scholarly journals The use of eDNA and DNA metabarcoding in monitoring the ecological condition of Norwegian lakes

2021 ◽  
Vol 4 ◽  
Author(s):  
Sara Atienza Casas ◽  
Markus Majaneva ◽  
Thomas Jensen ◽  
Marie Davey ◽  
Frode Fossøy ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Ecological Status ◽  
Ecological Condition ◽  
Molecular Techniques ◽  
Taxonomic Resolution ◽  
Potential Cost ◽  
Running Water ◽  
National Monitoring ◽  
Monitoring Programs ◽  
Dna Metabarcoding

Biodiversity assessments using molecular identification of organisms through high-throughput sequencing techniques have been a game changer in ecosystem monitoring, providing increased taxonomic resolution, more objective identifications, potential cost reductions, and reduced processing times. The use of DNA metabarcoding of bulk samples and environmental DNA (eDNA) is now widespread but is not yet universally implemented in national monitoring programs. While bulk sample metabarcoding involves extraction of DNA from organisms in a sample, eDNA analysis involves obtaining DNA directly from environmental samples, which can include microorganisms, meiofauna-size taxa and macrofauna traces such as larval stages, skin and hair cells, gametes, faeces and free DNA bound to particles. In Norway, freshwater biomonitoring in compliance with the EU Water Framework Directive (WFD) is conducted on several administrative levels, including national monitoring programs for running water, small and large lakes. These programs typically focus on a fraction of the actual biodiversity present in the monitored habitats (Weigand 2019). DNA metabarcoding of both bulk samples and eDNA samples are relevant tools for future freshwater biomonitoring in Norway. The aim of this PhD project is to develop assessment protocols based on DNA-metabarcoding and eDNA of benthic invertebrates, microcrustaceans and fish that can be used as standard biomonitoring tools to assess the ecological condition of lakes. The main topics addressed will be: - Development of protocols throughout the eDNA-metabarcoding workflow (i.e. sampling, filtration, preservation, extraction, amplification and sequencing) suitable to execute biodiversity assessments and determine the ecological status of lakes. - Comparison of the results obtained using molecular tools and traditional morphology-based approaches in order to assess the feasibility of such techniques to be incorporated as standard biomonitoring tools, such as the ones implemented under the provisions of the WFD. - Evaluate the effect of improved taxonomic resolution from molecular techniques on determining the ecological status of lakes, both by broadening the number of taxa analyzed and by identifying more taxa to species level. - Assess the feasibility of using eDNA extracted from water samples, taken at different depths and fish densities, to measure fish abundance/biomass as a proxy to calculate the ecological quality indices regulated in the WFD. - Analyze the coverage and resolution provided by reference libraries for certain taxa, such as crustacea, in order to assess the reliability and precision of taxonomic assignments.

scholarly journals Assessing pollution of aquatic environments with diatoms’ DNA metabarcoding: experience and developments from France water framework directive networks

2019 ◽  
Vol 3 ◽  
Author(s):  
Vasselon Valentin ◽  
Rimet Frédéric ◽  
Domaizon Isabelle ◽  
Monnier Olivier ◽  
Reyjol Yorick ◽  
...  
Keyword(s):  
Water Framework Directive ◽  
High Throughput Sequencing ◽  
Ecological Status ◽  
Aquatic Environments ◽  
Morphological Identification ◽  
The Past ◽  
Sequencing Technologies ◽  
Dna Metabarcoding ◽  
Morphological Approach ◽  
Status Assessment

