The room temperature preservation of filtered environmental <scp>DNA</scp> samples and assimilation into a phenol–chloroform–isoamyl alcohol <scp>DNA</scp> extraction

Renshaw, Mark A.; Olds, Brett P.; Jerde, Christopher L.; McVeigh, Margaret M.; Lodge, David M.

doi:10.1111/1755-0998.12281

Cited by 283 publications

(357 citation statements)

References 38 publications

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“…DNA was isolated following a modified chloroform -isoamyl alcohol (24:1; Amresco) extraction and an isopropanol precipitation. (Renshaw et al 2015; see full details in the Supplementary material, Appendix S1 1 ). To remove potential inhibitors, resuspended DNA was treated with the OneStepPCR Inhibitor Removal Kit (Zymo Research, Irvine, California, USA).…”

Section: Sample Processing and Extractionmentioning

confidence: 99%

Fish community assessment with eDNA metabarcoding: effects of sampling design and bioinformatic filtering

Evans

Renshaw

et al. 2017

Can. J. Fish. Aquat. Sci.

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164

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Species richness is a metric of biodiversity that represents the number of species present in a community. Traditional fisheries assessments that rely on capture of organisms often underestimate true species richness. Environmental DNA (eDNA) metabarcoding is an alternative tool that infers species richness by collecting and sequencing DNA present in the ecosystem. Our objective was to determine how spatial distribution of samples and "bioinformatic stringency" affected eDNA-metabarcoding estimates of species richness compared with capture-based estimates in a 2.2 ha reservoir. When bioinformatic criteria required species to be detected only in a single sample, eDNA metabarcoding detected all species captured with traditional methods plus an additional 11 noncaptured species. However, when we required species to be detected with multiple markers and in multiple samples, eDNA metabarcoding detected only seven of the captured species. Our analysis of the spatial patterns of species detection indicated that eDNA was distributed relatively homogeneously throughout the reservoir, except near the inflowing stream. We suggest that interpretation of eDNA metabarcoding data must consider the potential effects of water body type, spatial resolution, and bioinformatic stringency.Résumé : La richesse spécifique est une mesure de la biodiversité qui représente le nombre d'espèces présentes dans une communauté. Les évaluations traditionnelles des ressources halieutiques qui reposent sur la capture d'organismes sousestiment souvent la richesse spécifique réelle. Les méta-codes à barres d'ADN environnemental) (ADNe) constituent un autre outil qui permet d'inférer la richesse spécifique en recueillant et en séquençant l'ADN présent dans l'écosystème. Notre objectif consistait à déterminer comment la répartition spatiale des échantillons et la « rigueur bioinformatique » influent sur les estimations de la richesse spécifique reposant sur les méta-codes à barres d'ADNe par rapport aux estimations reposant sur la capture, dans un réservoir de 2,2 ha. Quand les critères bioinformatiques exigeaient la détection d'une espèce dans un seul échantillon, la méthode des méta-codes à barres d'ADNe a détecté toutes les espèces capturées par les méthodes traditionnelles en plus de 11 autres espèces non capturées. Toutefois, quand il fallait que les espèces soient détectées sur la base de plus d'un marqueur et dans plus d'un échantillon, les méta-codes à barres d'ADNe n'ont détecté que sept des espèces capturées. Notre analyse de la répartition spatiale de la détection d'espèces indique que l'ADNe était réparti de manière assez uniforme dans tout le réservoir, sauf près de l'embouchure du cours d'eau qui l'alimente. Nous proposons que l'interprétation des données obtenues par la méthode des méta-codes à barres d'ADNe doit tenir compte des effets potentiels du type de plan d'eau, de la résolution spatiale et de la rigueur bioinformatique. [Traduit par la Rédaction]

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Section: Sample Processing and Extractionmentioning

confidence: 99%

Fish community assessment with eDNA metabarcoding: effects of sampling design and bioinformatic filtering

Evans

Renshaw

et al. 2017

Can. J. Fish. Aquat. Sci.

