scholarly journals Improved freshwater macroinvertebrate detection from environmental DNA through minimized nontarget amplification

2021 ◽  
Vol 4 ◽  
Author(s):  
Mandy Sander ◽  
Arne Beermann ◽  
Dominik Buchner ◽  
Vasco Elbrecht ◽  
Peter Haase ◽  
...  
Keyword(s):  
Benthic Invertebrates ◽  
Environmental Dna ◽  
Benthic Invertebrate ◽  
Coi Gene ◽  
Taxonomic Resolution ◽  
Universal Primers ◽  
Reference Database ◽  
Mountain Range ◽  
Degenerate Primers ◽  
Universal Primer

Environmental DNA (eDNA) metabarcoding is a new, promising, and non-invasive method to detect biodiversity in aquatic environments. So far, it has mainly been used to screen for fish and amphibian diversity and rarely to detect macroinvertebrates. Typically, DNA metabarcoding relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase I (COI) gene with degenerate primers. In comparison to other genes like 16S, COI has a greater taxonomic resolution and availability of an extensive reference database. Benthic stream invertebrates are of critical importance for regulatory biomonitoring, but when using universal primers on eDNA isolated from water, the number of reads and OTUs is “watered down”. This means the target taxa, macroinvertebrates, are underrepresented in comparison to other nontarget taxa, e. g. algae, bacteria, and fungi. The aim of the project was to design an insect-specific primer, which minimizes nontarget amplification. Therefore, data from a time series of 15 months at the Kinzig (Hesse), a silica-rich low-mountain-range stream, which is part of the Rhine‑Main‑Observatory (LTER site) was generated using the universal primers BF2/BR2. With this data we identified the most abundant nontarget taxa and designed a new reverse primer (EPTDr2n) with 3’ ‐ specificity toward benthic invertebrate taxa. Primer specificity was validated in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. 20 eDNA samples from the Kinzig River and its tributaries were then used to test the new primer in situ together with primer fwhF2. The new primer combination showed a much higher amplification of benthic invertebrates, insects in particular, than two other universal primer pairs for both, number of target reads (fwhF2/EPTDr2n: 99.6% versus BF2/BR2: 25.89% and fwhF2/fwhR2n: 39.04%; Fig. 1) and number of target species (fwhF2/EPTDr2n: 305 versus BF2/BR2: 113 and fwhF2/fwhR2n: 185). Additionally, the number of benthic invertebrate species exceeded even the number of 153 species identified by expert taxonomists at nearby sites across two decades of sampling. While several taxa reported, like a few trichopteran genera, flatworms, and some crustaceans, were not found, the primer shows greatly improved results for eDNA metabarcoding of benthic invertebrates(Leese et al. 2021).

scholarly journals Improved freshwater macroinvertebrate detection from eDNA through minimized non-target amplification

2020 ◽  
Author(s):  
Florian Leese ◽  
Mandy Sander ◽  
Dominik Buchner ◽  
Vasco Elbrecht ◽  
Peter Haase ◽  
...  
Keyword(s):  
Stream Water ◽  
Pcr Amplification ◽  
Environmental Dna ◽  
Coi Gene ◽  
Taxonomic Resolution ◽  
Universal Primers ◽  
Reference Database ◽  
Degenerate Primers ◽  
Long Term Ecological Research

AbstractDNA metabarcoding of freshwater communities typically relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase (COI) gene with degenerate primers. The advantage of COI is its taxonomic resolution and the availability of an extensive reference database. However, when universal primers are used on environmental DNA (eDNA) isolated from stream water, macroinvertebrate read and OTU numbers are typically “watered down”, i.e. diluted, compared to whole specimen ‘bulk samples’ due to greater co-amplification of abundant non-target taxa such as algae and bacteria. Because stream macroinvertebrate taxa are of prime importance for regulatory biomonitoring, more effective ways to capture their diversity via eDNA isolated from water are important. In this study, we aimed to improve macroinvertebrate assessment from eDNA by minimizing non-target amplification. Therefore, we generated data using universal primers BF2/BR2 throughout 15 months from a German Long-Term Ecological Research (LTER) site, the River Kinzig, to identify most abundant non-target taxa. Based on these data, we designed a new reverse primer (EPTDr2n) with 3’-specificity towards macrozoobenthic taxa and validated its specificity in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. We then performed in vitro tests using 20 eDNA samples taken in the Kinzig catchment. We found that the percentage of target reads was much higher for the new primer combination compared to two universal macrozoobenthic primer pairs, BF2/BR2 and fwhF2/fwhR2n (>99 % vs. 21.4 % and 41.25 %, respectively). Likewise, number of detected macroinvertebrate taxa was substantially higher (351 vs. 46 and 170, respectively) and exceeded the number of 257 taxa identified by expert taxonomists at nearby sites across two decades of sampling. While few taxa such as Turbellaria were not detected, we show that the optimized primer avoids the dilution problem and thus significantly improves macroinvertebrate detection for bioassessment and -monitoring.

