Optimizing an enclosed bead beating extraction method for microbial and fish environmental DNA

Anderson, Sean R.; Thompson, Luke R.

doi:10.1002/edn3.251

Cited by 11 publications

(12 citation statements)

References 68 publications

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“…More water needed to be DNA extracted from Bedok samples in order to obtain enough DNA for all experiments (Lim et al, 2016; Loh, 2019). DNA extraction utilized a conventional phenol–chloroform protocol (see Appendix S2: Materials and Methods [Sambrook & Russell, 2006]) although the literature illustrates that bead‐based and commercial kits are also suitable for the extraction of eDNA (for example, bead‐based DNA extraction: Anderson & Thompson, 2022; commercial kits: Kumar et al, 2022), and we are now also using such kits in follow‐up studies. Negative controls were included in each round of DNA extraction (“extraction negatives”, nine in total), whereby empty Falcon tubes were used in place of sample tubes.…”

Section: Methodsmentioning

confidence: 99%

Toward eDNA‐based bioassessment of freshwater reservoirs with small volumes of water: Robust molecular protocols

et al. 2022

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Bioassessment of freshwater quality with eDNA is a powerful alternative to traditional methods involving collecting, sorting, and identifying metazoan taxa with morphology (e.g., macroinvertebrates). Particularly attractive for routine monitoring would be an eDNA method that uses a remote-controlled boat for collecting small volumes of water without filtration in order to simplify sampling. Such a method would not likely capture eDNA signals for all metazoan species, but may nevertheless allow for cost-effective, and frequent monitoring of water quality, which is important for tropical waterbodies that require year-round surveillance. We here optimize a molecular protocol for capturing metazoan signatures based on eDNA obtained from 15 ml of water. To test the robustness of the method, we used samples from two tropical reservoirs with known differences in water quality to optimize molecular procedures so that they yield repeatable results. Each reservoir was sampled at three sites ("biological replicates") and each water sample was subsampled twice before extracting the eDNA using ethanol precipitation for both subsamples ("technical replicates"). We then tested how much DNA (0.1 ng to 15 ng) and how many PCR cycles (25 or 35) minimized the variance of the metazoan eDNA signal between biological and technical replicates. For this purpose, we amplified a 313 bp COI minibarcode using a universal metazoan primer pair. We found that regardless of template amounts or PCR cycle numbers, the eDNA signatures for both reservoirs were distinct because only 17 of 59 mOTUs (mainly planktonic crustaceans and rotifers) were shared. We also found that template amounts between 0.5 and 15 ng yielded overall similar results, but the use of 35 PCR cycles significantly depressed the number of detected species (p-value < 0.05). Fortunately, the differences between the detected metazoan community of the reservoirs were so strong that all biological and technical replicates could be assigned unambiguously to their source reservoir although the variance between technical replicates remained high for all PCR experiments (Bray-Curtis dissimilarity: 5%-20%; Jaccard distance: 10%-40%).

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Section: Methodsmentioning

confidence: 99%

Toward eDNA‐based bioassessment of freshwater reservoirs with small volumes of water: Robust molecular protocols

et al. 2022

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“…In any case, most commercially available extraction kits are designed for liquid cultures or tissues that fully dissolve. This circumstance has resulted in a wide array of “standard” modifications to manufacturers’ protocols (e.g., Cruaud et al, 2017 ; Ushio, 2019 ; Anderson and Thompson, 2022 ), further complicating the quest for harmonized practice.…”

Section: Filtration Strategymentioning

confidence: 99%

“…In comparisons of three tested kits offered by Qiagen (Qiagen GmbH, Hilden, Germany), the DNeasy Blood & Tissue kit provided the best results for DNA yield ( Djurhuus et al, 2017 ; Hinlo et al, 2017 ) and equaled or outperformed the PowerWater kit in metabarcoding data quality and consistency ( Jeunen et al, 2019 ), particularly when modified with a bead-beating step ( Djurhuus et al, 2017 ). Other less commonly used kits described in the literature we reviewed ( Supplementary Table S1 ) include the ZymoBIOMICS 96 DNA/RNA MagBead (e.g., Anderson and Thompson, 2022 ), Epicentre MasterPure DNA purification (e.g., Geerts et al, 2018 ), Qiagen AllPrep DNA/RNA mini (e.g., Cruaud et al, 2017 ), and Presto Mini gDNA (e.g., Jeunen et al, 2019 ), but we found too few studies describing the performance of these kits to comment further.…”

