Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment
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Hypocreales
Library
Pyrosequencing
Soil microbiology
The objectives of this thesis were the establishment of molecular approaches in the diversity investigation of eukaryotic protists in the Southern Ocean, the comparison of different approaches and the delivery of a comprehensive and taxon detailed overview of protist assemblages in the Pacific sector of the Southern Ocean, especially in the Amundsen Sea. The molecular approaches used to achieve these goals were automated ribosomal intergenic spacer analysis (ARISA), sequencing of 18S rRNA gene clone libraries and 454-pyrosequencing.
The comparison of 18S rRNA clone library sequences with the results of 454-pyrosequencing was conducted with four Arctic water samples, focusing on picoplankton (0.4-3 μm), and with one Antarctic water sample, covering the whole size spectrum (>0.2 μm). It turned out that the two methods delivered different results. Both approaches discovered phylotypes that were not found with the other approach. The abundant biosphere, defined by the 454-pyrosequencing approach, was not fully recovered by the clone library approach. The cloning approach was biased against several groups, e.g. haptophytes in the Arctic samples and diatoms in the Antarctic sample. In summary, prior cloning data have to be handled with care, when compared with 454-pyrosequencing data. Additionally, cloning data are only of limited suitability to serve as a backbone for phylogenetic analysis of 454-pyrosequencing data.
The results of this comparison led to the decision to use ARISA and 454-pyrosequencing for the further protist diversity investigations. First, the hypothesis that distinct protist community assemblages characterize large-scale water masses was tested. The composition and biogeography of late summer eukaryotic protist assemblages along a transect from the coast of New Zealand to the eastern Ross Sea was determined. ARISA and 454-pyrosequencing were used in combination with flow cytometry and pigment measurements via high performance liquid chromatography (HPLC) to study the protist assemblage. Distinct biogeographic patterns defined by the different oceanic regions were revealed. Different water masses harboured different microbial communities, and environmental gradients limited their dispersal. Picoeukaryotes were of minor importance throughout the investigated transect and were nearly absent south of the Polar Front. Dinoflagellates, Syndiniales, and small stramenopiles dominated the Subantarctic Zone, whereas the importance of diatoms increased southwards, in the Polar Frontal Zone, the Antarctic Zone and the Subpolar Region. South of the Polar Front, haptophytes were the dominating group.
Second, the investigation focused on the Amundsen Sea to see if the protist community assemblages vary in different areas of a single large-scale water mass. The composition and structure of late summer eukaryotic protist assemblages along a west-east transect in the Amundsen Sea were analysed. ARISA and 454-pyrosequencing were combined with HPLC. Characteristic communities offshore and inshore were revealed, but the differences were weaker, compared to those found along the north-south transect. In general, total chlorophyll a and microeukaryotic contribution were higher in inshore samples. Picoeukaryotes were also of minor importance. Diatoms were the dominating group across the entire area, at which Eucampia sp. and Pseudo-nitzschia sp. were dominating inshore and Chaetoceros sp. was dominating offshore. At the eastern most station, the assemblage was dominated by Phaeocystis sp. Under the ice, ciliates showed their highest and haptophytes their lowest abundance.
This thesis sheds light on the use and applicability of several molecular methods for the investigation of protist assemblages in polar waters. It delivers a comprehensive and taxon detailed overview of the eukaryotic protist composition during the austral summer in the Pacific sector of the Southern Ocean, especially in the Amundsen Sea. This thesis constitutes as groundwork for future investigations of protist assemblage changes in this area.
Pyrosequencing
Protist
Library
Phylotype
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Pyrosequencing
Library
Phylotype
clone (Java method)
Operational taxonomic unit
Sphingomonas
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We analyzed the potential of pmoA amplicon pyrosequencing compared to that of Sanger sequencing with paddy soils as a model environment. We defined operational taxonomic unit (OTU) cutoff values of 7% and 18%, reflecting methanotrophic species and major phylogenetic pmoA lineages, respectively. Major lineages were already well covered by clone libraries; nevertheless, pyrosequencing provided a higher level of diversity at the species level.
