ITS all right mama: investigating the formation of chimeric sequences in the ITS2 region by DNA metabarcoding analyses of fungal mock communities of different complexities

2016 ◽  
Vol 17 (4) ◽  
pp. 730-741 ◽  
Author(s):  
Anders Bjørnsgaard Aas ◽  
Marie Louise Davey ◽  
Håvard Kauserud
Keyword(s):  
Its2 Region ◽  
Dna Metabarcoding ◽  
Chimeric Sequences ◽  
Mock Communities

scholarly journals Diversity of indoor fungi as revealed by DNA metabarcoding

Genome ◽  
2017 ◽  
Vol 60 (1) ◽  
pp. 55-64 ◽  
Author(s):  
Helena Korpelainen ◽  
Maria Pietiläinen
Keyword(s):  
Air Quality ◽  
Similarity Index ◽  
Taxonomic Composition ◽  
Its2 Region ◽  
Nursery Schools ◽  
Fungal Identification ◽  
Dna Metabarcoding ◽  
Before And After ◽  
Indoor Fungi ◽  
University Buildings

In the present study, we conducted DNA metabarcoding (the nuclear ITS2 region) for indoor fungal samples originating from two nursery schools with a suspected mould problem (sampling before and after renovation), from two university buildings, and from an old farmhouse. Good-quality sequences were obtained, and the results showed that DNA metabarcoding provides high resolution in fungal identification. The pooled proportions of sequences representing filamentous ascomycetes, filamentous basidiomycetes, yeasts, and other fungi equalled 62.3%, 8.0%, 28.3%, and 1.4%, respectively, and the total number of fungal genera found during the study was 585. When comparing fungal diversities and taxonomic composition between different types of buildings, no obvious pattern was detected. The average pairwise values of SørensenChao indices that were used to compare similarities for taxon composition between samples among the samples from the two university buildings, two nurseries, and farmhouse equaled 0.693, 0.736, 0.852, 0.928, and 0.981, respectively, while the mean similarity index for all samples was 0.864. We discovered that making explicit conclusions on the relationship between the indoor air quality and mycoflora is complicated by the lack of appropriate indicators for air quality and by the occurrence of wide spatial and temporal changes in diversity and compositions among samples.

scholarly journals The indoor mycobiome of daycare centers is affected by occupancy and climate

2021 ◽  
Author(s):  
Eva Lena Estensmo ◽  
Synnøve Smebye Botnen ◽  
Sundy Maurice ◽  
Pedro Maria Martin-Sanchez ◽  
Luis Morgado ◽  
...  
Keyword(s):  
Citizen Science ◽  
Environmental Gradients ◽  
Its2 Region ◽  
Number Of Children ◽  
Daycare Centers ◽  
Outdoor Climate ◽  
Geographic Structure ◽  
Dna Metabarcoding ◽  
Yeasts And Molds ◽  
Indoor Conditions

Many children spend considerable time in daycare centers and may here be influenced by indoor microorganisms, including fungi. In this study, we investigate the indoor mycobiome of 125 daycare centers distributed along strong environmental gradients throughout Norway. Dust samples were collected from doorframes outside and inside buildings using a citizen science sampling approach. Fungal communities in the dust samples were analyzed using DNA metabarcoding of the ITS2 region. We observed a marked difference between the outdoor and indoor mycobiomes. The indoor mycobiome included considerably more yeasts and molds compared to the outdoor samples, with Saccharomyces, Mucor, Malassezia and Penicillium among the most dominant fungal genera. Changes in the indoor fungal richness and composition correlated to numerous variables related to both outdoor and indoor conditions; there was a clear geographic structure in the indoor mycobiome composition that mirrored the outdoor climate, ranging from humid areas in western Norway to drier and colder areas in eastern Norway. Moreover, the number of children in the daycare centers, as well as various building features, influenced the indoor mycobiome composition. We conclude that the indoor mycobiome in Norwegian daycare centers is structured by multiple factors and is dominated by yeasts and molds. This study exemplifies how citizen science sampling enables DNA-based analyses of a high number of samples covering wide geographic areas.

