcoding studies are critically affected by substrate selection

Promises and pitfalls of using high‐throughput sequencing for diet analysis

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