Ecological status assessment of watercourses is based on the calculation of quality indices using pollution sensitivity of targeted biological groups, including diatoms. The determination and quantification of diatom species is generally based on microscopic morphological identification, which requires expertise and is time-consuming and costly. In Europe, this morphological approach is legally imposed by standards and regulatory decrees by the Water Framework Directive (WFD). Over the past decade, a DNA-based molecular biology approach has newly been developed to identify species based on genetic criteria rather than morphological ones (i.e. DNA metabarcoding). In combination with high throughput sequencing technologies, metabarcoding makes it possible both to identify all species present in an environmental sample and to process several hundred samples in parallel. This article presents the results of two recent studies carried out on the WFD networks of rivers of Mayotte (2013–2018) and metropolitan France (2016–2018). These studies aimed at testing the potential application of metabarcoding for biomonitoring in the context of the WFD. We discuss the various methodological developments and optimisations that have been made to make the taxonomic inventories of diatoms produced by metabarcoding more reliable, particularly in terms of species quantification. We present the results of the application of this DNA approach on more than 500 river sites, comparing them with those obtained using the standardised morphological method. Finally, we discuss the potential of metabarcoding for routine application, its limits of application and propose some recommendations for future implementation in WFD.

scholarly journals How can integrated morphotaxonomy- and metabarcoding-based diatom assemblage analyses best contribute to the ecological assessment of streams?

2021 ◽  
Vol 5 ◽  
Author(s):  
Panayiota Pissaridou ◽  
Marco Cantonati ◽  
Agnes Bouchez ◽  
Iakovos Tziortzis ◽  
Gerald Dörflinger ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Environmental Changes ◽  
Eastern Mediterranean ◽  
Ecological Status ◽  
Monitoring Network ◽  
Bioinformatic Analysis ◽  
Nutrient Concentrations ◽  
Mediterranean Country ◽  
Dna Metabarcoding

Environmental conditions, such as nutrient concentrations, salinity, elevation etc., shape diatom assemblages of periphytic biofilms. These assemblages respond rapidly to environmental changes, a fact which makes diatoms valuable bioindicators. Hence, freshwater biomonitoring programmes currently use diatom indices (e.g. EU Water Framework Directive - WFD). To date, microscopy-based assessments require high taxonomic expertise for diatom identification at the species level. High-throughput technologies now provide cost-effective identification approaches that are promising, complementary or alternative tools for bioassessment. The suitability of the metabarcoding method is evaluated for the first time in the Cyprus streams WFD monitoring network, an eastern Mediterranean country with many endemic species and results are compared to the results acquired from the morphotaxonomic analysis. Morphotaxonomic identification was conducted microscopically, using the most updated taxonomic concepts, literature and online resources. At the same time, DNA metabarcoding involved the use of the rbcL 312 bp barcode, high-throughput sequencing and bioinformatic analysis. The ecological status was calculated using the IPS Index. Results show a positive correlation between morpho-taxonomic and molecular IPS scores. Discrepancies between the two methodologies are related to the limitations of both techniques. This study confirmed that Fistulifera saprophila can have a crucial role in key differences observed, as it negatively influences IPS scores and microscopy methods frequently overlook it. Importantly, gaps in the DNA barcoding reference databases lead to a positive overestimation in IPS scores. Overall, we conclude that DNA metabarcoding offsets the morphotaxonomic methodology for the ecological quality assessment of freshwaters.

scholarly journals Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Edith Vamos ◽  
Kristian Meissner ◽  
Jukka Aroviita ◽  
Florian Leese
Keyword(s):  
Large Scale ◽  
Monitoring Program ◽  
Ecological Status ◽  
Taxonomic Resolution ◽  
Great Promise ◽  
Morphological Identification ◽  
Full Potential ◽  
Stream Monitoring ◽  
Dna Metabarcoding ◽  
Primer Sets

1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 2) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Specimens were morphologically classified, following standardised protocols, to the lowest taxonomic level for which identification was feasible in the routine national monitoring. 3) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded a higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of specimens per taxon (a proxy for biomass) were significantly correlated in each sample, although the adjusted R2 were low. With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately as expensive as morphology-based identification. 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve comparable assessment results to current protocols relying on morphological identification. Thus, metabarcoding represents a feasible and reliable method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems with current laboratory protocols and reference databases.

scholarly journals Strengths and weaknesses of DNA-based monitoring: Assessing macroinvertebrates in 18 Finnish streams with metabarcoding and morphology

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Edith Vamos ◽  
Kristian Meissner ◽  
Jukka Aroviita ◽  
Florian Leese
Keyword(s):  
Large Scale ◽  
Monitoring Program ◽  
Ecological Status ◽  
Taxonomic Resolution ◽  
Great Promise ◽  
Morphological Identification ◽  
Full Potential ◽  
Stream Monitoring ◽  
Dna Metabarcoding ◽  
Primer Sets

