Latest Articles from Metabarcoding and Metagenomics Latest 22 Articles from Metabarcoding and Metagenomics https://mbmg.pensoft.net/ Fri, 29 Mar 2024 09:06:13 +0200 Pensoft FeedCreator https://mbmg.pensoft.net/i/logo.jpg Latest Articles from Metabarcoding and Metagenomics https://mbmg.pensoft.net/ Abundance estimation with DNA metabarcoding – recent advancements for terrestrial arthropods https://mbmg.pensoft.net/article/112290/ Metabarcoding and Metagenomics 7: e112290

DOI: 10.3897/mbmg.7.112290

Authors: Wiebke Sickel, Vera Zizka, Alice Scherges, Sarah J. Bourlat, Petra Dieker

Abstract: Biodiversity is declining at alarming rates worldwide and large-scale monitoring is urgently needed to understand changes and their drivers. While classical taxonomic identification of species is time and labour intensive, the combination with DNA-based methods could upscale monitoring activities to achieve larger spatial coverage and increased sampling effort. However, challenges remain for DNA-based methods when the number of individuals per species and/or biomass estimates are required. Several methodological advancements exist to improve the potential of DNA metabarcoding for abundance analysis, which however need further evaluation. Here, we discuss laboratory, as well as some bioinformatic adjustments to DNA metabarcoding workflows regarding their potential to achieve species abundance estimation from arthropod community samples. Our review includes pre-laboratory processing methods such as specimen photography, laboratory methods such as the use of spike-in DNA as an internal standard and bioinformatic advancements like correction factors. We conclude that specimen photography coupled with DNA metabarcoding currently promises the greatest potential to achieve estimates of the number of individuals per species and biomass estimates, but that approaches such as spike-ins and correction factors are promising methods to pursue further.

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Review Article Thu, 23 Nov 2023 17:41:15 +0200
Evaluating five primer pairs for environmental DNA metabarcoding of Central European fish species based on mock communities https://mbmg.pensoft.net/article/103856/ Metabarcoding and Metagenomics 7: e103856

DOI: 10.3897/mbmg.7.103856

Authors: Till-Hendrik Macher, Robin Schütz, Atakan Yildiz, Arne J. Beermann, Florian Leese

Abstract: Environmental DNA (eDNA) metabarcoding has become a powerful tool for examining fish communities. Prior to the introduction of eDNA-based assessments into regulatory monitoring contexts (e.g., EU Water Framework Directive), there is a demand for methodological standardization. To ensure methodical accuracy and to meet regulatory standards, various sampling, laboratory and bioinformatic workflows have been established. However, a crucial prerequisite for comprehensive fish monitoring is the choice of suitable primer pairs to accurately identify the fishes present in a given water body. Various fish-specific primer pairs targeting different genetic marker regions were published over the past decade. However, a dedicated study to evaluate the performance of frequently applied fish primer pairs to assess Central European fish species has not yet been conducted. Therefore, we created an artificial 'mock' community composed of DNA from 45 Central European fish species and examined the detection ability and reproducibility of five primer pairs. Our study highlights the effect of primer choice and bioinformatic filtering on the outcome of eDNA metabarcoding results. From the five primer pairs evaluated in our study the tele02 (12S gene) primer pair was the best choice for eDNA metabarcoding of Central European freshwater fish. Also, the MiFish-U (12S) and SeaDNA-mid (COI) primer pairs displayed good detection ability and reproducibility. However, less specific primer pairs (i.e., targeting vertebrates) were found to be less reliable and generated high numbers of false-positive and false-negative detections. Our study illustrates how the careful selection of primer pairs and bioinformatic pipelines can make eDNA metabarcoding a more reliable tool for fish monitoring.

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Primer Validation Tue, 5 Sep 2023 14:09:27 +0300
Recommendations for the preservation of environmental samples in diatom metabarcoding studies https://mbmg.pensoft.net/article/85844/ Metabarcoding and Metagenomics 6: e85844

DOI: 10.3897/mbmg.6.85844

Authors: Ana Baricevic, Cécile Chardon, Maria Kahlert, Satu Maaria Karjalainen, Daniela Maric Pfannkuchen, Martin Pfannkuchen, Frédéric Rimet, Mirta Smodlaka Tankovic, Rosa Trobajo, Valentin Vasselon, Jonas Zimmermann, Agnès Bouchez

Abstract: 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.

