scholarly journals New insights into Danube’s macroinvertebrate communities from DNA metabarcoding as part of the Joint Danube Survey 4 (JDS4)

2021 ◽  
Vol 4 ◽  
Author(s):  
Arne Beermann ◽  
Dominik Buchner ◽  
Florian Leese ◽  
Till-Hendrik Macher ◽  
Miroslav Ocadlik ◽  
...  
Keyword(s):  
Bulk Sample ◽  
Taxonomic Composition ◽  
Danube River ◽  
Taxonomic Resolution ◽  
Macroinvertebrate Communities ◽  
Operational Taxonomic Units ◽  
Sampling Campaign ◽  
Dna Metabarcoding ◽  
Assessment And Monitoring ◽  
Reference Databases

The Joint Danube Survey (JDS) is a multinational effort in monitoring Danube’s water quality, including its major tributaries. The Danube river stretches over a distance of 2,800 km and flows through or borders 10 different countries to which it is of utter importance as a source of potable water and hydrodynamic power. The JDS is conducted every 6 years and provides a unique opportunity to collect comprehensive data on both abiotic parameters and organisms and to raise awareness of the importance of water as a natural resource. As part of JDS and as a biological quality element in many monitoring programs worldwide, macroinvertebrates are monitored as indicators for various environmental conditions. However, due to their diverse taxonomic composition, associated difficulties with their morphology-based identification as well as their sheer abundance, macroinvertebrates are often analysed with a low taxonomic resolution (i.e., above species level). As an alternative, DNA metabarcoding offers a promising approach to capture this species diversity more accurately. Here, we used DNA metabarcoding to investigate the macrozoobenthic diversity of 46 sites from the latest JDS sampling campaign in 2019. To analyse macroinvertebrate diversity, bulk samples were taken by kick-net sampling and analysed using two different approaches, analysing the bulk sample fixative and analysing homogenised organisms from complete bulk samples. DNA metabarcoding of the sample fixative revealed 1,146 Operational Taxonomic Units (OTUs) and 231 species compared to 833 OTUs and 333 species from homogenised sample analysis. While more dipterans, in particular Chironomidae, were detected in fixative (136 species) than homogenised bulk (90 species) analyses, the latter picked up more Trichoptera (19 vs. 2), Amphipoda (10 vs. 4) and Bivalvia species (13 vs. 5). Even though these results of a DNA-based assessment deliver new insights into species richness and composition of Danube’s macroinvertebrate communities from the Danube source to its delta already, it is evident that the majority of OTUs was not assigned to species. While filling this lack of reference sequences poses a major challenge, the JDS consortium also offers a unique opportunity to complement reference databases in a multinational effort towards a more comprehensive Danube assessment and monitoring.

scholarly journals A new oomycete metabarcoding method using the rps10 gene

2021 ◽  
Author(s):  
Zachary S. L. Foster ◽  
Felipe E Albornoz ◽  
Valerie J Fieland ◽  
Meredith M Larsen ◽  
Frank Andrew Jones ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Illumina Miseq ◽  
Taxonomic Resolution ◽  
Reference Database ◽  
Mock Community ◽  
Operational Taxonomic Units ◽  
Wide Range ◽  
Dna Metabarcoding ◽  
Improved Methods

Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause diseases in plants and animals. Improved methods are needed for rapid and accurate characterization of oomycete communities using DNA metabarcoding. We have identified the mitochondrial 40S ribosomal protein S10 gene (rps10) as a locus useful for oomycete metabarcoding and provide primers predicted to amplify all oomycetes based on available reference sequences from a wide range of taxa. We evaluated its utility relative to a popular barcode, the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a mock community using Illumina MiSeq. Amplified sequence variants (ASVs) and operational taxonomic units (OTUs) were identified per community. Both the sequence and predicted taxonomy of ASVs and OTUs were compared to the known composition of the mock community. Both rps10 and ITS yielded ASVs with sequences matching 21 of the 24 species in the mock community and matching all 24 when allowing for a 1 bp difference. Taxonomic classifications of ASVs included 23 members of the mock community for rps10 and 17 for ITS1. Sequencing results for the environmental samples suggest the proposed rps10 locus results in substantially less amplification of non-target organisms than the ITS1 method. The amplified rps10 region also has higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete metabarcoding. The rps10 barcode and methods described herein provide an effective tool for metabarcoding oomycetes using short-read sequencing.

scholarly journals Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1966 ◽  
Author(s):  
Vasco Elbrecht ◽  
Pierre Taberlet ◽  
Tony Dejean ◽  
Alice Valentini ◽  
Philippe Usseglio-Polatera ◽  
...  
Keyword(s):  
Species Level ◽  
Coi Gene ◽  
Taxonomic Resolution ◽  
Reference Database ◽  
Bias Estimation ◽  
Knowing That ◽  
Dna Metabarcoding ◽  
Local Reference ◽  
Comprehensive Survey ◽  
Reference Databases

Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate.

