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 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 Ammonia-oxidizing Archaea (AOA) are Winners to Survive in Oxygen-limited Habitat Compared to Ammonia-oxidizing Bacteria (AOB)

2021 ◽  
Author(s):  
Peigang Dai ◽  
Wenjing Song ◽  
Zhao Che ◽  
Lili Zhang ◽  
Zhaorong Dong
Keyword(s):  
Intertidal Zone ◽  
High Throughput Sequencing ◽  
Ammonia Oxidation ◽  
Illumina Miseq ◽  
Gene Copy ◽  
Ammonia Oxidizing Bacteria ◽  
Niche Specialization ◽  
Ammonia Oxidizing Archaea ◽  
Copy Numbers ◽  
Oxidation Reduction

Abstract Purpose: Both ammonia oxidizing archaea (AOA) and bacteria (AOB) perform the ammonia oxidation together. These two kinds of microbes present a convenient model for studying niche specialization. To date, few surveys concentrated on the influence of oxygen concentration on niche specialization of AOA and AOB in intertidal zones. Methods: Here, high-throughput sequencing by Illumina MiSeq and qPCR were applied to detect the change of abundance, diversity as well as community structure of both AOA and AOB with 0-60 cm sediments depth in the intertidal zone in Qingdao.Results and Conclusion: The AOA/AOB amoA gene copy numbers and AOA/AOB OTU numbers were all increased as sediment depth went deeper, which indicated that AOA were more adaptive to oxygen-limited niches compared to AOB and oxygen indeed led to the niche specialization of AOA and AOB in intertidal sediments. The dominant AOA and AOB were the Nitrosopumilus and Nitrosospira clusters, respectively, which indicated an ecological success in intertidal zone. Oxidation-reduction potential (ORP) was significantly positively correlated with AOB abundance and AOB OTU numbers (P < 0.01). In addition, both TN (P < 0.01) and pH (P < 0.05) were significantly and negatively correlated with AOB abundance. TN was also significantly and negatively correlated with AOB OTU numbers (P < 0.05).

scholarly journals High-Throughput Sequencing of Environmental DNA as a Tool for Monitoring Eukaryotic Communities and Potential Pathogens in a Coastal Upwelling Ecosystem

2021 ◽  
Vol 8 ◽  
Author(s):  
Raquel Ríos-Castro ◽  
Alejandro Romero ◽  
Raquel Aranguren ◽  
Alberto Pallavicini ◽  
Elisa Banchi ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Coastal Upwelling ◽  
Marine Ecosystem ◽  
Environmental Dna ◽  
Functional Roles ◽  
Molecular Approaches ◽  
Eukaryotic Microorganisms ◽  
Causative Agents ◽  
Veterinary Importance ◽  
Water Fractions

The marine environment includes diverse microeukaryotic organisms that play important functional roles in the ecosystem. With molecular approaches, eukaryotic taxonomy has been improved, complementing classical analysis. In this study, DNA metabarcoding was performed to describe putative pathogenic eukaryotic microorganisms in sediment and marine water fractions collected in Galicia (NW Spain) from 2016 to 2018. The composition of eukaryotic communities was distinct between sediment and water fractions. Protists were the most diverse group, with the clade TSAR (Stramenopiles, Alveolata, Rhizaria, and Telonemida) as the primary representative organisms in the environment. Harmful algae and invasive species were frequently detected. Potential pathogens, invasive pathogenic organisms as well as the causative agents of harmful phytoplanktonic blooms were identified in this marine ecosystem. Most of the identified pathogens have a crucial impact on the aquacultural sector or affect to relevant species in the marine ecosystem, such as diatoms. Moreover, pathogens with medical and veterinary importance worldwide were also found, as well as pathogens that affect diatoms. The evaluation of the health of a marine ecosystem that directly affects the aquacultural sector with a zoonotic concern was performed with the metabarcoding assay.

scholarly journals GoFish: A Streamlined Environmental DNA Presence/Absence Assay for Marine Vertebrates

2018 ◽  
Author(s):  
Mark Y. Stoeckle ◽  
Mithun Das Mishu ◽  
Zachary Charlop-Powers
Keyword(s):  
New York ◽  
Fish Species ◽  
River Estuary ◽  
Environmental Dna ◽  
Hudson River ◽  
Illumina Miseq ◽  
Turnaround Time ◽  
Seasonal Abundance ◽  
Thermal Cycler ◽  
Species Specific

AbstractHere we describe GoFish, a streamlined environmental DNA (eDNA) presence/absence assay. The assay amplifies a 12S segment with broad-range vertebrate primers, followed by nested PCR with M13-tailed, species-specific primers. Sanger sequencing confirms positives detected by gel electrophoresis. We first obtained 12S sequences from 77 fish specimens representing 36 northwestern Atlantic taxa not well documented in GenBank. Using the newly obtained and published 12S records, we designed GoFish assays for 11 bony fish species common in the lower Hudson River estuary and tested seasonal abundance and habitat preference at two sites. Additional assays detected nine cartilaginous fish species and a marine mammal, bottlenose dolphin, in southern New York Bight. GoFish sensitivity was equivalent to Illumina MiSeq metabarcoding. Unlike quantitative PCR (qPCR), GoFish does not require tissues of target and related species for assay development and a basic thermal cycler is sufficient. Unlike Illumina metabarcoding, indexing and batching samples are unnecessary and advanced bioinformatics expertise is not needed. The assay can be carried out from water collection to result in three days. The main limitations so far are species with shared target sequences and inconsistent amplification of rarer eDNAs. We think this approach will be a useful addition to current eDNA methods when analyzing presence/absence of known species, when turnaround time is important, and in educational settings.

