Recommendations for the preservation of environmental samples in diatom metabarcoding studies

Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa

Recommendations for the preservation of environmental samples in diatom metabarcoding studies

Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa (< 100 reads) differed among preservation methods and storage durations. For biomonitoring purposes, freshwater ecological index values were not affected by the preservation method and storage duration tested (including ethanol preservation), all treatments returning the same ecological status for a site. This study contributes to consolidating diatom metabarcoding. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring

Recommendations for the preservation of environmental samples in diatom metabarcoding studies

Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa

Alpine freshwater fish biodiversity assessment: an inter-calibration test for metabarcoding method set up

The analysis of environmental DNA (eDNA) by high throughput sequencing (HTS) is proving to be a promising tool for freshwater fish biodiversity assessment in Europe within the Water Framework Directive (WFD, 2000/60/EC), especially for large rivers and lakes where current fish monitoring techniques have known shortcomings. These new biomonitoring methods based on eDNA show several advantages compared to classical morphological methods. The sampling procedures are easier and cheaper and eDNA metabarcoding is non-invasive and very sensitive, allowing for the detection of traces of DNA. However, eDNA metabarcoding methods need careful standardization to make the results of different surveys comparable. The aim of the EU project Eco-AlpsWater is to test and validate molecular biodiversity monitoring tools for aquatic ecosystems (i.e., eDNA metabarcoding) to improve the traditional WFD monitoring approaches in Alpine waterbodies. To this end, an inter-calibration test was performed using fish mock community samples containing either tissue-extracted DNA, eDNA collected from aquaculture tanks and eDNA samples collected from Lake Bourget (France). Samples were analysed using a DNA metabarcoding approach, relying on the amplification and HTS of a 12S rDNA marker, in two separate laboratories, to evaluate if different laboratory and bioinformatic protocols can provide a reliable and comparable description of the fish communities in both mock and natural samples. Our results highlight good replicability of the molecular laboratory protocols for HTS and good amplification success of selected primers, providing essential information concerning the taxonomic resolution of the 12S mitochondrial marker in describing the Alpine fish communities. Interestingly, different concentrations of species DNA in the mock samples were well represented by the relative DNA reads abundance. These tests confirm the reproducibility of eDNA metabarcoding analyses for the biomonitoring of freshwater fish inhabiting Alpine and peri-Alpine lakes and river

Benthic Diatom Communities in an Alpine River Impacted by Waste Water Treatment Effluents as Revealed Using DNA Metabarcoding

Freshwater ecosystems are continuously affected by anthropogenic pressure. One of the main sources of contamination comes from wastewater treatment plant (WWTP) effluents that contain wide range of micro- and macropollutants. Chemical composition, toxicity levels and impact of treated effluents (TEs) on the recipient aquatic ecosystems may strongly differ depending on the wastewater origin. Compared to urban TEs, hospital ones may contain more active pharmaceutical substances. Benthic diatoms are relevant ecological indicators because of their high species and ecological diversity and rapid response to human pressure. They are routinely used for water quality monitoring. However, there is a knowledge gap on diatom communities’ development and behavior in treated wastewater in relation to prevailing micro- and macropollutants. In this study, we aim to (1) investigate the response of diatom communities to urban and hospital TEs, and (2) evaluate TEs effect on communities in the recipient river. Environmental biofilms were colonized in TEs and the recipient river up- and downstream from the WWTP output to study benthic diatoms using DNA metabarcoding combined with high-throughput sequencing (HTS). In parallel, concentrations of nutrients, pharmaceuticals and seasonal conditions were recorded. Diatom metabarcoding showed that benthic communities differed strongly in their diversity and structure depending on the habitat. TE sites were generally dominated by few genera with polysaprobic preferences belonging to the motile guild, while river sites favored diverse communities from oligotrophic and oligosaprobic groups. Seasonal changes were visible to lower extent. To categorize parameters important for diatom changes we performed redundancy analysis which suggested that communities within TE sites were associated to higher concentrations of beta-blockers and non-steroidal anti-inflammatory drugs in urban effluents vs. antibiotics and orthophosphate in hospital effluents. Furthermore, indicator species analysis showed that 27% of OTUs detected in river downstream communities were indicator for urban or hospital TE sites and were absent in the river upstream. Finally, biological diatom index (BDI) calculated to evaluate the ecological status of the recipient river suggested water quality decrease linked to the release of TEs. Thus, in-depth assessment of diatom community composition using DNA metabarcoding is proposed as a promising technique to highlight the disturbing effect of pollutants in Alpine rivers