Self Cite

164

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“…We kept these samples on ice until they could be processed in the lab (within hours of collection). We filtered the total volume of the samples (1 L) onto cellulose acetate filters (47 mm diameter; 0.45 um pore size) under vacuum pressure, and preserved the filter at room temperature in Longmire's buffer following (Renshaw et al, 2015). Deionized water (1-L) served as a negative control for filtering.…”

Section: Environmental Setting and Field Collectionsmentioning

confidence: 99%

“…Deionized water (1-L) served as a negative control for filtering. We extracted total DNA from the filters using the phenol:chloroform:isoamyl alcohol protocol in Renshaw et al (2015), resuspended the eluate in 200 uL water, and used 1 uL of diluted DNA extract (1:100) as template for PCR. See Supplemental Methods for additional sampling details.…”

Section: Environmental Setting and Field Collectionsmentioning

confidence: 99%

Genetic and Manual Survey Methods Yield Different and Complementary Views of an Ecosystem

et al. 2017

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Given the rapid rise of environmental DNA (eDNA) surveys in ecology and environmental science, it is important to be able to compare the results of these surveys to traditional methods of measuring biodiversity. Here we compare samples from a traditional method (a manual tow-net) to companion eDNA samples sequenced at three different genetic loci. We find only partial taxonomic overlap among the resulting datasets, with each reflecting a portion of the larger suite of taxa present in the sampled nearshore marine environment. In the larger context of eDNA sequencing surveys, our results suggest that primer amplification bias drives much of the taxonomic bias in eDNA detection, and that the baseline probability of detecting any given taxon with a broad-spectrum primer set is likely to be low. Whether catching fish with different nets or using different PCR primer sets, multiple data types can provide complementary views of a common ecosystem. However, it remains difficult to cross-validate eDNA sequencing techniques in the field, either for presence/absence or for abundance, particularly for primer sets that target very wide taxonomic ranges. Finally, our results highlight the breadth of diversity in a single habitat, and although eDNA does capture a richer sample of the community than traditional methods of sampling, a large number of eDNA primer sets focusing on different subsets of the biota would be necessary to survey any ecological community in a reasonably comprehensive way.

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“…We used a standard cetyl trimethyl ammonium bromide (CTAB) extraction method that has been used in numerous eDNA studies [1,28,36] and verified by Renshaw et al [38]. Therefore, capture efficiency should not have been a concern.…”

Section: Dna Extractionmentioning

confidence: 99%

Modelling the transport of environmental DNA through a porous substrate using continuous flow-through column experiments

Shogren

Tank

Andruszkiewicz

et al. 2016

J. R. Soc. Interface.

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Detecting environmental DNA (eDNA) in water samples is a powerful tool in determining the presence of rare aquatic species. However, many open questions remain as to how biological and physical conditions in flowing waters influence eDNA. Motivated by what one might find in a stream/ river benthos we conducted experiments in continuous flow columns packed with porous substrates to explore eDNA transport and ask whether substrate type and the presence of colonized biofilms plays an important role for eDNA retention. To interpret our data, and for modelling purposes, we began with the assumption that eDNA could be treated as a classical tracer. Comparing our experimental data with traditional transport models, we found that eDNA behaves anomalously, displaying characteristics of a heterogeneous, polydisperse substance with particle-like behaviour that can be filtered by the substrate. Columns were quickly flushed of suspended eDNA particles while a significant amount of particles never made it through and were retained in the column, as calculated from a mass balance. Suspended eDNA was exported through the column, regardless of biofilm colonization. Our results indicate that the variable particle size of eDNA results in stochastic retention, release and transport, which may influence the interpretation eDNA detection in biological systems.

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The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction

Cited by 283 publications

References 38 publications

Fish community assessment with eDNA metabarcoding: effects of sampling design and bioinformatic filtering

Fish community assessment with eDNA metabarcoding: effects of sampling design and bioinformatic filtering

Genetic and Manual Survey Methods Yield Different and Complementary Views of an Ecosystem

Modelling the transport of environmental DNA through a porous substrate using continuous flow-through column experiments

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