The response of macroinvertebrate community taxa and functional groups to pollution along a heavily impacted river in Central Europe (Bílina River, Czech Republic)

Biologia ◽  
2012 ◽  
Vol 67 (1) ◽  
Author(s):  
Claus Orendt ◽  
Georg Wolfram ◽  
Zdeněk Adámek ◽  
Pavel Jurajda ◽  
Mechthild Schmitt-Jansen
Keyword(s):  
Czech Republic ◽  
Community Analysis ◽  
Organic Pollution ◽  
Taxonomic Composition ◽  
Macroinvertebrate Communities ◽  
Functional Community ◽  
Sampling Campaign ◽  
Physico Chemical ◽  
Polluted Sites ◽  
Bílina River

AbstractMacroinvertebrate communities were investigated along a gradient of heavy industrial and municipal pollution in the highland Bílina River (Czech Republic). Physico-chemical determinants and ions were monitored and community analysis performed focusing on taxonomic composition, ecological functioning (feeder and dweller guilds) and water quality metrics, including saprobity index, BMWP and diversity. Impacted sites differed significantly from reference and from recovered stretches. Chemical data revealed two main pollution factors, (1) a “salinity determinant”, described best by conductivity and SO42−, and (2) an “organic pollution determinant”, represented best by O2 concentrations and NO2−, all varying locally and temporally. Some metrics and taxa showed significant correlations to abiotic parameters. Functional communities showed a stronger relationship to the “organic pollution determinant”, suggesting that elevated organic pollution had a dominating influence on functional community metrics; though other variables may also have an influence in this multistress environment. On the other hand, there were indications that the taxonomic community was more influenced by ion concentrations (“salinity determinant”). The gradient from reference sites to polluted sites was weaker in the final sampling campaign. The results presented here can be used as a reference for assessing future changes in environmental impact from pollution, being finer and more detailed than assessment according to the EU’s WFD.

scholarly journals Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

2016 ◽  
Author(s):  
Vasco Elbrecht ◽  
Pierre Taberlet ◽  
Tony Dejean ◽  
Alice Valentini ◽  
Philippe Usseglio-polatera ◽  
...  
Keyword(s):  
Species Level ◽  
Coi Gene ◽  
Taxonomic Resolution ◽  
Reference Database ◽  
Knowing That ◽  
Freshwater Invertebrate ◽  
Dna Metabarcoding ◽  
Local Reference ◽  
Comprehensive Survey ◽  
Reference Databases

Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. In comparison to COI, the 16S marker amplified more insect species and amplified more equally, probably due to decreased primer bias. Rough estimation of biomass might thus be less biased with 16S than with COI. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of species will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey in a context where it is possible to build a local reference database, the 16S marker could be more appropriate.

scholarly journals Exact sequence variants should replace operational taxonomic units in marker gene data analysis

2017 ◽  
Author(s):  
Benjamin J Callahan ◽  
Paul J McMurdie ◽  
Susan P Holmes
Keyword(s):  
De Novo ◽  
Marker Gene ◽  
Taxonomic Resolution ◽  
Reference Database ◽  
Sequence Variants ◽  
Sequencing Data ◽  
Operational Taxonomic Units ◽  
The Status ◽  
Reference Databases ◽  
Gene Data

AbstractRecent advances have made it possible to analyze high-throughput marker-gene sequencing data without resorting to the customary construction of molecular operational taxonomic units (OTUs): clusters of sequencing reads that differ by less than a fixed dissimilarity threshold. New methods control errors sufficiently that sequence variants (SVs) can be resolved exactly, down to the level of single-nucleotide differences over the sequenced gene region. The benefits of finer taxonomic resolution are immediately apparent, and arguments for SV methods have focused on their improved resolution. Less obvious, but we believe more important, are the broad benefits deriving from the status of SVs as consistent labels with intrinsic biological meaning identified independently from a reference database. Here we discuss how those features grant SVs the combined advantages of closed-reference OTUs — including computational costs that scale linearly with study size, simple merging between independently processed datasets, and forward prediction — and of de novo OTUs — including accurate diversity measurement and applicability to communities lacking deep coverage in reference databases. We argue that the improvements in reusability, reproducibility and comprehensiveness are sufficiently great that SVs should replace OTUs as the standard unit of marker gene analysis and reporting.

