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 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 Validation of river typologies using molecular information from diatoms

2021 ◽  
Vol 4 ◽  
Author(s):  
Andreia Mortágua ◽  
Marco Teixeira ◽  
Manuela Sales ◽  
Maria Feio ◽  
Salomé Almeida
Keyword(s):  
Water Framework Directive ◽  
Reference Conditions ◽  
Ecological Indicators ◽  
Molecular Data ◽  
Reference Database ◽  
Depth Information ◽  
Morphological Identification ◽  
Body Type ◽  
Dna Metabarcoding ◽  
Total Environment

The European Water Framework Directive (2000/60/EC) includes biological assessment of water bodies that has been implemented for many years. Indicator organisms such as diatoms respond to geological and hydrological features of rivers by modifying their structure. Therefore, when implementing the WFD, it was necessary to establish type-specific reference conditions to be able to measure the deviations of sampled communities due to anthropogenic impact.HTS-related eDNA metabarcoding has been developed to complement or even replace traditional approaches for its rapid, low-cost and highly accurate identification of communities for assessment of rivers’ ecological status (e.g. Mortágua et al., 2019; Pérez-Burillo et al. 2020) and proved to provide even more in-depth information about biological elements. The use of this information without assignment to species is being addressed once it eliminates the limiting factor of the reference database incompleteness and may provide new ecological information (e.g. Feio et al., 2020; Rivera et al., 2020). Since WFD requires the establishment of reference conditions for each water body type, for eDNA methods’ implementation it will be essential to review, confirm or reformulate, and perhaps create new typologies. Hereupon, the aim of this study is to analyze diatom communities from different typologies of Portuguese rivers resulting from DNA metabarcoding data and compare it with current typology system. To do so, we will verify the consistency of biological groups included in each type, validate the molecular data, analyze the correspondence of OTU/ISU/ESV to environmental characteristics of rivers. A total of 154 sampling sites were selected from central Portugal and northern Portugal in 2017 and 2019. The biofilm was collected for morphological identification and DNA sequencing of diatoms. Reference sites were selected for 4 river types (mountain, littoral, small and medium-large northern rivers) based on a set of pressure information (water quality, hydromorphology, land use and riparian zones). Diatom inventories were obtained from molecular and morphological analysis. DNA sequences were treated using Mothur software which processed two bioinformatic strategies in order to obtain the final ISU and OTU tables, while ESVs were treated with DADA2 package from R. Identification and counting of diatom valves took place under the light microscope concerning the morphological approach. We expect results to validate the molecular data for each typology either when assigning to species or not, and to understand whether it is necessary to establish new typologies for future use of the molecular approach in ecological assessment of rivers. Directive, W. F. (2000). Water Framework Directive. Journal reference OJL, 327, 1-73. Feio, M. J., Serra, S. R., Mortágua, A., Bouchez, A., Rimet, F., Vasselon, V., & Almeida, S. F. P. (2020). A taxonomy-free approach based on machine learning to assess the quality of rivers with diatoms. Science of the Total Environment, 722, 137900. https://doi.org/10.1016/j.scitotenv.2020.137900 Mortágua, A., Vasselon, V., Oliveira, R., Elias, C., Chardon, C., Bouchez, A., ... & Almeida, S. F. P. (2019). Applicability of DNA metabarcoding approach in the bioassessment of Portuguese rivers using diatoms. Ecological indicators, 106, 105470. https://doi.org/10.1016/j.ecolind.2019.105470 Pérez-Burillo, J., Trobajo, R., Vasselon, V., Rimet, F., Bouchez, A., & Mann, D. G. (2020). Evaluation and sensitivity analysis of diatom DNA metabarcoding for WFD bioassessment of Mediterranean rivers. Science of the Total Environment, 727, 138445. https://doi.org/10.1016/j.scitotenv.2020.138445 Rivera, S. F., Vasselon, V., Bouchez, A., & Rimet, F. (2020). Diatom metabarcoding applied to large scale monitoring networks: Optimization of bioinformatics strategies using Mothur software. Ecological indicators, 109, 105775. https://doi.org/10.1016/j.ecolind.2019.105775

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.

scholarly journals ‘There and back again’: revisiting the pathophysiological roles of human endogenous retroviruses in the post-genomic era

2013 ◽  
Vol 368 (1626) ◽  
pp. 20120504 ◽  
Author(s):  
Gkikas Magiorkinis ◽  
Robert Belshaw ◽  
Aris Katzourakis
Keyword(s):  
Human Genome ◽  
High Throughput Sequencing ◽  
Immune Escape ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Endogenous Retroviruses ◽  
Human Endogenous Retroviruses ◽  
The Past ◽  
Sequencing Technologies ◽  
Research Questions

Almost 8% of the human genome comprises endogenous retroviruses (ERVs). While they have been shown to cause specific pathologies in animals, such as cancer, their association with disease in humans remains controversial. The limited evidence is partly due to the physical and bioethical restrictions surrounding the study of transposons in humans, coupled with the major experimental and bioinformatics challenges surrounding the association of ERVs with disease in general. Two biotechnological landmarks of the past decade provide us with unprecedented research artillery: (i) the ultra-fine sequencing of the human genome and (ii) the emergence of high-throughput sequencing technologies. Here, we critically assemble research about potential pathologies of ERVs in humans. We argue that the time is right to revisit the long-standing questions of human ERV pathogenesis within a robust and carefully structured framework that makes full use of genomic sequence data. We also pose two thought-provoking research questions on potential pathophysiological roles of ERVs with respect to immune escape and regulation.

scholarly journals Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

2018 ◽  
Author(s):  
Vasco Elbrecht ◽  
Dirk Steinke
Keyword(s):  
High Throughput ◽  
Illumina Sequencing ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Scaling Up ◽  
The Past ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
Fusion Primer ◽  
Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

scholarly journals Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

2018 ◽  
Author(s):  
Vasco Elbrecht ◽  
Dirk Steinke
Keyword(s):  
High Throughput ◽  
Illumina Sequencing ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Scaling Up ◽  
The Past ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
Fusion Primer ◽  
Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

scholarly journals Comparison of Read Mapping and Variant Calling Tools for the Analysis of Plant NGS Data

Plants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 439 ◽  
Author(s):  
Hanna Marie Schilbert ◽  
Andreas Rempel ◽  
Boas Pucker
Keyword(s):  
High Throughput Sequencing ◽  
Performance Metrics ◽  
Model Organism ◽  
Variant Calling ◽  
Reference Sequence ◽  
Read Mapping ◽  
The Past ◽  
Sequencing Technologies ◽  
Plant Sciences ◽  
Ngs Data

High-throughput sequencing technologies have rapidly developed during the past years and have become an essential tool in plant sciences. However, the analysis of genomic data remains challenging and relies mostly on the performance of automatic pipelines. Frequently applied pipelines involve the alignment of sequence reads against a reference sequence and the identification of sequence variants. Since most benchmarking studies of bioinformatics tools for this purpose have been conducted on human datasets, there is a lack of benchmarking studies in plant sciences. In this study, we evaluated the performance of 50 different variant calling pipelines, including five read mappers and ten variant callers, on six real plant datasets of the model organism Arabidopsis thaliana. Sets of variants were evaluated based on various parameters including sensitivity and specificity. We found that all investigated tools are suitable for analysis of NGS data in plant research. When looking at different performance metrics, BWA-MEM and Novoalign were the best mappers and GATK returned the best results in the variant calling step.

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