A new oomycete metabarcoding method using the rps10 gene

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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Electrochemical characterization of natural organic matter by direct voltammetry in an aprotic solvent

Environmental Science Processes & Impacts ◽

10.1039/c9em00313d ◽

2019 ◽

Vol 21 (10) ◽

pp. 1664-1683

Author(s):

Ania S. Pavitt ◽

Paul G. Tratnyek

Keyword(s):

Organic Matter ◽

Natural Organic Matter ◽

Environmental Samples ◽

Aprotic Solvent ◽

Electrochemical Characterization ◽

Wide Range ◽

Improved Methods ◽

Similar Peak

Improved methods for direct electrochemical characterization of a wide range of natural organic matter (NOM) samples dissolved in an aprotic solvent gives well defined peaks with similar peak breadths and potentials regardless of source, which probably reflects selectivity in the methods used to extract NOM from environmental samples.

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Taxonomic identification of airborne pollen from complex environmental samples by DNA metabarcoding: a methodological study for optimizing protocols

10.1101/099481 ◽

2017 ◽

Cited By ~ 1

Author(s):

Kleopatra Leontidou ◽

Cristiano Vernesi ◽

Johannes De Groeve ◽

Fabiana Cristofolini ◽

Despoina Vokou ◽

...

Keyword(s):

Sample Preparation ◽

Chloroplast Dna ◽

Dna Extraction ◽

Environmental Samples ◽

Airborne Pollen ◽

Reference Database ◽

Taxonomic Assignment ◽

Spore Trap ◽

Custom Made ◽

Dna Metabarcoding

AbstractMetabarcoding is a promising DNA-based method for identifying airborne pollen from environmental samples with advantages over microscopic methods. This method requires several preparatory steps of the samples, with the extraction protocol being of fundamental importance to obtain an optimal DNA yield. Currently, there is no consensus in sample preparation and DNA extraction, especially for gravimetric pollen samplers. Therefore, the aim of this study was to develop protocols to process environmental samples for pollen DNA extraction and further metabarcoding analysis, and to assess the efficacy of these protocols for the taxonomic assignment of airborne pollen, collected by gravimetric (Tauber trap) and volumetric samplers (Burkard spore trap). Protocols were tested across an increasing complexity of samples, from single-species pure pollen to environmental samples. A short fragment (about 150 base pair) of chloroplast DNA was amplified by universal primers for plants (trnL). After PCR amplification, amplicons were Sanger-sequenced and taxonomic assignment was accomplished by comparison to a custom-made reference database including chloroplast DNA sequences of 46 plant families, including most of the anemophilous taxa occurring in the study area (Trentino, Italy, Eastern Italian Alps). Using as a benchmark the classical morphological pollen analysis, it emerged that DNA metabarcoding is applicable efficiently across a complexity of samples, provided that sample preparation, DNA extraction and amplification protocols are specifically optimized.

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Accuracy of microbial community diversity estimated by closed- and open-reference OTUs

PeerJ ◽

10.7717/peerj.3889 ◽

2017 ◽

Vol 5 ◽

pp. e3889 ◽

Cited By ~ 69

Author(s):

Robert C. Edgar

Keyword(s):

Ribosomal Rna ◽

De Novo ◽

Community Diversity ◽

Reference Database ◽

Mock Community ◽

Variable Regions ◽

Operational Taxonomic Units ◽

Sequencing Technologies ◽

Generation Sequencing ◽

Mock Communities

Next-generation sequencing of 16S ribosomal RNA is widely used to survey microbial communities. Sequences are typically assigned to Operational Taxonomic Units (OTUs). Closed- and open-reference OTU assignment matches reads to a reference database at 97% identity (closed), then clusters unmatched reads using a de novo method (open). Implementations of these methods in the QIIME package were tested on several mock community datasets with 20 strains using different sequencing technologies and primers. Richness (number of reported OTUs) was often greatly exaggerated, with hundreds or thousands of OTUs generated on Illumina datasets. Between-sample diversity was also found to be highly exaggerated in many cases, with weighted Jaccard distances between identical mock samples often close to one, indicating very low similarity. Non-overlapping hyper-variable regions in 70% of species were assigned to different OTUs. On mock communities with Illumina V4 reads, 56% to 88% of predicted genus names were false positives. Biological inferences obtained using these methods are therefore not reliable.

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DNA metabarcode choice and design

10.1093/oso/9780198767220.003.0002 ◽

2018 ◽

Author(s):

Pierre Taberlet ◽

Aurélie Bonin ◽

Lucie Zinger ◽

Eric Coissac

Keyword(s):

In Silico ◽

Taxonomic Group ◽

Taxonomic Resolution ◽

Software Suite ◽

Resolution Power ◽

Wide Range ◽

Dna Metabarcoding ◽

Primer Annealing ◽

Taxonomic Groups ◽

The Ideal

Chapter “DNA metabarcode choice and design” develops the properties of the ideal metabarcode in a given context, including conservation of the primer annealing regions and resolution power across the target taxonomic group of interest. It also highlights the experimental constraints influencing the choice of a metabarcode in practice. A detailed tutorial illustrates how to design and test metabarcoding primers in silico with the programs ecoPrimers, ecoPCR, and the software suite OBITools. Command lines and example files are provided to design and test universal metabarcoding primers for Bacteria. Chapter 2 also gives statistics about the taxonomic resolution and primer conservation of more than 60 metabarcodes available for DNA metabarcoding analysis of a wide range of taxonomic groups.