scholarly journals Comprehensive coverage of human last meal components revealed by a forensic DNA metabarcoding approach

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Judith Schneider ◽  
Eduard Mas-Carrió ◽  
Catherine Jan ◽  
Christian Miquel ◽  
Pierre Taberlet ◽  
...  
Keyword(s):  
Stomach Content ◽  
Pcr Amplification ◽  
Taxonomic Resolution ◽  
Animal Origin ◽  
Forensic Dna ◽  
Wildlife Ecology ◽  
Operational Taxonomic Units ◽  
Molecular Approaches ◽  
Dna Metabarcoding ◽  
Crucial Information

AbstractStomach content analyses are a valuable tool in human forensic science to interpret perimortem events. While the identification of food components of plant and animal origin has traditionally been conducted by macro- and microscopical approaches in case of incomplete digestion, molecular methods provide the potential to increase sensitivity and taxonomic resolution. In particular, DNA metabarcoding (PCR-amplification and next generation sequencing of complex DNA mixtures) has seen a rapid growth in the field of wildlife ecology to assess species’ diets from faecal and gastric samples. Despite clear advantages, molecular approaches have not yet been established in routine human forensics to investigate the last meal components of deceased persons. In this pilot study we applied for the first time a DNA metabarcoding approach to assess both plant and vertebrate components of 48 human stomach content samples taken during medicolegal autopsies. We obtained a final dataset with 34 vertebrate and 124 vegetal unique sequences, that were clustered to 9 and 33 operational taxonomic units (OTUs), respectively. Our results suggest that this approach can provide crucial information about circumstances preceding death, and open promising perspectives for biomedical dietary surveys based on digested food items found in the gastrointestinal tract.

scholarly journals Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

2016 ◽  
Author(s):  
Vasco Elbrecht ◽  
Pierre Taberlet ◽  
Tony Dejean ◽  
Alice Valentini ◽  
Philippe Usseglio-polatera ◽  
...  
Keyword(s):  
Species Level ◽  
Coi Gene ◽  
Taxonomic Resolution ◽  
Reference Database ◽  
Knowing That ◽  
Freshwater Invertebrate ◽  
Dna Metabarcoding ◽  
Local Reference ◽  
Comprehensive Survey ◽  
Reference Databases

Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. In comparison to COI, the 16S marker amplified more insect species and amplified more equally, probably due to decreased primer bias. Rough estimation of biomass might thus be less biased with 16S than with COI. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of species will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey in a context where it is possible to build a local reference database, the 16S marker could be more appropriate.

scholarly journals Implications of taxonomic and numerical resolution on DNA metabarcoding-based inference of benthic macroinvertebrate responses to river restoration

2021 ◽  
Author(s):  
Joeselle M. Serrana ◽  
Bin Li ◽  
Tetsuya Sumi ◽  
Yasuhiro Takemon ◽  
Kozo Watanabe
Keyword(s):  
Taxonomic Resolution ◽  
Benthic Macroinvertebrate ◽  
Reference Database ◽  
Macroinvertebrate Communities ◽  
Numerical Resolution ◽  
Trinity River ◽  
Dna Metabarcoding ◽  
Gravel Bars ◽  
Biological Assessments