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Research Article Thu, 24 Nov 2022 18:40:59 +0200
Environmental DNA metabarcoding is a promising method for assaying fish diversity in cenotes of the Yucatán Peninsula, Mexico https://mbmg.pensoft.net/article/89857/ Metabarcoding and Metagenomics 6: e89857

DOI: 10.3897/mbmg.6.89857

Authors: S. Elizabeth Alter, Jairo Arroyave

Abstract: The karst aquifer of the Yucatán Peninsula (YP) in southeastern Mexico is a unique ecosystem in which water-filled sinkholes, locally known as cenotes, connect subterranean waters with the surface. This system is home to around 20 species of freshwater fishes, including several that are endemic and/or threatened. Studies on this unique ichthyofauna have been partially hampered by the technical difficulties associated with sampling these habitats, particularly submerged caves. In this proof-of-concept study, we use environmental DNA (eDNA) metabarcoding to survey the diversity of freshwater fishes associated with the YP karst aquifer by sampling six cenotes from across the Ring of Cenotes region in northwestern Yucatán, a 180-km-diameter semicircular band of abundant karst sinkholes. Through a combination of conventional sampling (direct observation, fishing) and eDNA metabarcoding, we detected eight species of freshwater fishes across the six sampled cenotes. Overall, our eDNA metabarcoding approach was effective at detecting the presence of fishes from cenote water samples, including one of the two endemic cave-dwelling fish species restricted to the subterranean section of the aquifer. Although our study was focused on detecting fishes via eDNA, we also recovered DNA from several other vertebrate groups, particularly bats. These results suggest that the eDNA metabarcoding approach represents a promising and largely noninvasive method to assay aquatic biodiversity in these vulnerable habitats, allowing more effective, frequent, and wide-ranging surveys. Our detection of DNA from aerial and terrestrial vertebrate fauna implies that eDNA from cenotes, besides being a means to survey aquatic fauna, may also offer an effective way to quickly survey non-aquatic biodiversity associated with these persistent water bodies.

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Research Article Mon, 24 Oct 2022 11:16:02 +0300
Monitoring harmful microalgal species and their appearance in Tokyo Bay, Japan, using metabarcoding https://mbmg.pensoft.net/article/79471/ Metabarcoding and Metagenomics 6: e79471

DOI: 10.3897/mbmg.6.79471

Authors: Sirje Sildever, Noriko Nishi, Nobuharu Inaba, Taiga Asakura, Jun Kikuchi, Yasuhito Asano, Takanori Kobayashi, Takashi Gojobori, Satoshi Nagai

Abstract: During the recent decade, high-throughput sequencing (HTS) techniques, in particular, DNA metabarcoding, have facilitated increased detection of biodiversity, including harmful algal bloom (HAB) species. In this study, the presence of HAB species and their appearance patterns were investigated by employing molecular and light microscopy-based monitoring in Tokyo Bay, Japan. The potential co-appearance patterns between the HAB species, as well as with other eukaryotes and prokaryotes were investigated using correlation and association rule-based time-series analysis. In total, 40 unique HAB species were detected, including 12 toxin-producing HAB species previously not reported from the area. More than half of the HAB species were present throughout the sampling season (summer to autumn) and no structuring or succession patterns associated with the environmental conditions could be detected. Statistically significant (p < 0.05, rS ranging from −0.88 to 0.90) associations were found amongst the HAB species and other eukaryotic and prokaryotic species, including genera containing growth-limiting bacteria. However, significant correlations between species differed amongst the years, indicating that variability in environmental conditions between the years may have a stronger influence on the microalgal community structure and interspecies interactions than the variability during the sampling season. The association rule-based time-series analysis allowed the detection of a previously reported negative relationship between Synechococcus sp. and Skeletonema sp. in nature. Overall, the results support the applicability of metabarcoding and HTS-based microalgae monitoring, as it facilitates more precise species identification compared to light microscopy, as well as provides input for investigating potential interactions amongst different species/groups through simultaneous detection of multiple species/genera.

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Research Article Tue, 23 Aug 2022 18:50:32 +0300
A strategy for successful integration of DNA-based methods in aquatic monitoring https://mbmg.pensoft.net/article/85652/ Metabarcoding and Metagenomics 6: e85652

DOI: 10.3897/mbmg.6.85652

Authors: Philippe Blancher, Estelle Lefrançois, Frédéric Rimet, Valentin Vasselon, Christine Argillier, Jens Arle, Pedro Beja, Pieter Boets, Jeanne Boughaba, Christian Chauvin, Michael Deacon, Willie Duncan, Gunilla Ejdung, Stefania Erba, Benoit Ferrari, Helmut Fischer, Bernd Hänfling, Michael Haldin, Daniel Hering, Nicolas Hette-Tronquart, Alice Hiley, Marko Järvinen, Benjamin Jeannot, Maria Kahlert, Martyn Kelly, Julia Kleinteich, Serdar Koyuncuoğlu, Sascha Krenek, Sidsel Langhein-Winther, Florian Leese, David Mann, Rémy Marcel, Stefania Marcheggiani, Kristian Meissner, Patricia Mergen, Olivier Monnier, Frank Narendja, Diane Neu, Veronica Onofre Pinto, Alina Pawlowska, Jan Pawlowski, Martin Petersen, Sandra Poikane, Didier Pont, Marie-Sophie Renevier, Steinar Sandoy, Jonas Svensson, Rosa Trobajo, Andrea Tünde Zagyva, Iakovos Tziortzis, Berry van der Hoorn, Marlen Ines Vasquez, Kerry Walsh, Alexander Weigand, Agnès Bouchez