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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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Random sampling causes the low reproducibility of rare eukaryotic OTUs in Illumina COI metabarcoding

PeerJ ◽

10.7717/peerj.3006 ◽

2017 ◽

Vol 5 ◽

pp. e3006 ◽

Cited By ~ 60

Author(s):

Matthieu Leray ◽

Nancy Knowlton

Keyword(s):

Random Sampling ◽

Marine Invertebrates ◽

Illumina Miseq ◽

Pcr Primers ◽

Biodiversity Monitoring ◽

Analysis Pipeline ◽

Operational Taxonomic Units ◽

Abundance Patterns ◽

Dna Metabarcoding ◽

Data Reproducibility

DNA metabarcoding, the PCR-based profiling of natural communities, is becoming the method of choice for biodiversity monitoring because it circumvents some of the limitations inherent to traditional ecological surveys. However, potential sources of bias that can affect the reproducibility of this method remain to be quantified. The interpretation of differences in patterns of sequence abundance and the ecological relevance of rare sequences remain particularly uncertain. Here we used one artificial mock community to explore the significance of abundance patterns and disentangle the effects of two potential biases on data reproducibility: indexed PCR primers and random sampling during Illumina MiSeq sequencing. We amplified a short fragment of the mitochondrial Cytochrome c Oxidase Subunit I (COI) for a single mock sample containing equimolar amounts of total genomic DNA from 34 marine invertebrates belonging to six phyla. We used seven indexed broad-range primers and sequenced the resulting library on two consecutive Illumina MiSeq runs. The total number of Operational Taxonomic Units (OTUs) was ∼4 times higher than expected based on the composition of the mock sample. Moreover, the total number of reads for the 34 components of the mock sample differed by up to three orders of magnitude. However, 79 out of 86 of the unexpected OTUs were represented by <10 sequences that did not appear consistently across replicates. Our data suggest that random sampling of rare OTUs (e.g., small associated fauna such as parasites) accounted for most of variation in OTU presence–absence, whereas biases associated with indexed PCRs accounted for a larger amount of variation in relative abundance patterns. These results suggest that random sampling during sequencing leads to the low reproducibility of rare OTUs. We suggest that the strategy for handling rare OTUs should depend on the objectives of the study. Systematic removal of rare OTUs may avoid inflating diversity based on commonβdescriptors but will exclude positive records of taxa that are functionally important. Our results further reinforce the need for technical replicates (parallel PCR and sequencing from the same sample) in metabarcoding experimental designs. Data reproducibility should be determined empirically as it will depend upon the sequencing depth, the type of sample, the sequence analysis pipeline, and the number of replicates. Moreover, estimating relative biomasses or abundances based on read counts remains elusive at the OTU level.

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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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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

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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Understudied, underrepresented, and unknown: methodological biases that limit detection of early diverging fungi from environmental samples

10.22541/au.161839272.27561225/v1 ◽

2021 ◽

Author(s):

Nicole Reynolds ◽

Michelle Jusino ◽

Jason Stajich ◽

Matthew Smith

Keyword(s):

Environmental Samples ◽

Large Subunit ◽

Phylogenetic Reconstruction ◽

Pcr Amplification ◽

Illumina Miseq ◽

Fungal Communities ◽

Reference Database ◽

Lsu Rdna ◽

Length Variation ◽

Taxonomic Assignments

Metabarcoding is an important tool for understanding fungal communities. The internal transcribed spacer (ITS) rDNA is the accepted fungal barcode but has known problems. The large subunit (LSU) rDNA has also been used to investigate fungal communities but available LSU metabarcoding primers were mostly designed to target Dikarya (Ascomycota + Basidiomycota) with little attention to early diverging fungi (EDF). However, evidence from multiple studies suggests that EDF comprise a large portion of unknown diversity in community sampling. Here we investigate how DNA marker choice and methodological biases impact recovery of EDF from environmental samples. We focused on one EDF lineage, Zoopagomycota, as an example. We evaluated three primer sets (ITS1F/ITS2, LROR/LR3, and LR3 paired with new primer LR22F) to amplify and sequence a Zoopagomycota mock community and a set of 146 environmental samples with Illumina MiSeq. We compared two taxonomy assignment methods and created an LSU reference database compatible with AMPtk software. The two taxonomy assignment methods recovered strikingly different communities of fungi and EDF. Target fragment length variation exacerbated PCR amplification biases and influenced downstream taxonomic assignments, but this effect was greater for EDF than Dikarya. To improve identification of LSU amplicons we performed phylogenetic reconstruction and illustrate the advantages of this critical tool for investigating identified and unidentified sequences. Our results suggest much of the EDF community may be missed or misidentified with “standard” metabarcoding approaches and modified techniques are needed to understand the role of these taxa in a broader ecological context.

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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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