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  • 1.
    Corcoll, Natalia
    et al.
    Univ Gothenburg, Dept Biol & Environm Sci, Box 461, SE-40530 Gothenburg, Sweden..
    Osterlund, Tobias
    Chalmers, Dept Math Sci, SE-41296 Gothenburg, Sweden..
    Sinclair, Lucas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eiler, Alexander
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Kristiansson, Erik
    Chalmers, Dept Math Sci, SE-41296 Gothenburg, Sweden..
    Backhaus, Thomas
    Univ Gothenburg, Dept Biol & Environm Sci, Box 461, SE-40530 Gothenburg, Sweden..
    Eriksson, K. Martin
    Chalmers, Dept Mech & Maritime Sci, SE-41296 Gothenburg, Sweden..
    Comparison of four DNA extraction methods for comprehensive assessment of 16S rRNA bacterial diversity in marine biofilms using high-throughput sequencing2017In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 364, no 14, article id fnx139Article in journal (Refereed)
    Abstract [en]

    High-throughput DNA sequencing technologies are increasingly used for the metagenomic characterisation of microbial biodiversity. However, basic issues, such as the choice of an appropriate DNA extraction method, are still not resolved for non-model microbial communities. This study evaluates four commonly used DNA extraction methods for marine periphyton biofilms in terms of DNA yield, efficiency, purity, integrity and resulting 16S rRNA bacterial diversity. Among the tested methods, the Plant DNAzol (R) Reagent (PlantDNAzol) and the FastDNA (R) SPIN Kit for Soil (FastDNA Soil) methods were best suited to extract high quantities of DNA (77-130 mu g g wet wt(-1)). Lower amounts of DNA were obtained (<37 mu g g wet wt(-1)) with the Power Plant (R) Pro DNA Isolation Kit (PowerPlant) and the Power Biofilm (R) DNA Isolation Kit (PowerBiofilm) methods, but integrity and purity of the extracted DNA were higher. Results from 16S rRNA amplicon sequencing demonstrate that the choice of a DNA extraction method significantly influences the bacterial community profiles generated. A higher number of bacterial OTUs were detected when DNA was extracted with the PowerBiofilm and the PlantDNAzol methods. Overall, this study demonstrates the potential bias in metagenomic diversity estimates associated with different DNA extraction methods.

  • 2.
    Eiler, Alexander
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mondav, Rhiannon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sinclair, Lucas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fernandez-Vidal, Leyden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Scofield, Douglas G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology.
    Schwientek, Patrick
    Martinez-Garcia, Manuel
    Torrents, David
    McMahon, Katherine D.
    Andersson, Siv G. E.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Stepanauskas, Ramunas
    Woyke, Tanja
    Bertilsson, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tuning fresh: radiation through rewiring of central metabolism in streamlined bacteria2016In: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 10, no 8, p. 1902-1914Article in journal (Refereed)
  • 3.
    Langenheder, Silke
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Comte, Jérôme
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Univ Laval, Ctr Etud Nord, UMI Takuvik, Dept Biol IBIS, Pavillon Charles Eugene Marchand,1030 Ave Med, Quebec City, PQ G1V 0A6, Canada.
    Zha, Yinghua
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Samad, Md Sainur
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Univ Otago, Dept Microbiol & Immunol, 720 Cumberland St North Dunedin, Dunedin 9016, New Zealand.
    Sinclair, Lucas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Eiler, Alexander
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Lindström, Eva S.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Remnants of marine bacterial communities can be retrieved from deep sediments in lakes of marine origin2016In: Environmental Microbiology Reports, ISSN 1758-2229, E-ISSN 1758-2229, Vol. 8, no 4, p. 479-485Article in journal (Refereed)
    Abstract [en]