Diatom DNA metabarcoding for ecological assessment: Comparison among bioinformatics pipelines used in six European countries reveals the need for standardization

Ecological assessment of lakes and rivers using benthic diatom assemblages currently requires considerable taxonomic expertise to identify species using light microscopy. This traditional approach is also time-consuming. Diatom metabarcoding is a promising alternative and there is increasing interest in using this approach for routine assessment. However, until now, analysis protocols for diatom metabarcoding have been developed and optimised by research groups working in isolation. The diversity of existing bioinformatics methods highlights the need for an assessment of the performance and comparability of results of different methods. The aim of this study was to test the correspondence of outputs from six bioinformatics pipelines currently in use for diatom metabarcoding in different European countries. Raw sequence data from 29 biofilm samples were treated by each of the bioinformatics pipelines, five of them using the same curated reference database. The outputs of the pipelines were compared in terms of sequence unit assemblages, taxonomic assignment, biotic index score and ecological assessment outcomes. The three last components were also compared to outputs from traditional light microscopy, which is currently accepted for ecological assessment of phytobenthos, as required by the Water Framework Directive. We also tested the performance of the pipelines on the two DNA markers (rbcL and 185-V4) that are currently used by the working groups participating in this study. The sequence unit assemblages produced by different pipelines showed significant differences in terms of assigned and unassigned read numbers and sequence unit numbers. When comparing the taxonomic assignments at genus and species level, correspondence of the taxonomic assemblages between pipelines was weak. Most discrepancies were linked to differential detection or quantification of taxa, despite the use of the same reference database. Subsequent calculation of biotic index scores also showed significant differences between approaches, which were reflected in the final ecological assessment. Use of the rbcL marker always resulted in better correlation among molecular datasets and also in results closer to these generated using traditional microscopy. This study shows that decisions made in pipeline design have implications for the dataset's structure and the taxonomic assemblage, which in turn may affect biotic index calculation and ecological assessment. There is a need to define best-practice bioinformatics parameters in order to ensure the best representation of diatom assemblages. Only the use of similar parameters will ensure the compatibility of data from different working groups. The future of diatom metabarcoding for ecological assessment may also lie in the development of new metrics using, for example, presence/absence instead of relative abundance data. (C) 2020 The Authors. Published by Elsevier B.V

Metadata standards and practical guidelines for specimen and DNA curation when building barcode reference libraries for aquatic life

DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data.info:eu-repo/semantics/publishedVersio

The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems

The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs.info:eu-repo/semantics/publishedVersio

Barcoding et bioindication : développement du metabarcoding des diatomées pour l'évaluation de la qualité des cours d'eau