scholarly journals The bacterial hitchhiker's guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind

2021 ◽  
Author(s):  
Sanni Hintikka ◽  
Jeanette E. L. Carlsson ◽  
Jens Carlsson
Keyword(s):  
Marker Gene ◽  
Environmental Dna ◽  
Taxonomic Resolution ◽  
Universal Primers ◽  
Great Promise ◽  
Universal Primer ◽  
Bacterial Dna ◽  
Amplification Protocol ◽  
Target Dna ◽  
High Level

Environmental DNA (eDNA) metabarcoding from water samples has, in recent years, shown great promise for biodiversity monitoring. However, universal primers targeting the cytochrome oxidase I (COI) marker gene popular in metazoan studies have displayed high levels of nontarget amplification. To date, enrichment methods bypassing amplification have not been able to match the detection levels of conventional metabarcoding. This study evaluated the use of universal metabarcoding primers as capture probes to either isolate target DNA, or to remove nontarget DNA, prior to amplification by using biotinylated versions of universal metazoan and bacterial barcoding primers, namely metazoan COI and bacterial 16S. Additionally, each step of the protocol was assessed by amplifying both COI and bacterial 16S to investigate the effect on the metazoan and bacterial communities. Bacterial abundance increased in response to the captures (COI library), while the quality of the captured DNA was improved. The metazoan-based probe captured bacterial DNA in a range that was also amplifiable with the 16S primers, demonstrating the ability of universal capture probes to isolate larger fragments of DNA from eDNA. This concept could be applied to metazoan metabarcoding, by using a truly conserved site without a high-level taxonomic resolution as a target of capture, to isolate DNA spanning over a nearby barcoding region, which can then be processed through conventional metabarcoding by amplification protocol.

scholarly journals Species detections in environmental DNA using metabarcoding: a useful tool in estuaries monitoring

2015 ◽  
Author(s):  
Adrián Mártinez-Marqués ◽  
Carlos Enrique Carleos ◽  
Eva García-Vazquez ◽  
Yaisel J. Borrell Pichs
Keyword(s):  
Dna Analysis ◽  
Anthropogenic Activities ◽  
Dna Barcode ◽  
Environmental Dna ◽  
Coi Gene ◽  
Universal Primers ◽  
Mitochondrial Coi ◽  
Mitochondrial Coi Gene ◽  
Cantabrian Coast ◽  
Taxonomic Groups

Estuaries are amongst the most productive habitats in Earth, producing more organic materia than forests, meadows or agricultural lands. In addition, estuaries exhibit high, and precious, biodiversity levels. In this study an environmental DNA analysis of the two most important estuaries in Asturias (Cantabrian Coast, north Iberia) in terms of food production (Ría del Eo and Ría de Villaviciosa) was carried out. The objective was to monitor aquatic biodiversity and also to detect alien species that can be associated with anthropogenic activities (e.g.: aquaculture). To achieve these objectives, a metabarcoding methodology based in NGS (next generation sequencing) and the mitochondrial COI gene as a DNA Barcode was used. Results showed that this methodology was useful to detect the presence of three different non-native genera (Crepidula, Lymnaea, Macrobrachium) that are probably parasitating species cultured in these estuaries. It is true that Metabarcoding has still unsolved problems such as the lack of 100% universal primers and paucity of referenced sequences for some taxonomic groups in the databases. However, it represents already a powerful tool to manage the resources of these important ecosystems and to guarantee their long-term sustainailibity.

scholarly journals Novel Universal Primers for Metabarcoding eDNA Surveys of Marine Mammals and Other Marine Vertebrates

