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

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

10.7287/peerj.preprints.1855 ◽

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

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

PeerJ ◽

10.7717/peerj.1966 ◽

2016 ◽

Vol 4 ◽

pp. e1966 ◽

Cited By ~ 66

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.

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Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding

10.7287/peerj.preprints.26667 ◽

2018 ◽

Author(s):

Aimee L van der Reis ◽

Olivier Laroche ◽

Andrew G Jeffs ◽

Shane D Lavery

Keyword(s):

New Zealand ◽

Preliminary Analysis ◽

18S Rrna ◽

18S Rrna Gene ◽

Coi Gene ◽

Taxonomic Resolution ◽

Gut Contents ◽

Rrna Gene ◽

Diverse Range ◽

Dna Metabarcoding

Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of the diet of the New Zealand scampi (Metanephrops challengeri) through the identification of gut contents, but a number of methodological concerns need to be overcome first to ensure optimum DNA metabarcoding results. In this study, a range of methodological parameters were tested to determine the optimum protocols for DNA metabarcoding, and provide a first view of M. challengeri diet. Several PCR protocols were tested, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from both frozen and ethanol preserved samples for both foregut and hindgut digesta. The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including the use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable, but metabarcoding outcomes indicated that the ethanol samples produced better results from the COI gene. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta, however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet of M. challengeri identified a range of species (13,541 reads identified as diet), which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctate), tall sea pen (Funiculina quadrangularis) and the salp (Ihlea racovitza), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.

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A new oomycete metabarcoding method using the rps10 gene

10.1101/2021.09.22.460084 ◽

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.

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

ARPHA Conference Abstracts ◽

10.3897/aca.4.e65206 ◽

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.

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Combining the 5.8S and ITS2 gene regions to improve classification of fungi

10.1101/532358 ◽

2019 ◽

Author(s):

Felix Heeger ◽

Christian Wurzbacher ◽

Elizabeth C. Bourne ◽

Camila J. Mazzoni ◽

Michael T. Monaghan

Keyword(s):

High Variability ◽

Species Level ◽

Reference Sequence ◽

Reference Database ◽

List Type ◽

Dna Metabarcoding ◽

Automated Pipeline ◽

Reference Sequences ◽

Significant Underestimation

SummaryThe internal transcribed spacer (ITS) is used in DNA metabarcoding of fungi. One disadvantage of its high variability may be a failure to classify OTUs when no similar reference sequence exists. We tested whether the 5.8S region, often sequenced with ITS2 but discarded before analysis, could provide OUT classifications when ITS fails.We usedin silicoevaluation to compare classification success of 5.8S and ITS from the UNITE database when reference sequences of the same species, genus, or family were removed. We then developed an automated pipeline for a combined 5.8S - ITS2 analysis and applied it to mixed environmental samples containing many lineages that are underrepresented in databases.ITS was clearlysuperior for species-level classifications with a complete reference database, but 5.8S outperformed ITS at higher level classifications with an incomplete database. Our combined 5.8S-ITS2 pipeline classified 3x more fungal OTUs compared to ITS2 alone, particularly within Chytridiomycota (10x) and Rozellamycota (3x).Missing reference sequences led to the failure of ITS to classify many fungal OTUs at all, and to a significant underestimation of environmental fungal diversity. Using 5.8S to complement ITS classification will likely provide better estimates of diversity in lineages for which database coverage is poor.

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Evaluation and application of mitochondrial CO І gene in identification of endangered wildlife in multi-species mixed biological samples

E3S Web of Conferences ◽

10.1051/e3sconf/202014501020 ◽

2020 ◽

Vol 145 ◽

pp. 01020

Author(s):

Chen Yun-xia ◽

Xue Xiao-ming ◽

Huang Ya-lin ◽

Zhou Yong-wu ◽

Hou Sen-lin ◽

...