Diatoms are microscopic unicellular algae which are excellent indicators of the ecological status of the environment in which they live. In the Water Framework Directive (WFD), diatom communities are used to assess the quality of rivers. For this purpose, quality indices based on the susceptibility of species to pollution are calculated from the composition and relative abundance of diatom taxa. Species identification is typically carried out under a microscope, which, in addition to being complex, can be time-consuming and costly when many samples are processed.A newly developed method allows species to be identified, not on the basis of their morphological variability, but on the basis of their genetic variability using short DNA sequences (or DNA barcodes). Combined with high-throughput sequencing technologies, this molecular approach, called metabarcoding, allows to identify all the species present in an environmental sample and to process several hundred samples in parallel. These advantages make metabarcoding an alternative method to those based on morphological identification, which is interesting for the WFD. Although several studies have demonstrated the ability of this approach to correctly identify diatom species found in environmental samples, the lack of reliability in the quantification of the species relative abundances limits the reliability of calculated quality indices and the use of metabarcoding as a tool for biomonitoring. The objectives of this thesis were (i) to identify and optimize the biases impacting the relative quantification of diatoms in metabarcoding; (ii) to apply the molecular approach at a larger scale on environmental samples from river networks in order to compare quality assessments obtained by morphological and molecular approaches.First, we quantified the bias associated with DNA extraction using pure diatom cultures and environmental samples. Although the choice of the extraction method affects the quality and the quantity of extracted DNA, as the relative abundances of some species identified with metabarcoding, the community composition and water quality index values are not significantly affected. Thus, we decided to use the GenElute method for other works realized during the thesis, as this method produces the largest quantities of DNA at a lower cost. In a second step, using qPCR experiments carried out on pure diatom cultures, we showed that the rbcL gene copy number (used as DNA barcode) is correlated to the cell biovolume, which results in an overestimation of large biovolume species with the metabarcoding. On the basis of this correlation, a correction factor was proposed and applied to metabarcoding data obtained from artificial diatom communities and environmental samples, allowing to obtain species relative abundances similar to those obtained with the morphological approach and to improve the reliability of water quality index values. Finally, the application of the molecular approach at the scale of the WFD rivers monitoring networks of Mayotte and France has enabled us to show that metabarcoding is a faster and cheaper alternative to the morphological approach, while allowing a good water quality assessment of streams.Our work confirms that the molecular approach can be used to assess the quality of rivers. However, further studies will have to be performed before considering a routine application and its implementation in the WFD, particularly in terms of standardization of the methods used in the molecular approach.Les diatomées sont des algues unicellulaires microscopiques qui sont d’excellents indicateurs de l’état écologique du milieu dans lequel elles se trouvent. Dans le cadre de la Directive Cadre sur l’Eau (DCE), les communautés de diatomées sont utilisées pour évaluer la qualité des cours d’eau. Pour cela, des indices de qualité basés sur la sensibilité des espèces à la pollution sont calculés à partir de la composition et de l’abondance relative des taxa de diatomées. L’identification des espèces est généralement réalisée au microscope, ce qui, en plus d’être complexe, peut être assez long et coûteux lorsqu’il s’agit de traiter de nombreux échantillons.Une nouvelle méthode développée récemment permet d’identifier les espèces, non plus sur la base de leur variabilité morphologique, mais sur la base de leur variabilité génétique en utilisant de courtes séquences ADN (ou barcodes ADN). Combinée aux technologies de séquençage à haut débit, cette approche moléculaire, appelée metabarcoding, permet d’identifier l’ensemble des espèces présentes au sein d’un échantillon environnemental et de traiter plusieurs centaines d’échantillons en parallèle. Ces avantages font du metabarcoding une alternative à la méthode d’identification morphologique, intéressante dans le cadre de la DCE. Bien que plusieurs études aient montré la capacité de cette approche à identifier correctement les espèces de diatomées retrouvées dans des échantillons environnementaux, le manque de fiabilité dans la quantification des abondances relatives des espèces limite le calcul d’indices fiable et l’utilisation du metabarcoding comme outil pour la bioindication. Les objectifs de ce travail de thèse ont donc été (i) d’identifier et d’optimiser les biais impactant la quantification relative des diatomées en metabarcoding ; (ii) d’appliquer l’approche moléculaire à large échelle, sur des échantillons environnementaux de réseaux de cours d’eau afin de comparer les évaluations de qualité obtenues par les approches morphologique et moléculaire.Dans un premier temps, nous avons évalué les biais de quantification liés à l’extraction de l’ADN sur des cultures pures de diatomées et des échantillons environnementaux. Bien que le choix de la méthode d’extraction affecte la qualité et la quantité des ADN extraits, ainsi que les abondances relatives de certaines espèces obtenues en metabarcoding, la composition de la communauté ainsi que les notes de qualités ne sont pas significativement affectées. Nous avons donc décidé d’utiliser pour la suite des travaux la méthode GenElute qui produit les plus grandes quantités d’ADN pour un moindre coût. Dans un deuxième temps, grâce à des expériences de qPCR réalisées sur des cultures pures de diatomées, nous avons montré que le nombre de copies du gène rbcL (utilisé comme barcode ADN) est proportionnel au biovolume des cellules, ce qui a pour conséquence une surestimation des espèces à gros biovolumes en metabarcoding. A partir de cette corrélation, un facteur de correction a été proposé et appliqué sur les données de metabarcoding issues de communautés artificielles et d’échantillons environnementaux, permettant d’obtenir des abondances relatives d’espèces comparables à celles obtenues en microscopie et d’améliorer la fiabilité des notes de qualité. Finalement, l’application de l’approche moléculaire à l’échelle des réseaux DCE de surveillance des cours d’eau de Mayotte et de France métropolitaine a permis de montrer que le metabarcoding est une alternative plus rapide et plus économique que l’approche morphologique, tout en permettant une bonne évaluation de la qualité des cours d’eau.Nos travaux confirment que l’approche moléculaire peut être utilisée pour évaluer la qualité des cours d’eau. Cependant d’autres études devront être réalisées avant d’envisager une application en routine et une implémentation dans la DCE, notamment en termes de standardisation et de normalisation des méthodes utilisées dans l’approche moléculaire