2019 ◽  
Author(s):  
Elena Valsecchi ◽  
Jonas Bylemans ◽  
Simon J. Goodman ◽  
Roberto Lombardi ◽  
Ian Carr ◽  
...  
Keyword(s):  
Marine Mammals ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
Universal Primers ◽  
Marine Biodiversity ◽  
Primer Sets ◽  
Marine Megafauna

ABSTRACTMetabarcoding studies using environmental DNA (eDNA) and high throughput sequencing (HTS) are rapidly becoming an important tool for assessing and monitoring marine biodiversity, detecting invasive species, and supporting basic ecological research. Several barcode loci targeting teleost fish and elasmobranchs have previously been developed, but to date primer sets focusing on other marine megafauna, such as marine mammals have received less attention. Similarly, there have been few attempts to identify potentially ‘universal’ barcode loci which may be informative across multiple marine vertebrate Orders. Here we describe the design and validation of four new sets of primers targeting hypervariable regions of the vertebrate mitochondrial 12S and 16S rRNA genes, which have conserved priming sites across virtually all cetaceans, pinnipeds, elasmobranchs, boney fish, sea turtles and birds, and amplify fragments with consistently high levels of taxonomically diagnostic sequence variation. ‘In silico’ validation using the OBITOOLS software showed our new barcode loci outperformed most existing vertebrate barcode loci for taxon detection and resolution. We also evaluated sequence diversity and taxonomic resolution of the new barcode loci in 680 complete marine mammal mitochondrial genomes demonstrating that they are effective at resolving amplicons for most taxa to the species level. Finally, we evaluated the performance of the primer sets with eDNA samples from aquarium communities with known species composition. These new primers will potentially allow surveys of complete marine vertebrate communities in single HTS metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators.

Implications of seasonal variation for biomonitoring with predictive models in the Fraser River catchment, British Columbia

2001 ◽  
Vol 58 (7) ◽  
pp. 1411-1417 ◽  
Author(s):  
Pamela F Reece ◽  
Trefor B Reynoldson ◽  
John S Richardson ◽  
David M Rosenberg
Keyword(s):  
Seasonal Variation ◽  
Test Site ◽  
Benthic Invertebrate ◽  
Large River ◽  
Reference Condition ◽  
Taxonomic Resolution ◽  
Reference Database ◽  
Fraser River ◽  
River Catchment ◽  
Invertebrate Assemblage

Reference-condition models for the Fraser River catchment were developed using samples collected during the autumn of 1994, 1995, and 1996. The goal of this study was to examine applicability to the reference-condition models of samples collected in other seasons and the effect of taxonomic resolution (genus and family) on model sensitivity to seasonal variation in the benthic invertebrate assemblage. Samples from eight streams representing interior, coastal, and large-river habitats were collected in spring, summer, autumn, and winter of 1995 and in spring of 1996. The benthic invertebrate assemblage changed seasonally such that the models could not be used for seasons other than autumn. The models were equally sensitive to seasonal variation when genus-level or family-level data were used. We recommend that test samples (i.e., samples collected from disturbed sites and meant for comparison with the reference database) be collected either during the autumn or over multiple sampling dates to reduce the possibility that seasonal shifts or stochastic events will lead to erroneous conclusions about the state of a test site.

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.

scholarly journals Species detections in environmental DNA using metabarcoding: a useful tool in estuaries monitoring

2015 ◽  
Author(s):  
Adrián Mártinez-Marqués ◽  
Carlos Enrique Carleos ◽  
Eva García-Vazquez ◽  
Yaisel J. Borrell Pichs
Keyword(s):  
Dna Analysis ◽  
Anthropogenic Activities ◽  
Dna Barcode ◽  
Environmental Dna ◽  
Coi Gene ◽  
Universal Primers ◽  
Mitochondrial Coi ◽  
Mitochondrial Coi Gene ◽  
Cantabrian Coast ◽  
Taxonomic Groups

