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 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 Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia

2021 ◽  
Author(s):  
Masayuki K. Sakata ◽  
Mone U. Kawata ◽  
Atsushi Kurabayashi ◽  
Takaki Kurita ◽  
Masatoshi Nakamura ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Environmental Dna ◽  
Pcr Primers ◽  
Taxonomic Resolution ◽  
Rrna Gene ◽  
Biodiversity Monitoring ◽  
Natural Ecosystems ◽  
Universal Primer ◽  
Primer Sets

Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians ecological traits (e.g., nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding-analysis of extra-organismal DNA released into the environment-allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160-311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata, and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set Amph16S had the highest resolution among the tested sets. Finally, we applied Amph16S to actual metabarcoding and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.

scholarly journals Bias in Template-to-Product Ratios in Multitemplate PCR

1998 ◽  
Vol 64 (10) ◽  
pp. 3724-3730 ◽  
Author(s):  
Martin F. Polz ◽  
Colleen M. Cavanaugh
Keyword(s):  
Bacterial Species ◽  
Gene Copy Number ◽  
Pcr Amplification ◽  
Binding Energies ◽  
Site Directed Mutagenesis ◽  
Universal Primers ◽  
Gene Copy ◽  
Degenerate Primers ◽  
Relative Product ◽  
Genomic Dnas

ABSTRACT Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.

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.

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 Diazotrophic Diversity and Distribution in the Tropical and Subtropical Atlantic Ocean

2005 ◽  
Vol 71 (12) ◽  
pp. 7910-7919 ◽  
Author(s):  
Rebecca J. Langlois ◽  
Julie LaRoche ◽  
Philipp A. Raab
Keyword(s):  
Pacific Ocean ◽  
Atlantic Ocean ◽  
Heterotrophic Bacteria ◽  
Pcr Amplification ◽  
Environmental Dna ◽  
Degenerate Primers ◽  
Community Shift ◽  
The Pacific ◽  
Group A ◽  
The Pacific Ocean

ABSTRACT To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3°N and 56.6 to 18.5°W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as γ-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and γ-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30°C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30°C, more often in waters with temperature of <26°C, and were sometimes recovered from waters with detectable nitrate concentrations.

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 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 Screening of plant growth-promoting rhizobacterial strains for the degradation and utilization of exogenous pectin as a soul carbon source

2021 ◽  
Author(s):  
Mohammad K. Hassan
Keyword(s):  
Carbon Source ◽  
Gene Sequencing ◽  
Pcr Amplification ◽  
Pectate Lyase ◽  
Universal Primers ◽  
Transporter Gene ◽  
Pcr Product ◽  
Plant Growth Promoting ◽  
Growth Promoting

AbstractUniversal primers for gyrB were used to screen 79 strains of Bacillus species. The genomic DNA from each strain was extracted, and the PCR product of gyrB was amplified and purified for gene sequencing. Gene sequences were edited, and aligned, and Bacillus amyloliquefaciens subsp. plantarum (Bap) strains identified based on the gyrB phylogenetic tree. Two primers (exuT and uxuB) were designed to detect pectin-associated transporter gene exuT and D-mannonate oxidoreductase gene uxuB. The Bap strains were then screened for the presence of the exuT and uxuB genes. These two pectin-utilizing genes were confirmed in 57 and 54 Bap strains by PCR amplification. Second, an in vitro tests were conducted to screen 59 Bap strains using pectin as a sole carbon source to confirm the pectate lyase and utilizing activity. Pectate Agar (PA) and Tris-Spizizen Salts (TSS) medium were used for the in vitro pectate lyase and degradation assays.

Sign in / Sign up

Export Citation Format

Share Document