scholarly journals Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
Ryohei Nakao ◽  
Hideyuki Doi ◽  
Toshifumi Minamoto
Keyword(s):  
Fish Species ◽  
High Throughput Sequencing ◽  
Correlation Coefficients ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Taqman Probe ◽  
Individual Species ◽  
Natural Environments ◽  
Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

scholarly journals Monitoring spawning migrations of potamodromous fish species via eDNA

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Bettina Thalinger ◽  
Elisabeth Wolf ◽  
Michael Traugott ◽  
Josef Wanzenböck
Keyword(s):  
Fish Species ◽  
Anthropogenic Impacts ◽  
Environmental Dna ◽  
Signal Strength ◽  
Digital Pcr ◽  
Freshwater Ecosystems ◽  
Non Invasive ◽  
River Stretch ◽  
Daily Discharge ◽  
Invasive Techniques

Abstract Potamodromous fish are considered important indicators of habitat connectivity in freshwater ecosystems, but they are globally threatened by anthropogenic impacts. Hence, non-invasive techniques are necessary for monitoring during spawning migrations. The use of environmental DNA (eDNA) potentially facilitates these efforts, albeit quantitative examinations of spawning migrations remain so far mostly uncharted. Here, we investigated spawning migrations of Danube bleak, Alburnus mento, and Vimba bream, Vimba vimba, and found a strong correlation between daily visual fish counts and downstream eDNA signals obtained from filtered water samples analysed with digital PCR and end-point PCR coupled with capillary electrophoresis. By accounting for daily discharge fluctuations, it was possible to predict eDNA signal strength from the number of migrating fish: first, the whole spawning reach was taken into account. Second, the model was validated using eDNA signals and fish counts obtained from the upper half of the examined river stretch. Consequently, fish counts and their day-to-day changes could be described via an eDNA-based time series model for the whole migration period. Our findings highlight the capability of eDNA beyond delivering simple presence/absence data towards efficient and informative monitoring of highly dynamic aquatic processes such as spawning migrations of potamodromous fish species.

scholarly journals Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 243 ◽  
Author(s):  
Teruaki Tozaki ◽  
Aoi Ohnuma ◽  
Masaki Takasu ◽  
Mio Kikuchi ◽  
Hironaga Kakoi ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Genetic Manipulation ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Taqman Probe ◽  
Gene Doping ◽  
Athletic Ability ◽  
Horse Plasma ◽  
Primer Sets

Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic manipulation are urgently needed. Here, we developed a highly sensitive method to detect horse erythropoietin (EPO) transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the EPO transgene was cloned into a plasmid for use as a model. We extracted the spiked EPO transgene from horse plasma and urine via magnetic beads, followed by ddPCR amplification for absolute quantification and transgene detection. The results indicated high recovery rates (at least ~60% and ~40% in plasma and urine, respectively), suggesting successful detection of the spiked transgene at concentrations of >130 and 200 copies/mL of plasma and urine, respectively. Additionally, successful detection was achieved following intramuscular injection of 20 mg of the EPO transgene. This represents the first study demonstrating a method for detecting the EPO transgene in horse plasma and urine, with our results demonstrating its efficacy for promoting the control of gene-doping in the horseracing industry.

scholarly journals Quantitative monitoring of multispecies fish environmental DNA using high-throughput sequencing

2017 ◽  
Author(s):  
Masayuki Ushio ◽  
Hiroaki Murakami ◽  
Reiji Masuda ◽  
Tetsuya Sado ◽  
Masaki Miya ◽  
...  
Keyword(s):  
Fish Species ◽  
High Throughput Sequencing ◽  
Marine Ecosystem ◽  
Internal Standard ◽  
Environmental Dna ◽  
Illumina Miseq ◽  
Pcr Inhibition ◽  
Copy Numbers ◽  
Quantitative Monitoring ◽  
Monitoring Of Biodiversity