1) DNA metabarcoding holds great promise for assessment of stream ecosystems with macroinvertebrates. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 2) We tested metabarcoding using 18 macroinvertebrate samples from Finland using four primer sets. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Morphological identification was performed to the taxonomic level at which identification was reliable following standardized protocols. 3) We identified over twice the number of taxa, with greater species-level resolution, using DNA metabarcoding than morphology-based identification. For each sample, we detected more taxa by metabarcoding than by previous morphological methods, and all four primer sets showed similarly good performance. There was a significant linear correlation between sequence abundance and the number of taxa in each sample, but the scatter was up to two orders of magnitude. Ecological status assessment indices calculated from morphological and DNA metabarcoding datasets were mostly similar, with a few exceptions. With the recent drop in sequencing costs per sample, both methods identification are currently equally expensive. 4) We used actual samples for monitoring to demonstrate that DNA metabarcoding can achieve similar results and better taxonomic resolution than current morphological identification methods. Metabarcoding has thus already become a viable and reliable invertebrate identification method for stream assessment. However, to unlock the full potential of DNA metabarcoding for ecosystem assessment key problems in current laboratory protocols and reference databases, specified in this work, will require further attention.

scholarly journals Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Edith Vamos ◽  
Kristian Meissner ◽  
Jukka Aroviita ◽  
Florian Leese
Keyword(s):  
Large Scale ◽  
Monitoring Program ◽  
Ecological Status ◽  
Taxonomic Resolution ◽  
Great Promise ◽  
Morphological Identification ◽  
Full Potential ◽  
Stream Monitoring ◽  
Dna Metabarcoding ◽  
Primer Sets

1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 2) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Specimens were morphologically classified, following standardised protocols, to the lowest taxonomic level for which identification was feasible in the routine national monitoring. 3) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded a higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of specimens per taxon (a proxy for biomass) were significantly correlated in each sample, although the adjusted R2 were low. With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately as expensive as morphology-based identification. 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve comparable assessment results to current protocols relying on morphological identification. Thus, metabarcoding represents a feasible and reliable method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems with current laboratory protocols and reference databases.

scholarly journals Studying ecosystems with DNA metabarcoding: lessons from aquatic biomonitoring

2019 ◽  
Author(s):  
Alex Bush ◽  
Zacchaeus Compson ◽  
Wendy Monk ◽  
Teresita M. Porter ◽  
Royce Steeves ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
New Technology ◽  
Rapid Development ◽  
Compositional Analysis ◽  
Taxonomic Resolution ◽  
Ecological Studies ◽  
Ecological Data ◽  
Efficient Detection ◽  
Dna Metabarcoding

AbstractAn ongoing challenge for ecological studies has been the collection of data with high precision and accuracy at a sufficient scale to detect effects relevant to management of critical global change processes. A major hurdle for many workflows has been the time-consuming and challenging process of sorting and identification of organisms, but the rapid development of DNA metabarcoding as a biodiversity observation tool provides a potential solution. As high-throughput sequencing becomes more rapid and cost-effective, a ‘big data’ revolution is anticipated, based on higher and more accurate taxonomic resolution, more efficient detection, and greater sample processing capacity. These advances have the potential to amplify the power of ecological studies to detect change and diagnose its cause, through a methodology termed ‘Biomonitoring 2.0’.Despite its promise, the unfamiliar terminology and pace of development in high-throughput sequencing technologies has contributed to a growing concern that an unproven technology is supplanting tried and tested approaches, lowering trust among potential users, and reducing uptake by ecologists and environmental management practitioners. While it is reasonable to exercise caution, we argue that any criticism of new methods must also acknowledge the shortcomings and lower capacity of current observation methods. Broader understanding of the statistical properties of metabarcoding data will help ecologists to design, test and review evidence for new hypotheses.We highlight the uncertainties and challenges underlying DNA metabarcoding and traditional methods for compositional analysis, focusing on issues of taxonomic resolution, sample similarity, taxon misidentification, sample contamination, and taxon abundance. Using the example of freshwater benthic ecosystems, one of the most widely-applied non-microbial applications of DNA metabarcoding to date, we explore the ability of this new technology to improve the quality and utility of ecological data, recognising that the issues raised have widespread applicability across all ecosystem types.