Abstract: Recent advances in molecular biomonitoring open new horizons for aquatic ecosystem assessment. Rapid and cost-effective methods based on organismal DNA or environmental DNA (eDNA) now offer the opportunity to produce inventories of indicator taxa that can subsequently be used to assess biodiversity and ecological quality. However, the integration of these new DNA-based methods into current monitoring practices is not straightforward, and will require coordinated actions in the coming years at national and international levels. To plan and stimulate such an integration, the European network DNAqua-Net (COST Action CA15219) brought together international experts from academia, as well as key environmental biomonitoring stakeholders from different European countries. Together, this transdisciplinary consortium developed a roadmap for implementing DNA-based methods with a focus on inland waters assessed by the EU Water Framework Directive (2000/60/EC). This was done through a series of online workshops held in April 2020, which included fifty participants, followed by extensive synthesis work. The roadmap is organised around six objectives: 1) to highlight the effectiveness and benefits of DNA-based methods, 2) develop an adaptive approach for the implementation of new methods, 3) provide guidelines and standards for best practice, 4) engage stakeholders and ensure effective knowledge transfer, 5) support the environmental biomonitoring sector to achieve the required changes, 6) steer the process and harmonise efforts at the European level. This paper provides an overview of the forum discussions and the common European views that have emerged from them, while reflecting the diversity of situations in different countries. It highlights important actions required for a successful implementation of DNA-based biomonitoring of aquatic ecosystems by 2030.

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Forum Paper Wed, 20 Jul 2022 13:22:09 +0300
Evaluation of non-destructive DNA extraction protocols for insect metabarcoding: gentler and shorter is better https://mbmg.pensoft.net/article/78871/ Metabarcoding and Metagenomics 6: e78871

DOI: 10.3897/mbmg.6.78871

Authors: Daniel Marquina, Tomas Roslin, Piotr Łukasik, Fredrik Ronquist

Abstract: DNA metabarcoding can accelerate research on insect diversity, as it is cheap and fast compared to manual sorting and identification. Most metabarcoding protocols require homogenisation of the sample, preventing further work on the specimens. Mild digestion of the tissue by incubation in a lysis buffer has been proposed as an alternative, and, although some mild lysis protocols have already been presented, they have so far not been evaluated against each other. Here, we analyse how two mild lysis buffers (one more aggressive, one gentler in terms of tissue degradation), two different incubation times, and two DNA purification methods (a manual precipitation and an automated protocol) affect the accuracy of retrieving the true composition of mock communities using two mitochondrial markers (COI and 16S). We found that protocol-specific variation in concentration and purity of the DNA extracts produced had little effect on the recovery of species. However, the two lysis treatments differed in quantification of species abundances. Digestion in the gentler buffer and for a shorter time yielded better representation of original sample composition. Digestion in a more aggressive buffer or longer incubation time yielded lower alpha diversity values and increased differences between metabarcoding results and the true species-abundance distribution. We conclude that the details of non-destructive protocols can have a significant effect on metabarcoding performance. A short and mild lysis treatment appears the best choice for recovering the true composition of the sample. This not only improves accuracy, but also comes with a faster processing time than the other treatments.

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Research Article Thu, 16 Jun 2022 19:07:36 +0300
A triad of kicknet sampling, eDNA metabarcoding, and predictive modeling to assess richness of mayflies, stoneflies and caddisflies in rivers https://mbmg.pensoft.net/article/79351/ Metabarcoding and Metagenomics 6: e79351

DOI: 10.3897/mbmg.6.79351

Authors: François Keck, Samuel Hürlemann, Nadine Locher, Christian Stamm, Kristy Deiner, Florian Altermatt