Diatom DNA Metabarcoding for Biomonitoring: Strategies to Avoid Major Taxonomical and Bioinformatical Biases Limiting Molecular Indices Capacities

Recent years provided intense progression in the implementation of molecular techniques in a wide variety of research fields in ecology. Biomonitoring and bioassessment can greatly benefit from DNA metabarcoding and High-Throughput Sequencing (HTS) methods that potentially provide reliable, high quantity and quality standardized data in a cost- and time-efficient way. However, DNA metabarcoding has its drawbacks, introducing biases at all the steps of the process, particularly during bioinformatics treatments used to prepare HTS data for ecological analyses. The high diversity of bioinformatics methods (e.g., OTU clustering, chimera detection, taxonomic assignment) and parameters (e.g., percentage similarity threshold used to define OTUs) make inter-studies comparison difficult, limiting the development of standardized and easy-accessible bioassessment procedures for routine freshwater monitoring. In order to study and overcome these drawbacks, we constructed four de novo indices to assess river ecological status based on the same biological samples of diatoms analyzed with morphological and molecular methods. The biological inventories produced are (i) morphospecies identified by microscopy, (ii) OTUs provided via metabarcoding and hierarchical clustering of sequences using a 95% similarity threshold, (iii) individual sequence units (ISUs) via metabarcoding and only minimal bioinformatical quality filtering, and (iv) exact sequence variants (ESVs) using DADA2 denoising algorithm. The indices based on molecular data operated directly with ecological values estimated for OTUs/ISUs/ESVs. Our study used an approach of bypassing taxonomic assignment, so bias related to unclassified sequences missing from reference libraries could be handled and no information on ecology of sequences is lost. Additionally, we showed that the indices based on ISUs and ESVs were equivalent, outperforming the OTU-based one in terms of predictive power and accuracy by revealing the hidden ecological information of sequences that are otherwise clustered in the same OTU (intra-species/intra-population variability). Furthermore, ISUs, ESVs, and morphospecies indices provided similar estimation of site ecological status, validating that ISUs with limited bioinformatics treatments may be used for DNA freshwater monitoring. Our study is a proof of concept where taxonomy- and clustering-free approach is presented, that we believe is a step forward a standardized and comparable DNA bioassessment, complementary to morphological methods