scholarly journals Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Edith Vamos ◽  
Kristian Meissner ◽  
Jukka Aroviita ◽  
Florian Leese
Keyword(s):  
Large Scale ◽  
Monitoring Program ◽  
Ecological Status ◽  
Taxonomic Resolution ◽  
Great Promise ◽  
Morphological Identification ◽  
Full Potential ◽  
Stream Monitoring ◽  
Dna Metabarcoding ◽  
Primer Sets

1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 2) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Specimens were morphologically classified, following standardized protocols, to the lowest taxonomic level for which identification was feasible in the routine national monitoring. 3) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of specimens per taxon (proxy for biomass) were significantly correlated in each sample, although adjusted R2 were low. With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately equal priced per sample to morphology-based identification. 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve similar assessment results as those of current protocols for morphological identification. Thus, metabarcoding represents a feasible and reliable method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems with current laboratory protocols and reference databases.

scholarly journals Assessing and Maximizing Cultivated Diversity with Plate-Wash PCR and High Throughput Sequencing

2020 ◽  
Author(s):  
Emily N. Junkins ◽  
Bradley S. Stevenson
Keyword(s):  
High Throughput ◽  
Drug Targets ◽  
High Throughput Sequencing ◽  
Multidrug Resistant ◽  
Molecular Techniques ◽  
Bioactive Metabolites ◽  
Plate Count ◽  
Molecular Tools ◽  
Vast Number ◽  
Sequencing Studies

AbstractMolecular techniques continue to reveal a growing disparity between the immense diversity of microbial life and the small proportion that is in pure culture. The disparity, originally dubbed “the great plate count anomaly” by Staley and Konopka, has become even more vexing given our increased understanding of the importance of microbiomes to a host and the role of microorganisms in the vital biogeochemical functions of our biosphere. Searching for novel antimicrobial drug targets often focuses on screening a broad diversity of microorganisms. If diverse microorganisms are to be screened, they need to be cultivated. Recent innovative research has used molecular techniques to assess the efficacy of cultivation efforts, providing invaluable feedback to cultivation strategies for isolating targeted and/or novel microorganisms. Here, we aimed to determine the efficiency of cultivating representative microorganisms from a non-human, mammalian microbiome, identify those microorganisms, and determine the bioactivity of isolates. Molecular methods indicated that around 57% of the ASVs detected in the original inoculum were cultivated in our experiments, but nearly 53% of the total ASVs that were present in our cultivation experiments were not detected in the original inoculum. In light of our controls, our data suggests that when molecular tools were used to characterize our cultivation efforts, they provided a more complete, albeit more complex, understanding of which organisms were present compared to what was eventually cultivated. Lastly, about 3% of the isolates collected from our cultivation experiments showed inhibitory bioactivity against a multidrug-resistant pathogen panel, further highlighting the importance of informing and directing future cultivation efforts with molecular tools.ImportanceCultivation is the definitive tool to understand a microorganism’s physiology, metabolism, and ecological role(s). Despite continuous efforts to hone this skill, researchers are still observing yet-to-be cultivated organisms through high-throughput sequencing studies. Here, we use the very same tool that highlights biodiversity to assess cultivation efficiency. When applied to drug discovery, where screening a vast number of isolates for bioactive metabolites is common, cultivating redundant organisms is a hindrance. However, we observed that cultivating in combination with molecular tools can expand the observed diversity of an environment and its community, potentially increasing the number of microorganisms to be screened for natural products.

scholarly journals Its present state overmoistened landscape systems of Lviv Region

2014 ◽  
pp. 347-354
Author(s):  
N. Blazhko
Keyword(s):  
Key Words ◽  
Present State ◽  
Environmental Conditions ◽  
Ecological Status ◽  
Ecological Condition ◽  
Wetland Landscape ◽  
Current State ◽  
Destabilizing Factors ◽  
Good Ecological Status ◽  
Stabilizing Factors