Abstract: Monitoring biodiversity is essential to understand the impacts of human activities and for effective management of ecosystems. Thereby, biodiversity can be assessed through direct collection of targeted organisms, through indirect evidence of their presence (e.g. signs, environmental DNA, camera trap, etc.), or through extrapolations from species distribution and species richness models. Differences in approaches used in biodiversity assessment, however, may come with individual challenges and hinder cross-study comparability. In the context of rapidly developing techniques, we compared three different approaches in order to better understand assessments of aquatic macroinvertebrate diversity. Specifically, we compared the community composition and species richness of three orders of aquatic macroinvertebrates (mayflies, stoneflies, and caddisflies, hereafter EPT) obtained via eDNA metabarcoding and via traditional in situ kicknet sampling to catchment-level based predictions of a species richness model. We used kicknet data from 24 sites in Switzerland and compared taxonomic lists to those obtained using eDNA amplified with two different primer sets. Richness detected by these methods was compared to the independent predictions made by a statistical species richness model, that is, a generalized linear model using landscape-level features to estimate EPT diversity. Despite the ability of eDNA to consistently detect some EPT species found by traditional sampling, we found important discrepancies in community composition between the kicknet and eDNA approaches, particularly at a local scale. We found the EPT-specific primer set fwhF2/EPTDr2n, detected a greater number of targeted EPT species compared to the more general primer set mlCOIintF/HCO2198. Moreover, we found that the species richness measured by eDNA from either primer set was poorly correlated to the richness measured by kicknet sampling (Pearson correlation = 0.27) and that the richness estimated by eDNA and kicknet were poorly correlated with the prediction of the species richness model (Pearson correlation = 0.30 and 0.44, respectively). The weak relationships between the traditional kicknet sampling and eDNA with this model indicates inherent limitations in upscaling species richness estimates, and possibly a limited ability of the model to meet real world expectations. It is also possible that the number of replicates was not sufficient to detect ambiguous correlations. Future challenges include improving the accuracy and sensitivity of each approach individually, yet also acknowledging their respective limitations, in order to best meet stakeholder demands and address the biodiversity crisis we are facing.

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Research Article Tue, 10 May 2022 17:31:17 +0300
Comparing species detection success between molecular markers in DNA metabarcoding of coastal macroinvertebrates https://mbmg.pensoft.net/article/70063/ Metabarcoding and Metagenomics 5: e70063

DOI: 10.3897/mbmg.5.70063

Authors: Barbara R. Leite, Pedro E. Vieira, Jesús S. Troncoso, Filipe O. Costa

Abstract: DNA metabarcoding has great potential to improve marine biomonitoring programs by providing a rapid and accurate assessment of species composition in zoobenthic communities. However, some methodological improvements are still required, especially regarding failed detections, primers efficiency and incompleteness of databases. Here we assessed the efficiency of two different marker loci (COI and 18S) and three primer pairs in marine species detection through DNA metabarcoding of the macrozoobenthic communities colonizing three types of artificial substrates (slate, PVC and granite), sampled between 3 and 15 months of deployment. To accurately compare detection success between markers, we also compared the representativeness of the detected species in public databases and revised the reliability of the taxonomic assignments. Globally, we recorded extensive complementarity in the species detected by each marker, with 69% of the species exclusively detected by either 18S or COI. Individually, each of the three primer pairs recovered, at most, 52% of all species detected on the samples, showing also different abilities to amplify specific taxonomic groups. Most of the detected species have reliable reference sequences in their respective databases (82% for COI and 72% for 18S), meaning that when a species was detected by one marker and not by the other, it was most likely due to faulty amplification, and not by lack of matching sequences in the database. Overall, results showed the impact of marker and primer applied on species detection ability and indicated that, currently, if only a single marker or primer pair is employed in marine zoobenthos metabarcoding, a fair portion of the diversity may be overlooked.

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Methods Wed, 29 Dec 2021 17:51:59 +0200
Phytool, a ShinyApp to homogenise taxonomy of freshwater microalgae from DNA barcodes and microscopic observations https://mbmg.pensoft.net/article/74096/ Metabarcoding and Metagenomics 5: e74096

DOI: 10.3897/mbmg.5.74096

Authors: Alexis Canino, Agnès Bouchez, Christophe Laplace-Treyture, Isabelle Domaizon, Frédéric Rimet

Abstract: Methods for biomonitoring of freshwater phytoplankton are evolving rapidly with eDNA-based methods, offering great complementarity with microscopy. Metabarcoding approaches have been more commonly used over the last years, with a continuous increase in the amount of data generated. Depending on the researchers and the way they assigned barcodes to species (bioinformatic pipelines and molecular reference databases), the taxonomic assignment obtained for HTS DNA reads might vary. This is also true for traditional taxonomic studies by microscopy with regular adjustments of the classification and taxonomy. For those reasons (leading to non-homogeneous taxonomies), gap-analyses and comparisons between studies become even more challenging and the curation processes to find potential consensus names are time-consuming. Here, we present a web-based application (Phytool), developed with ShinyApp (Rstudio), that aims to make the harmonisation of taxonomy easier and in a more efficient way, using a complete and up-to-date taxonomy reference database for freshwater microalgae. Phytool allows users to homogenise and update freshwater phytoplankton taxonomical names from sequence files and data tables directly uploaded in the application. It also gathers barcodes from curated references in a user-friendly way in which it is possible to search for specific organisms. All the data provided are downloadable with the possibility to apply filters in order to select only the required taxa and fields (e.g. specific taxonomic ranks). The main goal is to make accessible to a broad range of users the connection between microscopy and molecular biology and taxonomy through different ready-to-use functions. This study estimates that only 25% of species of freshwater phytoplankton in Phytobs are associated with a barcode. We plead for an increased effort to enrich reference databases by coupling taxonomy and molecular methods. Phytool should make this crucial work more efficient. The application is available at https://caninuzzo.shinyapps.io/phytool_v1/