Effective ecosystem conservation and resource management require quantitative monitoring of biodiversity, including accurate descriptions of species composition and temporal variations of species abundance. Therefore, quantitative monitoring of biodiversity has been performed for many ecosystems, but it is often time- and effort-consuming and costly. Recent studies have shown that environmental DNA (eDNA), which is released to the environment from macro-organisms living in a habitat, contains information about species identity and abundance. Thus, analyzing eDNA would be a promising approach for more efficient biodiversity monitoring. In the present study, we added internal standard DNAs (i.e., known amounts of short DNA fragments from fish species that have never been observed in a sampling area) to eDNA samples, which were collected weekly from a coastal marine ecosystem in Maizuru-Bay, Kyoto, Japan (from April 2015 to March 2016), and performed metabarcoding analysis using Illumina MiSeq to simultaneously identify fish species and quantify fish eDNA copy numbers. A correction equation was obtained for each sample using the relationship between the number of sequence reads and the added amount of the standard DNAs, and this equation was used to estimate the copy numbers from the sequence reads of non-standard fish eDNA. The calculated copy numbers showed significant positive correlation with those determined by quantitative PCR, suggesting that eDNA metabarcoding with standard DNA enabled useful quantification of eDNA. Furthermore, for samples that show a high level of PCR inhibition, our method might allow more accurate quantification than qPCR because the correction equations generated using internal standard DNAs would include the effect of PCR inhibition. A single run of Illumina MiSeq produced > 70 quantitative fish eDNA time series in our study, showing that our method could contribute to more efficient and quantitative monitoring of biodiversity.

scholarly journals Environmental DNA methods for the analysis of macroorganismal populations

2021 ◽  
Vol 4 ◽  
Author(s):  
Hideyuki Doi
Keyword(s):  
Fish Species ◽  
Population Analysis ◽  
Species Distributions ◽  
Environmental Dna ◽  
Terrestrial Ecosystems ◽  
Measurement Methods ◽  
Natural Environments ◽  
New Methods ◽  
Dominant Fish ◽  
Last 300 Years

Environmental DNA (eDNA) methods have been widely used to investigate the distribution and abundance/biomass of macroorganisms. eDNA methods analyze DNA collected directly from the environment, such as from water, soil, and air. The techniques have been applied to many taxa inhabiting various aquatic and terrestrial ecosystems. The recent development of eDNA methods has revolutionized the way we assess macroorganisms in natural environments. In this talk, I will present current developments of eDNA methodology, especially with regard to population analysis using various DNA measurement methods. For example, 1) eDNA was used to assess fish species distributions and abundance/biomass (Takahara et al. 2012, Doi et al. 2017a), 2) quantitative PCR of sedimentary DNA was applied to sediment core samples to detect the DNA of three dominant fish species spanning the last 300 years (Kuwae et al. 2020), and 3) new methods for sampling eDNA from water (Doi et al. 2017b) and on-site measurement (Doi et al. 2020) will be presented. I end by addressing the need for standardized protocols for eDNA monitoring to enable broader uptake of eDNA technology (Minamoto et al. 2021).

scholarly journals Standards for Methods Utilizing Environmental DNA for Detection of Fish Species

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 296 ◽  
Author(s):  
Lu Shu ◽  
Arne Ludwig ◽  
Zuogang Peng
Keyword(s):  
Fish Species ◽  
High Throughput Sequencing ◽  
Quantitative Polymerase Chain Reaction ◽  
Environmental Dna ◽  
Aquatic Organism ◽  
Negative Control ◽  
Community Diversity ◽  
Gene Markers ◽  
Mitochondrial Cytochrome B ◽  
Fish Detection

Environmental DNA (eDNA) techniques are gaining attention as cost-effective, non-invasive strategies for acquiring information on fish and other aquatic organisms from water samples. Currently, eDNA approaches are used to detect specific fish species and determine fish community diversity. Various protocols used with eDNA methods for aquatic organism detection have been reported in different eDNA studies, but there are no general recommendations for fish detection. Herein, we reviewed 168 papers to supplement and highlight the key criteria for each step of eDNA technology in fish detection and provide general suggestions for eliminating detection errors. Although there is no unified recommendation for the application of diverse eDNA in detecting fish species, in most cases, 1 or 2 L surface water collection and eDNA capture on 0.7-μm glass fiber filters followed by extraction with a DNeasy Blood and Tissue Kit or PowerWater DNA Isolation Kit are useful for obtaining high-quality eDNA. Subsequently, species-specific quantitative polymerase chain reaction (qPCR) assays based on mitochondrial cytochrome b gene markers or eDNA metabarcoding based on both 12S and 16S rRNA markers via high-throughput sequencing can effectively detect target DNA or estimate species richness. Furthermore, detection errors can be minimized by mitigating contamination, negative control, PCR replication, and using multiple genetic markers. Our aim is to provide a useful strategy for fish eDNA technology that can be applied by researchers, advisors, and managers.