    Some bacteria can be preserved over time in deep sediments where they persist either in dormant or slow-growing vegetative stages. Here, we hypothesized that such cells can be revived when exposed to environmental conditions similar to those before they were buried in the sediments. To test this hypothesis, we collected bacteria from sediment samples of different ages (140–8500 calibrated years before present, cal BP) from three lakes that differed in the timing of their physical isolation from the Baltic Sea following postglacial uplift. After these bacterial communities were grown in sterile water from the Baltic Sea, we determined the proportion of 16S rRNA sequence reads associated with marine habitats by extracting the environment descriptive terms of homologous sequences retrieved from public databases. We found that the proportion of reads associated with marine descriptive term was significantly higher in cultures inoculated with sediment layers formed under Baltic conditions and where salinities were expected to be similar to current levels. Moreover, a similar pattern was found in the original sediment layers. Our study, therefore, suggests that remnants of marine bacterial communities can be preserved in sediments over thousands of years and can be revived from deep sediments in lakes of marine origin.

  • 4.
    Peura, Sari
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sinclair, Lucas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bertilsson, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eiler, Alexander
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Metagenomic insights into strategies of aerobic and anaerobic carbon and nitrogen transformation in boreal lakes2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 12102Article in journal (Refereed)
    Abstract [en]

    Thousands of net-heterotrophic and strongly stratifying lakes dominate the boreal landscape. Besides their central role as emitters of greenhouse gases, we have only recently begun to understand the microbial systems driving the metabolic processes and elemental cycles in these lakes. Using shotgun metagenomics, we show that the functional potential differs among lake types, with humic lakes being particularly enriched in carbon degradation genes. Most of the metabolic pathways exhibit oxygen- and temperature-dependent stratification over depth, coinciding with shifts in bacterial community composition, implying that stratification is a major factor controlling lake metabolism. In the bottom waters, rare and poorly characterized taxa, such as epsilon-Proteobacteria, but also autotrophs, such as photolithotrophic Chlorobia were abundant. These oxygen-depleted layers exhibited high genetic potential for mineralization, but also for fixation of carbon and nitrogen, and genetic markers for both methane production and oxidation were present. Our study provides a first glimpse of the genetic versatility of freshwater anoxic zones, and demonstrates the potential for complete turnover of carbon compounds within the water column.

  • 5.
    Savio, Domenico
    et al.
    Vienna Univ Technol, CWRS, A-1040 Vienna, Austria.;Vienna Univ Technol, Inst Chem Engn, Res Grp Environm Microbiol & Mol Ecol, A-1040 Vienna, Austria..
    Sinclair, Lucas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Ijaz, Umer Z.
    Univ Glasgow, Sch Engn, Glasgow, Lanark, Scotland..
    Parajka, Juraj
    Vienna Univ Technol, CWRS, A-1040 Vienna, Austria.;Vienna Univ Technol, Inst Hydraul Engn & Water Resource Management, A-1040 Vienna, Austria..
    Reischer, Georg H.
    Vienna Univ Technol, Inst Chem Engn, Res Grp Environm Microbiol & Mol Ecol, A-1040 Vienna, Austria.;Interuniv Cooperat Ctr Water & Hlth, London, England..
    Stadler, Philipp
    Vienna Univ Technol, CWRS, A-1040 Vienna, Austria.;Vienna Univ Technol, Inst Water Qual Resource & Waste Management, A-1040 Vienna, Austria..
    Blaschke, Alfred P.
    Vienna Univ Technol, CWRS, A-1040 Vienna, Austria.;Vienna Univ Technol, Inst Hydraul Engn & Water Resource Management, A-1040 Vienna, Austria..
    Bloeschl, Guenter
    Vienna Univ Technol, CWRS, A-1040 Vienna, Austria.;Vienna Univ Technol, Inst Hydraul Engn & Water Resource Management, A-1040 Vienna, Austria..
    Mach, Robert L.
    Vienna Univ Technol, Inst Chem Engn, Res Grp Environm Microbiol & Mol Ecol, A-1040 Vienna, Austria..
    Kirschner, Alexander K. T.
    Interuniv Cooperat Ctr Water & Hlth, London, England.;Med Univ Vienna, Inst Hyg & Appl Immunol Water Hyg, Vienna, Austria..
    Farnleitner, Andreas H.
    Vienna Univ Technol, CWRS, A-1040 Vienna, Austria.;Vienna Univ Technol, Inst Chem Engn, Res Grp Environm Microbiol & Mol Ecol, A-1040 Vienna, Austria.;Interuniv Cooperat Ctr Water & Hlth, London, England..
    Eiler, Alexander
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Plant Ecology and Evolution.
    Bacterial diversity along a 2600 km river continuum2015In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 17, no 12, p. 4994-5007Article in journal (Refereed)
    Abstract [en]