scholarly journals Fungal microbiome of barley grain revealed by NGS and mycological analysis

2020 ◽  
pp. 286-297
Author(s):  
Igor Kazartsev ◽  
Tatiana Gagkaeva ◽  
Olga Gavrilova ◽  
Philipp Gannibal
Keyword(s):  
Agar Plate ◽  
Barley Grain ◽  
Species Level ◽  
Its2 Region ◽  
Fungal Community Structure ◽  
Dna Metabarcoding ◽  
Nextgeneration Sequencing ◽  
Broad Variety ◽  
Modern Vision ◽  
Barley Grains

Introduction. Barley can be infected with a broad variety of fungi, which can cause considerable loss of crop yield and reduce the quality of grain. Modern vision on the geographical and ecological distribution and biodiversity of micromycetes has been established by traditional, cultivation-based methods. However, more recently, molecular methods have shifted microbiological research to a new level, making it possible to investigate hidden taxonomical biodiversity. Study objects and methods. For this study, we determined the fungal biome on the surface and inside of barley grains using the traditional mycological method and the contemporary molecular method, which employed DNA metabarcoding based on NGS (nextgeneration sequencing) of the ITS2 region. We analyzed five cultivars that were collected in two subsequent crop seasons (2014, 2015). Results and discussion. DNA metabarcoding revealed 43 operational taxonomic units, while 17 taxa of genus or species level were recovered by the traditional method. DNA metabarcoding revealed several minor species and one predominant, presumably plantpathogenic Phaeosphaeria sp., which were not detected in the agar plate-based assay. Traditionally, Fusarium fungi were identified by mycological assay. However, the resolution of DNA metabarcoding was sufficient to determine main Fusarium groups divided by ability to produce toxic secondary metabolites. The combined list of Ascomycetes consisted of 15 genera, including 14 fungi identified to species level. The list of Basidiomycota derived from DNA metabarcoding data alone included 8 genera. Conclusion. It was found that crop season predetermines the fungal community structure; mycobiota on the surface and inside of grain was significantly different.

scholarly journals Profiling grapevine trunk pathogens in planta: A case for community-targeted DNA metabarcoding

2018 ◽  
Author(s):  
Abraham Morales-Cruz ◽  
Rosa Figueroa-Balderas ◽  
Jadran F. García ◽  
Eric Tran ◽  
Philippe E. Rolshausen ◽  
...  
Keyword(s):  
Microbial Ecology ◽  
Time Course ◽  
Universal Primers ◽  
In Planta ◽  
Universal Primer ◽  
Grapevine Trunk Diseases ◽  
Field Samples ◽  
Dna Metabarcoding ◽  
Trunk Diseases ◽  
Mock Communities

ABSTRACTDNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Their detection is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the Fungal Kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated Ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology. We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock communities, (ii) time-course experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were greatly over- or under-estimated with universal primers. Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.

A quantitative protocol for DNA metabarcoding of springtails (Collembola)

Genome ◽  
2016 ◽  
Vol 59 (9) ◽  
pp. 705-723 ◽  
Author(s):  
Seikoh Saitoh ◽  
Hiroaki Aoyama ◽  
Saori Fujii ◽  
Haruki Sunagawa ◽  
Hideki Nagahama ◽  
...  
Keyword(s):  
Internal Control ◽  
Sequence Similarity ◽  
Community Sample ◽  
Pcr Primers ◽  
Dna Metabarcoding ◽  
Species Abundances ◽  
Collembolan Species ◽  
Order Degenerate ◽  
Number Of Individuals ◽  
Mock Communities