Keyword(s):

Species Identification ◽

Biological Samples ◽

Animal Species ◽

Simultaneous Detection ◽

Species Level ◽

Reference Database ◽

Target Classification ◽

Identification Rate ◽

Dna Metabarcoding ◽

Mixed Samples

In this study, the second-generation high-throughput sequencing and DNA barcoding were combined to manually prepare multi-species mixed samples, and the mitochondrial gene CO І was used as a barcode to simultaneously identify the animal species in the mixed samples and identify endangered species. The results showed that under the family and genus level, the simultaneous detection rate of the species in the mixed samples was as high as 100%, and the species identification rate was as high as 89% at the species level, and with high sensitivity, as little as 1% of the trace species could be detected. However, nearly 30% of non-target classification annotations appeared at the species level. It can be concluded that the mini CO I barcoding can be applied to the simultaneous identification of animal species in mixed biological samples, and the species identification rate is high. Non-target classification match existing at the species level can be further improved by increasing the length of the barcoding, improving the sequencing technology, reference database and so on. In this study, DNA metabarcoding technology was used to evaluate the feasibility of identification of endangered animals in multi-species mixed biological samples with CO І, in order to lay a preliminary foundation for the advancement of DNA metabarcoding method in the field of wildlife forensic identification.

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

10.1101/113597 ◽

2017 ◽

Cited By ~ 7

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.

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Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding

PeerJ ◽

10.7717/peerj.5641 ◽

2018 ◽

Vol 6 ◽

pp. e5641 ◽

Cited By ~ 5

Author(s):

Aimee L. van der Reis ◽

Olivier Laroche ◽

Andrew G. Jeffs ◽

Shane D. Lavery

Keyword(s):

New Zealand ◽

Preliminary Analysis ◽

18S Rrna ◽

18S Rrna Gene ◽

Coi Gene ◽

Taxonomic Resolution ◽

Gut Contents ◽

Rrna Gene ◽

Diverse Range ◽

Dna Metabarcoding

Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of the diet of the New Zealand scampi (Metanephrops challengeri) through the identification of gut contents, but a number of methodological concerns need to be overcome first to ensure optimum DNA metabarcoding results. In this study, a range of methodological parameters were tested to determine the optimum protocols for DNA metabarcoding, and provide a first view ofM.challengeridiet. Several PCR protocols were tested, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from both frozen and ethanol preserved samples for both foregut and hindgut digesta. The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including the use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta; however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet ofM.challengeriidentified a range of species (13,541 reads identified as diet), which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctata), tall sea pen (Funiculina quadrangularis) and the salp (Ihlea racovitzai), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.

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Analyzing multiple stressor effects on EPT taxa in a mesocosm experiment with DNA metabarcoding

ARPHA Conference Abstracts ◽

10.3897/aca.4.e64752 ◽

2021 ◽

Vol 4 ◽

Author(s):

Marie-Thérése Werner ◽

Arne Beermann ◽

Vasco Elbrecht ◽

Vera Zizka ◽

Florian Leese

Keyword(s):

Water Quality ◽

Flow Velocity ◽

Fine Sediment ◽

Sediment Deposition ◽

Species Level ◽

Taxonomic Resolution ◽

Response Patterns ◽

Multiple Stressor ◽

Dna Metabarcoding ◽

Ept Taxa

Multiple stressors diversely and often adversely affect stream ecosystems around the globe. Therefore, understanding multiple stressor effects on different organisms is essential for a better ecosystem understanding, an accurate water quality assessment and improved ecosystem management. However, while multiple stressor effects should be assessed at species level this taxonomic resolution is often not achieved e.g. for stream macroinvertebrates. Due to their high abundance and diversity, species-level identification is often not feasible with morphology‑based approaches. DNA metabarcoding represents an alternative approach for studying multiple stressor interactions at species level. In an outdoor experiment over 10,000 specimens from the insect orders Ephemeroptera, Plecoptera and Trichoptera (EPT), which are routinely used as bioindicators, and their responses to stressors were studied. In the experiment salinity, fine sediment deposition and flow velocity were manipulated in a full‑factorial design in 64 mesocosms with two microhabitats each (streambed and leaf litter), resulting in eight replicates per treatment. DNA metabarcoding revealed 122 EPT Operational Taxonomic Units (OTUs), from which the most abundant 27 alone showed 14 different response patterns to the applied stressors. The high taxonomic resolution achieved by DNA metabarcoding revealed species specific stressor responses that were hidden at a lower taxonomic resolution. As a prominent example, Rhithrogena semicolorata responded negatively to fine sediment deposition and flow velocity reduction, while Ecdyonurus torrentis (both Heptageniidae, Ephemeroptera) was insensitive to experimental manipulation, highlighting different stressor responses among species within the same family (Fig. 1, Beermann et al. 2020). Even for well-studied organisms such as EPT taxa, this study shows that DNA metabarcoding has the potential to depict response patterns at species or OTU level despite high specimen abundance. Consequently, DNA metabarcoding promises to be a rewarding method when investigating and assessing multiple stressor effects on stream water quality.

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