Pyrosequencing
Amplicon
Sanger sequencing
Methanotroph
Operational taxonomic unit
clone (Java method)
Library
Phylogenetic diversity
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Bioremediation is a cost-effective and sustainable approach for treating polluted soils, but our ability to improve on current bioremediation strategies depends on our ability to isolate microorganisms from these soils. Although culturing is widely used in bioremediation research and applications, it is unknown whether the composition of cultured isolates closely mirrors the indigenous microbial community from contaminated soils. To assess this, we paired culture-independent (454-pyrosequencing of total soil DNA) with culture-dependent (isolation using seven different growth media) techniques to analyse the bacterial and fungal communities from hydrocarbon-contaminated soils. Although bacterial and fungal rarefaction curves were saturated for both methods, only 2.4% and 8.2% of the bacterial and fungal OTUs, respectively, were shared between datasets. Isolated taxa increased the total recovered species richness by only 2% for bacteria and 5% for fungi. Interestingly, none of the bacteria that we isolated were representative of the major bacterial OTUs recovered by 454-pyrosequencing. Isolation of fungi was moderately more effective at capturing the dominant OTUs observed by culture-independent analysis, as 3 of 31 cultured fungal strains ranked among the 20 most abundant fungal OTUs in the 454-pyrosequencing dataset. This study is one of the most comprehensive comparisons of microbial communities from hydrocarbon-contaminated soils using both isolation and high-throughput sequencing methods.
Pyrosequencing
Soil microbiology
Enrichment culture
Isolation
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Corroborative data collected from 16S rRNA clone libraries, intergenic transcribed spacer (ITS) region clone libraries, and 16S rRNA hypervariable region tag pyrosequencing demonstrate microdiversity within single-species archaeal biofilms of the Lost City Hydrothermal Field. Both 16S rRNA clone libraries and pyrosequencing of the V6 hypervariable region show that Lost City Methanosarcinales (LCMS) biofilms are dominated by a single sequence, but the pyrosequencing data set also reveals the presence of an additional 1654 rare sequences. Clone libraries constructed with DNA spanning the V6 hypervariable region and ITS show that multiple ITS sequences are associated with the same dominant V6 sequence. Furthermore, ITS variability differed among three chimney samples, and the sample with the highest ITS diversity also contained the highest V6 diversity as measured by clone libraries as well as tag pyrosequencing. These results indicate that the extensive microdiversity detected in V6 tag sequences is an underestimate of genetic diversity within the archaeal biofilms.
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Hypervariable region
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clone (Java method)
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Sequencing of 18S rDNA clone libraries and 454-pyrosequencing are valuable methods used to describe microbial diversity. The massively parallel 454-pyrosequencing generates vast amounts of ribosomal sequence data and has the potential to uncover more organisms, even rare species. However, the relatively short sequence lengths of ∼500 bp are suboptimal for taxonomic annotation and phylogenetic analyses. In this study, we assessed the potential of 18S ribosomal clone libraries to complement corresponding 454-pyrosequencing data with near full-length sequence information. This involved a comparison of protist community compositions in five polar samples suggested by 18S rDNA clone libraries, with the corresponding community compositions suggested by 454-pyrosequencing. The study was conducted with four Arctic water samples, focusing on the eukaryotic picoplankton (0.4–3 µm), and with one sample collected in the Southern Ocean, examining the entire size spectrum (> 0.4 µm). For all individual samples, the protist community compositions suggested by the two different approaches showed significant similarities. Around 70% of the sequences detected by sequencing of clone libraries were also present in the 454-pyrosequencing data set. However, the clone library sequences reflected only ∼20% of the abundant biosphere identified by 454-pyrosequencing and identified ribosomal sequences that were not detected in the 454-pyrosequencing data sets.