We developed a novel protocol with superior quantitative analysis results for DNA metabarcoding of Collembola, a major soil microarthropod order. Degenerate PCR primers were designed for conserved regions in the mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on published collembolan mitogenomes. The best primer pair was selected based on its ability to amplify each gene, irrespective of the species. DNA was extracted from 10 natural communities sampled in a temperate forest (with typically 25–30 collembolan species per 10 soil samples) and 10 mock communities (with seven cultured collembolan species). The two gene regions were then amplified using the selected primers, ligated with adapters for 454 technology, and sequenced. Examination of the natural community samples showed that 32 and 36 operational taxonomic units (defined at a 90% sequence similarity threshold) were recovered from the mtCOI and mt16S data, respectively, which were comparable to the results of the microscopic identification of 25 morphospecies. Further, sequence abundances for each collembolan species from the mtCOI and mt16S data of the mock communities, after normalization by using a species as the internal control, showed good correlation with the number of individuals in the samples (R = 0.91–0.99), although relative species abundances within a mock community sample estimated from sequences were skewed from community composition in terms of the number of individuals or biomass of the species. Thus, this protocol enables the comparison of collembolan communities in a quantitative manner by metabarcoding.

scholarly journals Internal transcribed spacer primer evaluation for vascular plant metabarcoding

2021 ◽  
Vol 5 ◽  
Author(s):  
Andreas Kolter ◽  
Birgit Gemeinholzer
Keyword(s):  
Vascular Plant ◽  
Widespread Application ◽  
Continuous Quality ◽  
Dna Metabarcoding ◽  
Underlying Causes ◽  
Continuous Quality Assurance ◽  
Biodiversity Decline ◽  
Its Primers ◽  
Mock Communities

The unprecedented ongoing biodiversity decline necessitates scalable means of monitoring in order to fully understand the underlying causes. DNA metabarcoding has the potential to provide a powerful tool for accurate and rapid biodiversity monitoring. Unfortunately, in many cases, a lack of universal standards undermines the widespread application of metabarcoding. One of the most important considerations in metabarcoding of plants, aside from selecting a potent barcode marker, is primer choice. Our study evaluates published ITS primers in silico and in vitro, through mock communities and presents newly designed primers. We were able to show that a large proportion of previously available ITS primers have unfavourable attributes. Our combined results support the recommendation of the introduced primers ITS-3p62plF1 and ITS-4unR1 as the best current universal plant specific ITS2 primer combination. We also found that PCR optimisation, such as the addition of 5% DMSO, is essential to obtain meaningful results in ITS2 metabarcoding. Finally, we conclude that continuous quality assurance is indispensable for reliable metabarcoding results.

scholarly journals Enhancing DNA metabarcoding performance and applicability with bait capture enrichment and DNA from conservative ethanol

2019 ◽  
Author(s):  
M. Gauthier ◽  
L. Konecny-Dupré ◽  
A. Nguyen ◽  
V. Elbrecht ◽  
T. Datry ◽  
...  
Keyword(s):  
Taxonomic Resolution ◽  
Detection Rates ◽  
Accurate Representation ◽  
Dna Metabarcoding ◽  
Efficient Alternative ◽  
Alternative Identification ◽  
Species Occurrences ◽  
Enrichment Step ◽  
Mock Communities ◽  
Identification Tool

ABSTRACTMetabarcoding is often presented as an alternative identification tool to compensate for coarse taxonomic resolution and misidentification encountered with traditional morphological approaches. However, metabarcoding comes with two major impediments which slow down its adoption. First, the picking and destruction of organisms for DNA extraction are time and cost consuming and do not allow organism conservation for further evaluations. Second, current metabarcoding protocols include a PCR enrichment step which induces errors in the estimation of species diversity and relative biomasses. In this study, we first evaluated the capacity of capture enrichment to replace PCR enrichment using controlled freshwater macrozoobenthos mock communities. Then, we tested if DNA extracted from the fixative ethanol (etDNA) of the same mock communities can be used as an alternative to DNA extracted from pools of whole organisms (bulk DNA). We show that capture enrichment provides more reliable and accurate representation of species occurrences and relative biomasses in comparison with PCR enrichment for bulk DNA. While etDNA does not permit to estimate relative biomasses, etDNA and bulk DNA provide equivalent species detection rates. Thanks to its robustness to mismatches, capture enrichment is already an efficient alternative to PCR enrichment for metabarcoding and, if coupled to etDNA, is a time-saver option in studies where presence information only is sufficient.