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Software Description Wed, 15 Dec 2021 08:44:20 +0200
Plankton biodiversity and species co-occurrence based on environmental DNA – a multiple marker study https://mbmg.pensoft.net/article/72371/ Metabarcoding and Metagenomics 5: e72371

DOI: 10.3897/mbmg.5.72371

Authors: Sirje Sildever, Peeter Laas, Natalja Kolesova, Inga Lips, Urmas Lips, Satoshi Nagai

Abstract: Metabarcoding in combination with high-throughput sequencing (HTS) allows simultaneous detection of multiple taxa by targeting single or several taxonomically informative gene regions from environmental DNA samples. In this study, a multiple-marker HTS approach was applied to investigate the plankton diversity and seasonal succession in the Baltic Sea from winter to autumn. Four different markers targeting the 16S, 18S, and 28S ribosomal RNA genes were employed, including a marker for more efficient dinoflagellate detection. Typical seasonal changes were observed in phyto- and bacterioplankton communities. In phytoplankton, the appearance patterns of selected common, dominant, or harmful species followed the patterns also confirmed based on 20 years of phytoplankton monitoring data. In the case of zooplankton, both macro- and microzooplankton species were detected. However, no seasonal patterns were detected in their appearance. In total, 15 and 2 new zoo- and phytoplankton species were detected from the Baltic Sea. HTS approach was especially useful for detecting microzooplankton species as well as for investigating the co-occurrence and potential interactions of different taxa. The results of this study further exemplify the efficiency of metabarcoding for biodiversity monitoring and the advantage of employing multiple markers through the detection of species not identifiable based on a single marker survey and/or by traditional morphology-based methods.

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Research Article Wed, 3 Nov 2021 13:48:46 +0200
18S rRNA amplicon sequence data (V1–V3) of the Bronx river estuary, New York https://mbmg.pensoft.net/article/69691/ Metabarcoding and Metagenomics 5: e69691

DOI: 10.3897/mbmg.5.69691

Authors: Melissa R. Ingala, Irena E. Werner, Allison M. Fitzgerald, Eugenia Naro-Maciel

Abstract: Characterising and monitoring biological diversity to foster sustainable ecosystems is highly recommended as urban centres rapidly expand. However, much of New York City’s biodiversity remains undescribed, including in the historically degraded, but recovering Bronx River Estuary. In a pilot study to identify organisms and characterise biodiversity patterns there, 18S rRNA gene amplicons (V1–V3 region), obtained from river sediments and surface waters of Hunts Point Riverside and Soundview Parks, were sequenced. Across 48 environmental samples collected over three seasons in 2015 and 2016, following quality control and contaminant removal, 2,763 Amplicon Sequence Variants (ASVs) were identified from 1,918,463 sequences. Rarefaction analysis showed sufficient sampling depth, and community composition varied over time and by substrate at the study sites over the sampling period. Protists, plants, fungi and animals, including organisms of management concern, such as Eastern oysters (Crassostrea virginica), wildlife pathogens and groups related to Harmful Algal Blooms, were detected. The most common taxa identified in river sediments were annelid worms, nematodes and diatoms. In the water column, the most commonly observed organisms were diatoms, algae of the phylum Cryptophyceae, ciliates and dinoflagellates. The presented dataset demonstrates the reach of 18S rRNA metabarcoding for characterising biodiversity in an urban estuary.

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Data Paper Tue, 7 Sep 2021 08:22:23 +0300
How can integrated morphotaxonomy- and metabarcoding-based diatom assemblage analyses best contribute to the ecological assessment of streams? https://mbmg.pensoft.net/article/68438/ Metabarcoding and Metagenomics 5: e68438

DOI: 10.3897/mbmg.5.68438

Authors: Panayiota Pissaridou, Marco Cantonati, Agnes Bouchez, Iakovos Tziortzis, Gerald Dörflinger, Marlen I. Vasquez

Abstract: 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.