    The bacterioplankton diversity in large rivers has thus far been under-sampled despite the importance of streams and rivers as components of continental landscapes. Here, we present a comprehensive dataset detailing the bacterioplankton diversity along the midstream of the Danube River and its tributaries. Using 16S rRNA-gene amplicon sequencing, our analysis revealed that bacterial richness and evenness gradually declined downriver in both the free-living and particle-associated bacterial communities. These shifts were also supported by beta diversity analysis, where the effects of tributaries were negligible in regards to the overall variation. In addition, the river was largely dominated by bacteria that are commonly observed in freshwaters. Dominated by the acI lineage, the freshwater SAR11 (LD12) and the Polynucleobacter group, typical freshwater taxa increased in proportion downriver and were accompanied by a decrease in soil and groundwater-affiliated bacteria. Based on views of the meta-community and River Continuum Concept, we interpret the observed taxonomic patterns and accompanying changes in alpha and beta diversity with the intention of laying the foundation for a unified concept for river bacterioplankton diversity.

  • 6.
    Sinclair, Lucas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ahmed Osman, Omneya
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Bertilsson, Stefan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Eiler, Alexander
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Microbial Community Composition and Diversity via 16S rRNA Gene Amplicons: Evaluating the Illumina Platform2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 2, article id e0116955Article in journal (Refereed)
    Abstract [en]

    As new sequencing technologies become cheaper and older ones disappear, laboratories switch vendors and platforms. Validating the new setups is a crucial part of conducting rigorous scientific research. Here we report on the reliability and biases of performing bacterial 16S rRNA gene amplicon paired-end sequencing on the MiSeq Illumina platform. We designed a protocol using 50 barcode pairs to run samples in parallel and coded a pipeline to process the data. Sequencing the same sediment sample in 248 replicates as well as 70 samples from alkaline soda lakes, we evaluated the performance of the method with regards to estimates of alpha and beta diversity. Using different purification and DNA quantification procedures we always found up to 5-fold differences in the yield of sequences between individually barcodes samples. Using either a one-step or a two-step PCR preparation resulted in significantly different estimates in both alpha and beta diversity. Comparing with a previous method based on 454 pyrosequencing, we found that our Illumina protocol performed in a similar manner – with the exception for evenness estimates where correspondence between the methods was low. We further quantified the data loss at every processing step eventually accumulating to 50% of the raw reads. When evaluating different OTU clustering methods, we observed a stark contrast between the results of QIIME with default settings and the more recent UPARSE algorithm when it comes to the number of OTUs generated. Still, overall trends in alpha and beta diversity corresponded highly using both clustering methods. Our procedure performed well considering the precisions of alpha and beta diversity estimates, with insignificant effects of individual barcodes. Comparative analyses suggest that 454 and Illumina sequence data can be combined if the same PCR protocol and bioinformatic workflows are used for describing patterns in richness, beta-diversity and taxonomic composition.

