Assessing the performance and efficiency of environmental DNA/RNA capture methodologies under controlled experimental conditions

Zaiko, Anastasija; Ammon, Ulla von; Stuart, Jacqui; Smith, Kirsty F.; Yao, Richard; Welsh, Melissa; Pochon, Xavier; Bowers, Holly A.

doi:10.1111/2041-210x.13879

Cited by 19 publications

(20 citation statements)

References 89 publications

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“…In addition to non-specific amplification, extraction efficiency, reverse transcription efficiency of cDNA, and PCR bias should be validated to compare the copy numbers between DNA and RNA; however, avoiding these experimental biases is very difficult. Co-extraction, employed to minimize bias, is one of the best approaches to compare eDNA and eRNA and helps to reduce sampling bias. ,, Over the study period, fish DNA and RNA copy numbers in the influent and the effluent per sampling day were similar (Figure S2). The number of positive droplets in negative controls (negative filtration controls, netative co-extraction controls, and negative cDNA synthesis controls) was similar to that in negative controls (sterile distilled water).…”

Section: Resultsmentioning

confidence: 99%

Environmental Nucleic Acid Pollution: Characterization of Wastewater Generating False Positives in Molecular Ecological Surveys

et al. 2023

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Environmental nucleic acids (eDNA and eRNA) metabarcoding analyses have attracted considerable research attention as they non-invasively and cost-effectively monitor ecosystems. For heretofore unknown reasons, however, they often detect false positives. Here, we focused on domestic wastewater as a possible source of false positives and characterized fish nucleic acids in the influent and effluent of a wastewater treatment plant (WWTP). The metabarcoding analysis revealed that DNA and RNA detected in the influent wastewater originated from over 120 fish species, and most of these were consumed fish. The nucleic acid content was significantly higher in the influent than in the effluent, and the copy numbers of DNA in the influent were significantly higher than those of RNA. These findings were corroborated by the current literature and indicated that eRNA could effectively mitigate false positives. The present study demonstrated that rivers may be extensively contaminated by fish nucleic acids, and WWTPs substantially alter the quantities and ratios of fish DNA and RNA. Therefore, analyses of the wastewater near sampling points and coverage of the sewage system enable us to select and use the appropriate nucleic acid type (DNA or RNA) for metabarcoding analysis and improve the accuracy of fish species detection and identification.

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Section: Resultsmentioning

confidence: 99%

Environmental Nucleic Acid Pollution: Characterization of Wastewater Generating False Positives in Molecular Ecological Surveys

et al. 2023

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“…Similar validation techniques can be used to develop eRNA metabarcoding as a quantitative or semi-quantitative method. There is growing interest in optimizing eRNA extraction protocols from different types of environmental media to standardize the use of eRNA in downstream molecular applications 55 . Thus, standardizing eRNA protocols will help with integration into environmental management toolkits for regulatory purposes.…”

Section: Discussionmentioning

confidence: 99%

Environmental RNA as a Tool for Marine Community Biodiversity Assessments

Giroux

Reichman

Langknecht

et al. 2022

Sci Rep

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Microscopic organisms are often overlooked in traditional diversity assessments due to the difficulty of identifying them based on morphology. Metabarcoding is a method for rapidly identifying organisms where Environmental DNA (eDNA) is used as a template. However, legacy DNA is problematically detected from organisms no longer in the environment during sampling. Environmental RNA (eRNA), which is only produced by living organisms, can also be collected from environmental samples and used for metabarcoding. The aim of this study was to determine differences in community composition and diversity between eRNA and eDNA templates for metabarcoding. Using mesocosms containing field-collected communities from an estuary, RNA and DNA were co-extracted from sediment, libraries were prepared for two loci (18S and COI), and sequenced using an Illumina MiSeq. Results show a higher number of unique sequences detected from eRNA in both markers and higher α-diversity compared to eDNA. Significant differences between eRNA and eDNA for all β-diversity metrics were also detected. This study is the first to demonstrate community differences detected with eRNA compared to eDNA from an estuarine system and illustrates the broad applications of eRNA as a tool for assessing benthic community diversity, particularly for environmental conservation and management applications.

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“…Studies on human microbiome samples ( Gorzelak et al, 2015 ; Song et al, 2016 ) and eukaryotic eDNA ( Thomsen et al, 2012 ; Maruyama et al, 2014 ; Yamanaka et al, 2016 ) have shown changes in DNA yield and composition over relatively short time scales; however, other reports offer conflicting results on microbiome stability (e.g., Lauber et al, 2010 ). Environmental RNA is more prone to degradation than eDNA ( Marshall et al, 2021 ; Zaiko et al, 2022 ); thus, shorter filtration times are needed. If RNA is targeted for collection, limit filtration to 20 minutes to provide a practical timeframe to process multiple samples ( Zaiko et al, 2022 ) or 15 minutes to be in accordance with AtlantECO protocols ( Pesant et al, 2022 ).…”

Section: Logistic and Resource Considerationsmentioning

confidence: 99%

“…Environmental RNA is more prone to degradation than eDNA ( Marshall et al, 2021 ; Zaiko et al, 2022 ); thus, shorter filtration times are needed. If RNA is targeted for collection, limit filtration to 20 minutes to provide a practical timeframe to process multiple samples ( Zaiko et al, 2022 ) or 15 minutes to be in accordance with AtlantECO protocols ( Pesant et al, 2022 ).…”

Section: Logistic and Resource Considerationsmentioning

confidence: 99%

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Capturing marine microbiomes and environmental DNA: A field sampling guide

Patin

Goodwin

2023

Front. Microbiol.

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The expanding interest in marine microbiome and eDNA sequence data has led to a demand for sample collection and preservation standard practices to enable comparative assessments of results across studies and facilitate meta-analyses. We support this effort by providing guidelines based on a review of published methods and field sampling experiences. The major components considered here are environmental and resource considerations, sample processing strategies, sample storage options, and eDNA extraction protocols. It is impossible to provide universal recommendations considering the wide range of eDNA applications; rather, we provide information to design fit-for-purpose protocols. To manage scope, the focus here is on sampling collection and preservation of prokaryotic and microeukaryotic eDNA. Even with a focused view, the practical utility of any approach depends on multiple factors, including habitat type, available resources, and experimental goals. We broadly recommend enacting rigorous decontamination protocols, pilot studies to guide the filtration volume needed to characterize the target(s) of interest and minimize PCR inhibitor collection, and prioritizing sample freezing over (only) the addition of preservation buffer. An annotated list of studies that test these parameters is included for more detailed investigation on specific steps. To illustrate an approach that demonstrates fit-for-purpose methodologies, we provide a protocol for eDNA sampling aboard an oceanographic vessel. These guidelines can aid the decision-making process for scientists interested in sampling and sequencing marine microbiomes and/or eDNA.

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Assessing the performance and efficiency of environmental DNA/RNA capture methodologies under controlled experimental conditions

Cited by 19 publications

References 89 publications

Environmental Nucleic Acid Pollution: Characterization of Wastewater Generating False Positives in Molecular Ecological Surveys

Environmental Nucleic Acid Pollution: Characterization of Wastewater Generating False Positives in Molecular Ecological Surveys

Environmental RNA as a Tool for Marine Community Biodiversity Assessments

Capturing marine microbiomes and environmental DNA: A field sampling guide

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