Section: Dna Extractionmentioning

confidence: 99%

“…This mechanical lysis step improved microbial community DNA yield over both an internal extraction protocol without beads and a protocol based on opening the cartridge, while maintaining the enclosed filter environment ( Ushio, 2019 ). Anderson and Thompson (2022) adapted this protocol in several useful ways, including adding beads after sample collection, optimization of bead composition to maximize recovery of hard-to-lyse organisms, and demonstrating high-throughput extractions performed on a magnetic bead-handling robot [KingFisher™ Flex Purification System (Thermo Fisher Scientific, Waltham, MA)].…”

Section: Dna Extractionmentioning

confidence: 99%

“… Trigodet et al (2022) found that column-based commercial kits with enzyme modifications were equally as, if not more, effective than PCI protocols for generating high-quality long read sequences. In addition, robotic sampling and processing instruments, such as the KingFisher™ Flex Purification System mentioned above, can reduce the time and effort required to process eDNA samples ( Marotz et al, 2017 ; Anderson and Thompson, 2022 ). Autonomous samplers, like the Monterey Bay Aquarium Research Institute Long-Range Autonomous Underwater Vehicle containing an Environmental Sample Processor (3G ESP-LRAUV), can filter marine water in situ and preserve filters equivalent to traditional shipboard sampling for eDNA analysis by qPCR ( Yamahara et al, 2019 ) or DNA metabarcoding ( Truelove et al, 2022 ).…”

Section: Dna Extractionmentioning

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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A Guide to Environmental DNA Extractions for Non‐Molecular Trained Biologists, Ecologists, and Conservation Scientists

Rieder,

Jemmi,

Hunter

et al. 2024

Environmental DNA

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Ecologists, biologists, and conservation scientists are increasingly interested in the use of environmental DNA (eDNA) data for research and potentially decision‐making. While commercial DNA extraction kits are typically user‐friendly and accessible, they may fail to deliver the desired results with inherently complex eDNA samples, necessitating protocol optimization or educated selection of alternative approaches. To this end, knowledge of the basic steps and principles of DNA extractions is essential, but traditional education tracks in ecology, conservation, and environmental management typically do not include in‐depth training in molecular methods. The primary objective of this paper is to enable scientists with an ecological background and limited molecular training to understand the four key steps of eDNA isolations, and to use this expertise to their advantage. We describe the purpose of commonly used reagents and chemicals, point out alternatives for each key step, explain the impact of certain choices regarding isolation approaches on DNA integrity and purity, and highlight the possibility of a tailor‐made “mix and match” approach. We anticipate that this paper will enable field ecologists to develop a deeper understanding of the mechanisms and chemistry underlying eDNA extractions, thus allowing them to make informed decisions regarding the best eDNA extraction method for their research goals. Our intention is not to provide comprehensive, step‐by‐step protocols, but to offer guiding principles while highlighting alternative solutions. Finally, we hope that this paper will act as a useful resource to support knowledge transfer and teaching.

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Optimizing an enclosed bead beating extraction method for microbial and fish environmental DNA

Cited by 11 publications

References 68 publications

Toward eDNA‐based bioassessment of freshwater reservoirs with small volumes of water: Robust molecular protocols

Toward eDNA‐based bioassessment of freshwater reservoirs with small volumes of water: Robust molecular protocols

Capturing marine microbiomes and environmental DNA: A field sampling guide

A Guide to Environmental DNA Extractions for Non‐Molecular Trained Biologists, Ecologists, and Conservation Scientists

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