Barcoding the food chain: from Sanger to high-throughput sequencing

Genome ◽  
2016 ◽  
Vol 59 (11) ◽  
pp. 946-958 ◽  
Author(s):  
Joanne E. Littlefair ◽  
Elizabeth L. Clare
Keyword(s):  
Dna Barcoding ◽  
High Throughput ◽  
Food Chain ◽  
High Throughput Sequencing ◽  
Human Mobility ◽  
Environmental Dna ◽  
Universal Primers ◽  
Individual Species ◽  
Parasitic Infections ◽  
Forensic Evaluation

Society faces the complex challenge of supporting biodiversity and ecosystem functioning, while ensuring food security by providing safe traceable food through an ever-more-complex global food chain. The increase in human mobility brings the added threat of pests, parasites, and invaders that further complicate our agro-industrial efforts. DNA barcoding technologies allow researchers to identify both individual species, and, when combined with universal primers and high-throughput sequencing techniques, the diversity within mixed samples (metabarcoding). These tools are already being employed to detect market substitutions, trace pests through the forensic evaluation of trace “environmental DNA”, and to track parasitic infections in livestock. The potential of DNA barcoding to contribute to increased security of the food chain is clear, but challenges remain in regulation and the need for validation of experimental analysis. Here, we present an overview of the current uses and challenges of applied DNA barcoding in agriculture, from agro-ecosystems within farmland to the kitchen table.

scholarly journals Relative versus absolute RNA quantification: a comparative analysis based on the example of endothelial expression of vasoactive receptors

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Kevin Kuhlmann ◽  
Melanie Cieselski ◽  
Julia Schumann
Keyword(s):  
Dynamic Range ◽  
Genetic Material ◽  
Housekeeping Genes ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Test Procedures ◽  
Experimental Conditions ◽  
Rna Quantification ◽  
The Stability ◽  
Inflammatory Milieu

Abstract Background In the present study, two distinct PCR methods were used for the quantification of genetic material and their results were compared: real-time-PCR (qPCR; relative quantification) and droplet digital PCR (ddPCR; absolute quantification). The comparison of the qPCR and the ddPCR was based on a stimulation approach of microvascular endothelial cells in which the effect of a pro-inflammatory milieu on the expression of vasoactive receptors was investigated. Results There was consistency in directions of effects for the majority of genes tested. With regard to the indicated dimension of the effects, the overall picture was more differentiated. It was striking that deviations were more pronounced if the measured values were on the extreme edges of the dynamic range of the test procedures. Conclusions To obtain valid and reliable results, dilution series are recommended, which should be carried out initially. In case of ddPCR the number of copies per µl should be adjusted to the low three-digit range. With regard to qPCR it is essential that the stability and reliability of the reference genes used is guaranteed. Here, ddPCR offers the advantage that housekeeping genes are not required. Furthermore, an absolute quantification of the sample can be easily performed by means of ddPCR. Before using ddPCR, however, care should be taken to optimize the experimental conditions. Strict indications for this methodology should also be made with regard to economic and timing factors.

Exploring the plant environmental DNA diversity in soil from two sites on Deception Island (Antarctica, South Shetland Islands) using metabarcoding

2021 ◽  
pp. 1-10
Author(s):  
Micheline Carvalho-Silva ◽  
Luiz Henrique Rosa ◽  
Otávio H.B. Pinto ◽  
Thamar Holanda Da Silva ◽  
Diego Knop Henriques ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Crater Lake ◽  
Environmental Dna ◽  
Food Sources ◽  
South Shetland Islands ◽  
Deception Island ◽  
Shetland Islands ◽  
Operational Taxonomic Units ◽  
First Time ◽  
Impacted Site

Abstract The few Antarctic studies to date to have applied metabarcoding in Antarctica have primarily focused on microorganisms. In this study, for the first time, we apply high-throughput sequencing of environmental DNA to investigate the diversity of Embryophyta (Viridiplantae) DNA present in soil samples from two contrasting locations on Deception Island. The first was a relatively undisturbed site within an Antarctic Specially Protected Area at Crater Lake, and the second was a heavily human-impacted site in Whalers Bay. In samples obtained at Crater Lake, 84% of DNA reads represented fungi, 14% represented Chlorophyta and 2% represented Streptophyta, while at Whalers Bay, 79% of reads represented fungi, 20% represented Chlorophyta and < 1% represented Streptophyta, with ~1% of reads being unassigned. Among the Embryophyta we found 16 plant operational taxonomic units from three Divisions, including one Marchantiophyta, eight Bryophyta and seven Magnoliophyta. Sequences of six taxa were detected at both sampling sites, eight only at Whalers Bay and two only at Crater Lake. All of the Magnoliophyta sequences (flowering plants) represent species that are exotic to Antarctica, with most being plausibly linked to human food sources originating from local national research operator and tourism facilities.

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