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Research Article Wed, 25 Aug 2021 14:32:09 +0300
Wet grinding of invertebrate bulk samples – a scalable and cost-efficient protocol for metabarcoding and metagenomics https://mbmg.pensoft.net/article/67533/ Metabarcoding and Metagenomics 5: e67533

DOI: 10.3897/mbmg.5.67533

Authors: Dominik Buchner, Peter Haase, Florian Leese

Abstract: Most metabarcoding protocols for invertebrate bulk samples start with sample homogenisation, followed by DNA extraction, amplification of a specific marker region, and sequencing. Many of the above-mentioned laboratory steps have been verified thoroughly and best practice strategies exist, yet, no clear recommendation for the basis of almost all metabarcoding studies exists: the homogenisation of samples itself. Two different categories of devices are typically used for homogenisation: bead mills or blenders. Both have upsides and downsides. Bead mills rely on single-use plastics and therefore produce a lot of waste and are expensive. In addition to that, processing times can go up to 30 minutes making them unsuitable for large-scale studies. Blenders can handle larger sample volumes in a shorter time, and be cleaned – yet suffer from an increased risk of cross-contamination. We aimed to develop a fast, robust, cheap, and reliable sample homogenisation protocol that overcomes limitations of both approaches, i.e. does not produce difficult to discard waste and avoid single-use plastics while reducing overall costs. We tested the performance of the new protocol using six size-sorted Malaise trap samples and six unsorted stream macroinvertebrate kick-net samples. We used 14 replicates per sample and included many negative controls at different steps of the protocol to quantify the impacts of i) insufficient homogenisation and ii) cross-contamination. Our results show that 3-min homogenisation is sufficient to recover about 80% of OTUs per sample in each replicate and that a non-hazardous DIY cleaning solution provides an effective and efficient way of cleaning. The improvements of the protocol in terms of speed, ease of handling, an overall reduction of costs as well as the documented reliability and robustness make it an important candidate for sample homogenisation after sampling in particular for large-scale and regulatory metabarcoding but also metagenomics biodiversity assessments and monitoring.

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Methods Tue, 20 Jul 2021 13:27:58 +0300
Application of propylene glycol in DNA-based studies of invertebrates https://mbmg.pensoft.net/article/57278/ Metabarcoding and Metagenomics 5: e57278

DOI: 10.3897/mbmg.5.57278

Authors: Alexander M. Weigand, Noah Desquiotz, Hannah Weigand, Nikolaus Szucsich

Abstract: High-throughput sequencing (HTS) studies on invertebrates commonly use ethanol as the main sample fixative (upon collection) and preservative (for storage and curation). However, alternative agents exists, which should not be automatically neglected when studies are newly designed. This review provides an overview of the application of propylene glycol (PG) in DNA-based studies of invertebrates, thus to stimulate an evidence-based discussion. The use of PG in DNA-based studies of invertebrates is still limited (n = 79), but a steady increase has been visible since 2011. Most studies used PG as a fixative for passive trapping (73%) and performed Sanger sequencing (66%; e.g. DNA barcoding). More recently, HTS setups joined the field (11%). Terrestrial Coleoptera (30%) and Diptera (20%) were the most studied groups. Very often, information on the grade of PG used (75%) or storage conditions (duration, temperature) were lacking. This rendered direct comparisons of study results difficult, and highlight the need for further systematic studies on these subjects. When compared to absolute ethanol, PG can be more widely and cheaply acquired (e.g. as an antifreeze, 13% of studies). It also enables longer trapping intervals, being especially relevant at remote or hard-to-reach places. Shipping of PG-conserved samples is regarded as risk-free and is authorised, pinpointing its potential for larger trapping programs or citizen science projects. Its property to retain flexibility of morphological characters as well as to lead to a reduced shrinkage effect was especially appraised by integrative study designs. Finally, the so far limited application of PG in the context of HTS showed promising results for short read amplicon sequencing and reduced representation methods. Knowledge of the influence of PG fixation and storage for long(er) read HTS setups is currently unavailable. Given our review results and taking difficulties of direct methodological comparisons into account, future DNA-based studies of invertebrates should on a case-by-case basis critically scrutinise if the application of PG in their anticipated study design can be of benefit.

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Review Article Mon, 4 Jan 2021 15:31:58 +0200
Can metabarcoding resolve intraspecific genetic diversity changes to environmental stressors? A test case using river macrozoobenthos https://mbmg.pensoft.net/article/51925/ Metabarcoding and Metagenomics 4: e51925