scholarly journals Quantitative assessment of multiple fish species around artificial reefs combining environmental DNA metabarcoding and acoustic survey

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masaaki Sato ◽  
Nariaki Inoue ◽  
Ryogen Nambu ◽  
Naoki Furuichi ◽  
Tomohito Imaizumi ◽  
...  
Keyword(s):  
Fish Species ◽  
Internal Standard ◽  
Environmental Dna ◽  
Artificial Reefs ◽  
Sampling Station ◽  
Biodiversity Monitoring ◽  
Dna Metabarcoding ◽  
Positive Correlations ◽  
Multiple Species ◽  
Echo Sounder

AbstractSince the early 1970s, many artificial reefs (ARs) have been deployed in Japanese coastal waters to create fisheries grounds. Recently, researchers began to use environmental DNA (eDNA) methods for biodiversity monitoring of aquatic species. A metabarcoding approach using internal standard DNAs [i.e., quantitative MiSeq sequencing (qMiSeq)] makes it possible to monitor eDNA concentrations of multiple species simultaneously. This method can improve the efficiency of monitoring AR effects on fishes. Our study investigated distributions of marine fishes at ARs and surrounding stations in the open oceanographic environment of Tateyama Bay, central Japan, using qMiSeq and echo sounder survey. Using the qMiSeq with 12S primers, we found higher quantities of fish eDNAs at the ARs than at surrounding stations and different fish species compositions between them. Comparisons with echo sounder survey also showed positive correlations between fish eDNA concentration and echo intensity, which indicated a highly localized signal of eDNA at each sampling station. These results suggest that qMiSeq is a promising technique to complement conventional methods to monitor distributions of multiple fish species.

scholarly journals Assessment of Passive Traps Combined with High-Throughput Sequencing To Study Airborne Fungal Communities

2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Jaime Aguayo ◽  
Céline Fourrier-Jeandel ◽  
Claude Husson ◽  
Renaud Ioos
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Fungal Diversity ◽  
High Throughput Sequencing ◽  
Linear Models ◽  
Environmental Dna ◽  
Illumina Miseq ◽  
Fungal Communities ◽  
Correct Identification ◽  
Spore Trap

ABSTRACTTechniques based on high-throughput sequencing (HTS) of environmental DNA have provided a new way of studying fungal diversity. However, these techniques suffer from a number of methodological biases which may appear at any of the steps involved in a metabarcoding study. Air is one of the most important environments where fungi can be found, because it is the primary medium of dispersal for many species. Looking ahead to future developments, it was decided to test 20 protocols, including different passive spore traps, spore recovery procedures, DNA extraction kits, and barcode loci. HTS was performed with the Illumina MiSeq platform targeting two subloci of the fungal internal transcribed spacer. Multivariate analysis and generalized linear models showed that the type of passive spore trap, the spore recovery procedure, and the barcode all impact the description of fungal communities in terms of richness and diversity when assessed by HTS metabarcoding. In contrast, DNA extraction kits did not significantly impact these results. Although passive traps may be used to describe airborne fungal communities, a study using specific real-time PCR and a mock community showed that these kinds of traps are affected by environmental conditions that may induce losses of biological material, impacting diversity and community composition results.IMPORTANCEThe advent of high-throughput sequencing (HTS) methods, such as those offered by next-generation sequencing (NGS) techniques, has opened a new era in the study of fungal diversity in different environmental substrates. In this study, we show that an assessment of the diversity of airborne fungal communities can reliably be achieved by the use of simple and robust passive spore traps. However, a comparison of sample processing protocols showed that several methodological biases may impact the results of fungal diversity when assessed by metabarcoding. Our data suggest that identifying these biases is of paramount importance to enable a correct identification and relative quantification of community members.

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.

scholarly journals The Fecal Bacterial Microbiota in Horses with Equine Recurrent Uveitis

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 745
Author(s):  
Michelle Martin de Bustamante ◽  
Diego Gomez ◽  
Jennifer MacNicol ◽  
Ralph Hamor ◽  
Caryn Plummer
Keyword(s):  
Beta Diversity ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Diversity Indices ◽  
Rrna Gene ◽  
Linear Discriminant ◽  
Alpha And Beta Diversity ◽  
Bacterial Microbiota ◽  
Healthy Control ◽  
Equine Recurrent Uveitis

The objective of this study was to describe and compare the fecal bacterial microbiota of horses with equine recurrent uveitis (ERU) and healthy horses using next-generation sequencing techniques. Fecal samples were collected from 15 client-owned horses previously diagnosed with ERU on complete ophthalmic examination. For each fecal sample obtained from a horse with ERU, a sample was collected from an environmentally matched healthy control with no evidence of ocular disease. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene. The relative abundance of predominant taxa, and alpha and beta diversity indices were calculated and compared between groups. The phyla Firmicutes, Bacteroidetes, Verrucomicrobia, and Proteobacteria predominated in both ERU and control horses, accounting for greater than 60% of sequences. Based on linear discriminant analysis effect size (LEfSe), no taxa were found to be enriched in either group. No significant differences were observed in alpha and beta diversity indices between groups (p > 0.05 for all tests). Equine recurrent uveitis is not associated with alteration of the gastrointestinal bacterial microbiota when compared with healthy controls.

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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