Estuaries are amongst the most productive habitats in Earth, producing more organic materia than forests, meadows or agricultural lands. In addition, estuaries exhibit high, and precious, biodiversity levels. In this study an environmental DNA analysis of the two most important estuaries in Asturias (Cantabrian Coast, north Iberia) in terms of food production (Ría del Eo and Ría de Villaviciosa) was carried out. The objective was to monitor aquatic biodiversity and also to detect alien species that can be associated with anthropogenic activities (e.g.: aquaculture). To achieve these objectives, a metabarcoding methodology based in NGS (next generation sequencing) and the mitochondrial COI gene as a DNA Barcode was used. Results showed that this methodology was useful to detect the presence of three different non-native genera (Crepidula, Lymnaea, Macrobrachium) that are probably parasitating species cultured in these estuaries. It is true that Metabarcoding has still unsolved problems such as the lack of 100% universal primers and paucity of referenced sequences for some taxonomic groups in the databases. However, it represents already a powerful tool to manage the resources of these important ecosystems and to guarantee their long-term sustainailibity.

scholarly journals New 12S metabarcoding primers for enhanced Neotropical freshwater fish biodiversity assessment

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
David T. Milan ◽  
Izabela S. Mendes ◽  
Júnio S. Damasceno ◽  
Daniel F. Teixeira ◽  
Naiara G. Sales ◽  
...  
Keyword(s):  
Fish Species ◽  
Species Delimitation ◽  
Fish Fauna ◽  
Environmental Dna ◽  
Threshold Value ◽  
Full Length ◽  
Taxonomic Resolution ◽  
Reference Database ◽  
Biodiversity Assessment ◽  
Neotropical Fish

Abstract The megadiverse Neotropical fish fauna lacks a comprehensive and reliable DNA reference database, which hampers precise species identification and DNA based biodiversity assessment in the region. Here, we developed a mitochondrial 12S ribosomal DNA reference database for 67 fish species, representing 54 genera, 25 families, and six major Neotropical orders. We aimed to develop mini-barcode markers (i.e. amplicons with less than 200 bp) suitable for DNA metabarcoding by evaluating the taxonomic resolution of full-length and mini-barcodes and to determine a threshold value for fish species delimitation using 12S. Evaluation of the target amplicons demonstrated that both full-length library (565 bp) and mini-barcodes (193 bp) contain enough taxonomic resolution to differentiate all 67 fish species. For species delimitation, interspecific genetic distance threshold values of 0.4% and 0.55% were defined using full-length and mini-barcodes, respectively. A custom reference database and specific mini-barcode markers are important assets for ecoregion scale DNA based biodiversity assessments (such as environmental DNA) that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.

Identification of benthic invertebrate and diatom indicator taxa that distinguish different stream types as well as degraded from reference conditions in Luxembourg

2008 ◽  
Vol 58 (4) ◽  
pp. 419-472 ◽  
Author(s):  
Alain Dohet ◽  
Henry-Michel Cauchie ◽  
Luc Ector ◽  
Lucien Hoffmann
Keyword(s):  
Benthic Invertebrates ◽  
Reference Conditions ◽  
Ecological Status ◽  
Benthic Invertebrate ◽  
Taxonomic Resolution ◽  
Anthropogenic Pressures ◽  
Ecological Requirements ◽  
River Types ◽  
Quality Of Water ◽  
Species Being

AbstractThe main objective of the Water Framework Directive (WFD) is to achieve good ecological status for surface waters in Europe by 2015. The ecological status has to be defined based on near-natural reference conditions. Benthic invertebrates and diatoms are among the key biological elements recommended by the Directive to assess ecological quality of water bodies. The purpose of this study is to identify species associations of these biological elements that are characteristic of the different stream types occurring in Luxembourg and that distinguish degraded from reference conditions. In general, the results reveal that diatoms and invertebrates can be considered as complementary indicators with more diatom species being characteristic of small size stream types and more benthic invertebrate species being associated with larger stream types. Among invertebrates, Trichoptera, Hydrachnidia, Ephemeroptera and Diptera show high affinities for most stream types. Plecoptera, Oligochaeta appear as useful indicators for some particular types. If only reference sites are selected (all river types considered), the number of indicator species drops from 55 to 24 for diatoms and from 81 to 48 for benthic invertebrates. Moreover, for the larger stream type, no reference site was found at all. This trend is likely to be a consequence of the multiple anthropogenic pressures that have affected large parts of European lowland rivers for decades. Our results suggest that Trichoptera, Hydrachnidia, Diptera, Ephemeroptera and Oligochaeta could be considered as best candidate groups for a tiered-taxonomic resolution approach where only taxa which have narrow and specific ecological requirements would be identified to finer levels. In Central Europe, however, since taxonomic soundness and easy recognition are required, only Trichoptera and Ephemeroptera are the groups to be recommended at the present time.

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.

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