AbstractExploring and clearly defining the level of taxonomic identification and quantification approaches for diversity and biomonitoring studies are essential, given its potential influence on the assessment and interpretation of ecological outcomes. In this study, we evaluated the response of benthic macroinvertebrate communities to the restoration or construction of gravel bars conducted in the dam-impacted Trinity River, with the non-dam influenced tributaries serving as the reference sites. We aim to evaluate the performance of different taxonomic and numerical (i.e., abundance vs. presence/absence data) resolutions of DNA metabarcoding with consequent comparison to morphology-based identification and how it affects assessment outcomes. DNA metabarcoding detected 93% of the morphologically identified individuals and provided finer taxonomic resolution. We also detected significant correlations between morphological sample abundance, biomass, and DNA metabarcoding read abundance. We observed a relatively high and significant congruence in macroinvertebrate community structure and composition between different taxonomic and numerical resolutions of both methods, indicating a satisfactory surrogacy between the two approaches and their varying identification levels and data transformation. Additionally, the community-environmental association were significant for all datasets but showed varying significant associations against the physicochemical parameters. Furthermore, both methods identified Simulium spp. as significant indicators of the dam-impacted gravel bars. Still, only DNA metabarcoding showed significant false discovery rate proving the method’s robustness compared to morphology-based identification. Our observations imply that coarser taxonomic resolution could be highly advantageous to DNA metabarcoding-based applications in situations where the lack of taxonomic information, e.g., poor reference database, might severely affect the quality of biological assessments.

DNA metabarcoding data analysis

2018 ◽  
Author(s):  
Pierre Taberlet ◽  
Aurélie Bonin ◽  
Lucie Zinger ◽  
Eric Coissac
Keyword(s):  
Data Analysis ◽  
Dna Extraction ◽  
Illumina Sequencing ◽  
Molecular Methods ◽  
Sequencing Data ◽  
Ecological Processes ◽  
Operational Taxonomic Units ◽  
Dna Metabarcoding ◽  
Reference Databases ◽  
Illumina Sequencing Data

DNA metabarcoding generates huge amounts of data containing noise introduced by molecular methods. Chapter 8 “DNA metabarcoding data analysis” discusses the analytic steps and available software to curate and evaluate DNA metabarcoding data prior to final ecological analyses. It provides command lines to perform primary analyses of Illumina sequencing data with the OBITools, ranging from read assignment to samples to the formation of molecular operational taxonomic units (MOTUs) and their assignment to a taxon through comparison against reference databases. Chapter 8 also develops several methods to further curate sequencing data from contaminants or dysfunctional PCRs by using DNA extraction, PCR, and sequencing blank controls as well as PCR/biological replicates. It also presents several classical analyses to ensure that the diversity of the sample or the study site is appropriately covered. Finally, this chapter considers what conclusions on biodiversity and ecological processes can be really drawn from metabarcoding data.

scholarly journals Metabarcoding of Insect-Associated Fungal Communities: A Comparison of Internal Transcribed Spacer (ITS) and Large-Subunit (LSU) rRNA Markers

2021 ◽  
Author(s):  
Angelina Ceballos-Escalera ◽  
John Richards ◽  
Maria Belen Arias ◽  
Daegan J. G. Inward ◽  
Alfried Vogler
Keyword(s):  
Internal Transcribed Spacer ◽  
Bark Beetles ◽  
Forest Type ◽  
Phylogenetic Analyses ◽  
Large Subunit ◽  
Taxonomic Composition ◽  
Fungal Communities ◽  
Operational Taxonomic Units ◽  
The Face ◽  
Reference Databases

Abstract Complex communities of fungi are regularly characterised by deep sequencing with standard barcode markers, especially the Internal Transcribed Spacer (ITS) and the Large-Subunit (LSU) gene of the rRNA locus. Full taxonomic characterisation of fungal communities is necessary for establishing ecological associations or for early detection of pathogens and invasive species, but reliance on a single short sequence fragment may be problematic due to various technological and analytical obstacles. Here we conducted a side-by-side comparison of fungal communities associated with bark beetles (Scolytinae), the likely vectors of several tree pathogens, using ITS and LSU. Both markers revealed similar patterns of overall species richness and response to key variables (beetle species, forest type). Identification against the respective reference databases using various classifiers revealed similar higher-level taxonomic composition, but decreasing resolution towards lower levels, especially at the species level. Thus, Operational Taxonomic Units (OTUs) could not be linked via taxonomic classifiers across ITS and LSU fragments. However, a phylogenetic analysis (focused on the epidemiologically important Sordariomycetes) placed OTUs relative to reference sequences spanning both loci and demonstrated the largely similar phylogenetic distribution of ITS and LSU-derived OTUs. The analysis of congruence in both markers also suggested the biologically most defensible threshold values for OTU delimitation (98% for ITS, 99% for LSU). Studies of complex fungal communities using the canonical ITS metabarcode marker require corroboration across additional loci, and benefit from phylogenetic analyses and their greater precision compared to conventional taxonomic classifiers, even in the face of incomplete and partially identified reference databases.

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