COMPARATIVE STUDY BETWEEN FISH COLLECTION BY NATIONAL CENSUS ON RIVER ENVIRONMENT AND ENVIRONMENTAL DNA METABARCODING

2018 ◽  
Vol 74 (5) ◽  
pp. I_415-I_420 ◽  
Author(s):  
Yoshihisa AKAMATSU ◽  
Takayoshi TSUZUKI ◽  
Ryota YOKOYAMA ◽  
Yayoi FUNAHASHI ◽  
Munehiro OHTA ◽  
...  
Keyword(s):  
Comparative Study ◽  
Environmental Dna ◽  
Dna Metabarcoding ◽  
Fish Collection

Environmental DNA for functional diversity

2018 ◽  
Author(s):  
Pierre Taberlet ◽  
Aurélie Bonin ◽  
Lucie Zinger ◽  
Eric Coissac
Keyword(s):  
Functional Groups ◽  
Functional Diversity ◽  
Environmental Dna ◽  
Soil Organisms ◽  
Meaningful Information ◽  
Dna Metabarcoding ◽  
Pros And Cons ◽  
Target Community

Chapter 10 “Environmental DNA for functional diversity” discusses the potential of environmental DNA to assess functional diversity. It first focuses on DNA metabarcoding and discusses the extent to which this approach can be used and/or optimized to retrieve meaningful information on the functions of the target community. This knowledge usually involves coarsely defined functional groups (e.g., woody, leguminous, graminoid plants; shredders or decomposer soil organisms; pathogenicity or decomposition role of certain microorganisms). Chapter 10 then introduces metagenomics and metatranscriptomics approaches, their advantages, but also the challenges and solutions to appropriately sampling, sequencing these complex DNA/RNA populations. Chapter 10 finally presents several strategies and software to analyze metagenomes/metatranscriptomes, and discusses their pros and cons.

Environmental DNA

2018 ◽  
Author(s):  
Pierre Taberlet ◽  
Aurélie Bonin ◽  
Lucie Zinger ◽  
Eric Coissac
Keyword(s):  
Graduate Students ◽  
Feeding Habits ◽  
Environmental Dna ◽  
Research Field ◽  
Background Information ◽  
Ecological Processes ◽  
Biodiversity Research ◽  
Dna Metabarcoding ◽  
Standard Information

Environmental DNA (eDNA), i.e. DNA released in the environment by any living form, represents a formidable opportunity to gather high-throughput and standard information on the distribution or feeding habits of species. It has therefore great potential for applications in ecology and biodiversity management. However, this research field is fast-moving, involves different areas of expertise and currently lacks standard approaches, which calls for an up-to-date and comprehensive synthesis. Environmental DNA for biodiversity research and monitoring covers current methods based on eDNA, with a particular focus on “eDNA metabarcoding”. Intended for scientists and managers, it provides the background information to allow the design of sound experiments. It revisits all steps necessary to produce high-quality metabarcoding data such as sampling, metabarcode design, optimization of PCR and sequencing protocols, as well as analysis of large sequencing datasets. All these different steps are presented by discussing the potential and current challenges of eDNA-based approaches to infer parameters on biodiversity or ecological processes. The last chapters of this book review how DNA metabarcoding has been used so far to unravel novel patterns of diversity in space and time, to detect particular species, and to answer new ecological questions in various ecosystems and for various organisms. Environmental DNA for biodiversity research and monitoring constitutes an essential reading for all graduate students, researchers and practitioners who do not have a strong background in molecular genetics and who are willing to use eDNA approaches in ecology and biomonitoring.

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