DOI: 10.3897/mbmg.4.51925

Authors: Vera Marie Alida Zizka, Martina Weiss, Florian Leese

Abstract: Genetic diversity is the most basal level of biodiversity and determines the evolutionary capacity of species to adapt to changing environments, yet it is typically neglected in routine biomonitoring and stressor impact assessment. For a comprehensive analysis of stressor impacts on genetic diversity, it is necessary to assess genetic variants simultaneously in many individuals and species. Such an assessment is not as straightforward and usually limited to one or few focal species. However, nowadays species diversity can be assessed by analysing thousands of individuals of a community simultaneously with DNA metabarcoding. Recent bioinformatic advances also allow for the extraction of exact sequence variants (ESVs or haplotypes) in addition to Operational Taxonomic Units (OTUs). By using this new capability, we here evaluated if the analysis of intraspecific mitochondrial diversity in addition to species diversity can provide insights into responses of stream macrozoobenthic communities to environmental stressors. For this purpose, we analysed macroinvertebrate bulk samples of three German river systems with different stressor levels using DNA metabarcoding. While OTU and haplotype number were negatively correlated with stressor impact, this association was not as clear when studying haplotype diversity across all taxa. However, stressor responses were found for sensitive EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa and those exceedingly resistant to organic stress. An increase in haplotype number per OTU and haplotype diversity of sensitive taxa was observed with an increase in ecosystem quality and stability, while the opposite pattern was detected for pollution resistant taxa. However, this pattern was less prominent than expected based on the strong differences in stressor intensity between sites. To compare genetic diversity among communities in river systems, we focussed on OTUs, which were present in all systems. As OTU composition differed strongly between rivers, this led to the exclusion of a high number of OTUs, especially in diverse river systems of good quality, which potentially diminished the increase in intraspecific diversity. To better understand responses of intraspecific genetic diversity to environmental stressors, for example in river ecosystems, it would be important to increase OTU overlap between compared sites, e.g. by sampling a narrower stressor gradient, and to perform calibrated studies controlling for the number of individuals and their haplotypes. However, this pioneer study shows that the extraction of haplotypes from DNA metabarcoding datasets is a promising source of information to simultaneously assess intraspecific diversity changes in response to environmental impacts for a metacommunity.

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Research Article Fri, 24 Jul 2020 08:14:34 +0300
Assessing pollution of aquatic environments with diatoms’ DNA metabarcoding: experience and developments from France Water Framework Directive networks https://mbmg.pensoft.net/article/39646/ Metabarcoding and Metagenomics 3: e39646

DOI: 10.3897/mbmg.3.39646

Authors: Vasselon Valentin, Rimet Frédéric, Domaizon Isabelle, Monnier Olivier, Reyjol Yorick, Bouchez Agnès

Abstract: 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.

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Research Article Wed, 6 Nov 2019 13:35:23 +0200
Advancing the use of molecular methods for routine freshwater macroinvertebrate biomonitoring – the need for calibration experiments https://mbmg.pensoft.net/article/34735/ Metabarcoding and Metagenomics 3: e34735

DOI: 10.3897/mbmg.3.34735

Authors: Rosetta C. Blackman, Elvira Mächler, Florian Altermatt, Amanda Arnold, Pedro Beja, Pieter Boets, Bastian Egeter, Vasco Elbrecht, Ana Filipa Filipe, J. Iwan Jones, Jan Macher, Markus Majaneva, Filipa M. S. Martins, Cesc Múrria, Kristian Meissner, Jan Pawlowski, Paul L. Schmidt Yáñez, Vera M.A. Zizka, Florian Leese, Benjamin Price, Kristy Deiner

Abstract: Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates.

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Forum Paper Thu, 11 Jul 2019 17:29:00 +0300
Monitoring riverine fish communities through eDNA metabarcoding: determining optimal sampling strategies along an altitudinal and biodiversity gradient https://mbmg.pensoft.net/article/30457/ Metabarcoding and Metagenomics 2: e30457

DOI: 10.3897/mbmg.2.30457

Authors: Jonas Bylemans, Dianne M. Gleeson, Mark Lintermans, Christopher M. Hardy, Matthew Beitzel, Dean M. Gilligan, Elise M. Furlan

Abstract: Monitoring aquatic biodiversity through DNA extracted from environmental samples (eDNA) combined with high-throughput sequencing, commonly referred to as eDNA metabarcoding, is increasing in popularity within the scientific community. However, sampling strategies, laboratory protocols and analytical pipelines can influence the results of eDNA metabarcoding surveys. While the impact of laboratory protocols and analytical pipelines have been extensively studied, the importance of sampling strategies on eDNA metabarcoding surveys has not received the same attention. To avoid underestimating local biodiversity, adequate sampling strategies (i.e. sampling intensity and spatial sampling replication) need to be implemented. This study evaluated the impact of sampling strategies along an altitudinal and biodiversity gradient in the upper section of the Murrumbidgee River (Murray-Darling Basin, Australia). An eDNA metabarcoding survey was used to determine the local fish biodiversity and evaluate the influence of sampling intensity and spatial sampling replication on the biodiversity estimates. The results show that optimal eDNA sampling strategies varied between sites and indicate that river morphology, species richness and species abundance affect the optimal sampling intensity and spatial sampling replication needed to accurately assess the fish biodiversity. While the generality of the patterns will need to be confirmed through future studies, these findings provide a basis to guide future eDNA metabarcoding surveys in river systems.