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clone (Java method)
Protist
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Biofouling, the accumulation of microorganisms, is a major problem in paper mills processing paper and cardboard. This leads to the production of lower quality recycled products. Several studies have focused on the microbial content in the paper mill and the final products. Our aim was to determine the microbial biota in a bale of collected cardboard prior to entering the paper mill. Total genomic DNA was isolated and analyzed using two different methods for comparison purposes: 454 pyrosequencing and clone library. A total of 3268 V6-V8 454 pyrosequencing reads and 322 cloned V6-V8 16S rRNA nucleotide sequences were obtained. Both methods showed the presence of three major bacterial genera: Bacillus, Solibacillus and Paenibacillus, all members of the spore-forming phylum Firmicutes. Pyrosequencing, however, revealed a richer and more diverse bacterial community than clone library. It showed the presence of additional minor Firmicute genera and of a small number of Proteobacteria. The sorting at the recycling plant, the storing, and the processing at the paper mill, the end uses, will all contribute to the bacterial microbiota present in a bale of collected cardboard as revealed here.
Pyrosequencing
Library
cardboard
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Pyrosequencing
Euryarchaeota
Library
Sanger sequencing
Amplicon
Cloning (programming)
Thaumarchaeota
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We compared dideoxy sequencing of cloned chaperonin-60 universal target (cpn60 UT) amplicons to pyrosequencing of amplicons derived from vaginal microbial communities. In samples pooled from a number of individuals, the pyrosequencing method produced a data set that included virtually all of the sequences that were found within the clone library and revealed an additional level of taxonomic richness. However, the relative abundances of the sequences were different in the two datasets. These observations were expanded and confirmed by the analysis of paired clone library and pyrosequencing datasets from vaginal swabs taken from four individuals. Both for individuals with a normal vaginal microbiota and for those with bacterial vaginosis, the pyrosequencing method revealed a large number of low-abundance taxa that were missed by the clone library approach. In addition, we showed that the pyrosequencing method generates a reproducible profile of microbial community structure in replicate amplifications from the same community. We also compared the taxonomic composition of a vaginal microbial community determined by pyrosequencing of 16S rRNA amplicons to that obtained using cpn60 universal primers. We found that the profiles generated by the two molecular targets were highly similar, with slight differences in the proportional representation of the taxa detected. However, the number of operational taxonomic units was significantly higher in the cpn60 data set, suggesting that the protein-encoding gene provides improved species resolution over the 16S rRNA target. These observations demonstrate that pyrosequencing of cpn60 UT amplicons provides a robust, reliable method for deep sequencing of microbial communities.
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Amplicon
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Replicate
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Controlling microbial contamination of drinking water is critical to public health. However, understanding of the microbial ecology of drinking water remains incomplete. Representing the first application of high-throughput sequencing in drinking water microbiology, the objective of this study is to evaluate pyrosequencing as a high-throughput technique for the characterization of bacterial diversity in drinking water in comparison with conventional clone library analysis. Pyrosequencing and clone library analysis were performed in parallel to study the bacterial community composition in drinking water samples following the concentration of microbial biomass in drinking water with ultrafiltration. Validated by clone library analysis, pyrosequencing was confirmed as a highly efficient deep-sequencing technique to characterize the bacterial diversity in drinking water. Sequences of Alphaproteobacteria and Betaproteobacteria dominated the bacterial community in drinking water with Oxalobacteraceae and Methylobacteriaceae as the most abundant bacterial families, which is consistent with the prominent abundance of these populations frequently detected in various freshwater environments where source waters originate. Bacterial populations represented by the most abundant sequences in drinking water were closely related to cultures of metabolically versatile bacterial taxa widely distributed in the environment, suggesting a potential link between environmental distribution, metabolic characteristics, and abundance in drinking water.
Pyrosequencing
Alphaproteobacteria
Betaproteobacteria
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clone (Java method)
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Citations (12)