Reviewed the current state of wetland landscape systems (PLC) Lviv region. Highlighted areas PLC of good ecological status, with a satisfactory ecological condition and unsatisfactory environmental conditions. Key words: overmoistened landscape systems (OLS)peat, peat, drainage improvement, environmental stabilizing factors, environmental destabilizing factors.

scholarly journals The Fellowship of the Ring Test: DNAqua-Net WG2 initiative to compare diatom metabarcoding protocols used in routine freshwater biomonitoring for standardisation

2021 ◽  
Vol 4 ◽  
Author(s):  
Valentin Vasselon ◽  
Éva Ács ◽  
Salomé Almeida ◽  
Karl Andree ◽  
Laure Apothéloz-Perret-Gentil ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Aquatic Ecosystems ◽  
Quality Index ◽  
Ecological Status ◽  
Pcr Amplification ◽  
Ring Test ◽  
Dna Metabarcoding ◽  
Ecological Status Assessment ◽  
Status Assessment

During the past decade genetic approaches have been developed to monitor biodiversity in aquatic ecosystems. These enable access to taxonomic and genetic information from biological communities using DNA from environmental samples (e.g. water, biofilm, soil) and methods based on high-throughput sequencing technologies, such as DNA metabarcoding. Within the context of the Water Framework Directive (WFD), such approaches could be applied to assess Biological Quality Elements (BQE). These are used as indicators of the ecological status of aquatic ecosystems as part of national monitoring programs of the european network of 110,000 surface water monitoring sites with 79.5% rivers and 11% lake sites (Charles et al. 2020). A high-throughput method has the potential to increase our spatio-temporal monitoring capacity and to accelerate the transfer of information to water managers with the aim to increase protection of aquatic ecosystems. Good progress has been made with developing DNA metabarcoding approaches for benthic diatom assemblages. Technological innovation and protocol optimization have allowed robust taxonomic (species) and genetic (OTU, ESV) information to be obtained from which diatom quality indices can be calculated to infer ecological status to rivers and lakes. Diatom DNA metabarcoding has been successfully applied for biomonitoring at the scale of national river monitoring networks in several countries around the world and can now be considered technically ready for routine application (e.g. Apothéloz-Perret-Gentil et al. 2017, Bailet et al. 2019, Mortágua et al. 2019, Vasselon et al. 2019, Kelly et al. 2020, Pérez-Burillo et al. 2020, Pissaridou et al. 2021). However, protocols and methods used by each laboratory still vary between and within countries, limiting their operational transferability and the ability to compare results. Thus, routine use of DNA metabarcoding for diatom biomonitoring requires standardization of all steps of the metabarcoding procedure, from the sampling to the final ecological status assessment in order to define good practices and standards. Following previous initiatives which resulted in a CEN technical report for biofilm sampling and preservation (CEN 2018), a set of experiments was initiated during the DNAqua-Net WG2 diatom workshop (Cyprus, 2019) to focus on DNA extraction and PCR amplification steps in order to evaluate: i) the transferability and reproducibility of a protocol between different laboratories; ii) the variability introduced by different protocols currently applied by the scientific community. 19 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using i) the same fixed protocol and ii) their own protocol. Experiments were performed by each participant on a set of standardized DNA and biofilm samples (river, lake, mock community). In order to specifically test the variability of DNA extraction and PCR amplification steps, all other steps of the metabarcoding process were fixed and the preparation of the Miseq sequencing was performed by only one laboratory. The variability within and between participants will be evaluated on DNA extracts quantity, taxonomic (genus, species) and genetic richness, community structure comparison and diatom quality index scores (IPS). We will also evaluate the variability introduced by different DNA extraction and PCR amplification protocols on diatom quality index scores and the final ecological status assessment. The results from this collaborative work will not serve to define “one protocol to rule them all”, but will provide valuable information to define guidelines and minimum requirements that should be considered when performing diatom metabarcoding for biomonitoring.

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