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Research Article Mon, 31 Dec 2018 12:00:49 +0200
The influence of macroinvertebrate abundance on the assessment of freshwater quality in The Netherlands https://mbmg.pensoft.net/article/26744/ Metabarcoding and Metagenomics 2: e26744

DOI: 10.3897/mbmg.2.26744

Authors: Kevin K. Beentjes, Arjen G. C. L. Speksnijder, Menno Schilthuizen, Bartholomeus E.M. Schaub, Berry B. van der Hoorn

Abstract: The use of molecular tools for the detection and identification of invertebrate species enables the development of more easily standardisable inventories of biological elements for water quality assessments, as it circumvents human-based bias and errors in species identifications. Current Ecological Quality Ratio (EQR) assessments methods, however, often rely on abundance data. Translating metabarcoding sequence data into biomass or specimen abundances has proven difficult, as PCR amplification bias due to primer mismatching often provides skewed proportions of read abundances. While some potential solutions have been proposed in previous research, we instead looked at the necessity of abundance data in EQR assessments. In this study, we used historical monitoring data from natural (lakes, rivers and streams) and artificial (ditches and canals) water bodies to assess the impact of species abundances on the EQR scores for macroinvertebrates in the Water Framework Directive (WFD) monitoring programme of The Netherlands. By removing all the abundance data from the taxon observations, we simulated presence/absence-based monitoring, for which EQRs were calculated according to traditional methods. Our results showed a strong correlation between abundance-based and presence/absence-based EQRs. EQR scores were generally higher without abundances (75.8% of all samples), which resulted in 9.1% of samples being assigned to a higher quality class. The majority of the samples (89.7%) were assigned to the same quality class in both cases. These results are valuable for the incorporation of presence/absence metabarcoding data into water quality assessment methodology, potentially eliminating the need to translate metabarcoding data into biomass or absolute specimen counts for EQR assessments.

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Research Article Fri, 12 Oct 2018 13:32:20 +0300
A DNA metabarcoding protocol for hyporheic freshwater meiofauna: Evaluating highly degenerate COI primers and replication strategy https://mbmg.pensoft.net/article/26869/ Metabarcoding and Metagenomics 2: e26869

DOI: 10.3897/mbmg.2.26869

Authors: Alexander M. Weigand, Jan-Niklas Macher

Abstract: The hyporheic zone, i.e. the ecotone between surface water and the groundwater, is a rarely studied freshwater ecosystem. Hyporheic taxa are often meiofaunal (<1 mm) in size and difficult to identify based on morphology. Metabarcoding approaches are promising for the study of these environments and taxa, but it is yet unclear if commonly applied metabarcoding primers and replication strategies can be used. In this study, we took sediment cores from two near natural upstream (NNU) and two ecologically improved downstream (EID) sites in the Boye catchment (Emscher River, Germany), metabarcoding their meiofaunal communities. We evaluated the usability of a commonly used, highly degenerate COI primer pair (BF2/BR2) and tested how sequencing three PCR replicates per sample and removing MOTUs present in only one out of three replicates impacts the inferred community composition. A total of 22,514 MOTUs were detected, of which only 263 were identified as Metazoa. Our results highlight the gaps in reference databases for meiofaunal taxa and the potential problems of using highly degenerate primers for studying samples containing a high number of non-metazoan taxa. Alpha diversity was higher in EID sites and showed higher community similarity when compared to NNU sites. Beta diversity analyses showed that removing MOTUs detected in only one out of three replicates per site greatly increased community similarity in samples. Sequencing three sample replicates and removing rare MOTUs is seen as a good compromise between retaining too many false-positives and introducing too many false-negatives. We conclude that metabarcoding hyporheic communities using highly degenerate COI primers can provide valuable first insights into the diversity of these ecosystems and highlight some potential application scenarios.

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Methods Thu, 23 Aug 2018 09:49:07 +0300
Combining NCBI and BOLD databases for OTU assignment in metabarcoding and metagenomic datasets: The BOLD_NCBI _Merger https://mbmg.pensoft.net/article/22262/ Metabarcoding and Metagenomics 1: e22262

DOI: 10.3897/mbmg.1.22262

Authors: Jan-Niklas Macher, Till-Hendrik Macher, Florian Leese

Abstract: Metabarcoding and metagenomic approaches are becoming routine techniquesfor use in biodiversity assessment and in ecological studies. The assignment of taxonomic information to millions of sequences obtained via high-throughput sequencing is challenging, as many DNA reference libraries are lacking information on certain taxonomic groups and can contain erroneous sequences. Combining different reference databases is therefore a promising approach for maximising taxonomic coverage and reliability of results. The “BOLD_NCBI_Merger” bash script is introduced, which combines sequence data obtained from the National Centre for Biotechnology Information (NCBI) GenBank and the Barcode of Life Database (BOLD) and prepares it for taxonomic assignment with the software MEGAN.

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Software Description Fri, 15 Dec 2017 15:03:10 +0200