  • 7.
    Sinclair, Lucas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Ijaz, Umer Z.
    Univ Glasgow, Sch Engn, Infrastruct & Environm Res Div, Glasgow, Lanark, Scotland..
    Jensen, Lars Juhl
    Univ Copenhagen, Fac Hlth & Med Sci, Novo Nordisk Fdn Ctr Prot Res, Copenhagen, Denmark..
    Coolen, Marco J. L.
    Curtin Univ Technol, Dept Chem, WA OIGC, Bentley, WA, Australia..
    Gubry-Rangin, Cecile
    Univ Aberdeen, Inst Biol & Environm Sci, Aberdeen, Scotland..
    Chronakova, Alica
    Acad Sci Czech Republic, Ctr Biol, Inst Soil Biol, Ceske Budejovice, Czech Republic..
    Oulas, Anastasis
    Cyprus Inst Neurol & Genet, Bioinformat Grp, Nicosia, Cyprus.;Hellen Ctr Marine Res, Inst Marine Biol Biotechnol & Aquaculture IMBBC, Iraklion, Greece..
    Pavloudi, Christina
    Hellen Ctr Marine Res, Inst Marine Biol Biotechnol & Aquaculture IMBBC, Iraklion, Greece..
    Schnetzer, Julia
    Max Planck Inst Marine Microbiol, Microbial Genom & Bioinformat Grp, Dept Mol Ecol, Bremen, Germany..
    Weimann, Aaron
    Helmholtz Ctr Infect Res, Computat Biol Infect Res, Braunschweig, Germany..
    Ijaz, Ali
    Univ Western Sydney, Hawkesbury Inst Environm, Sydney, NSW, Australia..
    Eiler, Alexander
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Quince, Christopher
    Univ Warwick, Warwick Med Sch, Warwick, England..
    Pafilis, Evangelos
    Hellen Ctr Marine Res, Inst Marine Biol Biotechnol & Aquaculture IMBBC, Iraklion, Greece..
    Seqenv: linking sequences to environments through text mining2016In: PeerJ, ISSN 2167-8359, E-ISSN 2167-8359, Vol. 4, article id e2690Article in journal (Refereed)
    Abstract [en]

    Understanding the distribution of taxa and associated traits across different environments is one of the central questions in microbial ecology. High-throughput sequencing (HTS) studies are presently generating huge volumes of data to address this biogeographical topic. However, these studies are often focused on specific environment types or processes leading to the production of individual, unconnected datasets. The large amounts of legacy sequence data with associated metadata that exist can be harnessed to better place the genetic information found in these surveys into a wider environmental context. Here we introduce a software program, seqenv, to carry out precisely such a task. It automatically performs similarity searches of short sequences against the "nt" nucleotide database provided by NCBI and, out of every hit, extracts if it is available the textual metadata field. After collecting all the isolation sources from all the search results, we run a text mining algorithm to identify and parse words that are associated with the Environmental Ontology (EnvO) controlled vocabulary. This, n turn, enables us to determine both in which environments individual sequences or taxa have previously been observed and, by weighted summation of those results, to summarize complete samples. We present two demonstrative applications of seqenv to a survey of ammonia oxidizing archaea as well as to a plankton paleome dataset from the Black Sea. These demonstrate the ability of the tool to reveal novel patterns in HTS and its utility in the fields of environmental source tracking, paleontology, and s of microbial biogeography.

  • 8. Weissbrodt, David G.
    et al.
    Shani, Noam
    Sinclair, Lucas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Lefebvre, Gregory
    Rossi, Pierre
    Maillard, Julien
    Rougemont, Jacques
    Holliger, Christof
    PyroTRF-ID: a novel bioinformatics methodology for the affiliation of terminal-restriction fragments using 16S rRNA gene pyrosequencing data2012In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, p. 306-Article in journal (Refereed)
    Abstract [en]

    Background: In molecular microbial ecology, massive sequencing is gradually replacing classical fingerprinting techniques such as terminal-restriction fragment length polymorphism (T-RFLP) combined with cloning-sequencing for the characterization of microbiomes. Here, a bioinformatics methodology for pyrosequencing-based T-RF identification (PyroTRF-ID) was developed to combine pyrosequencing and T-RFLP approaches for the description of microbial communities. The strength of this methodology relies on the identification of T-RFs by comparison of experimental and digital T-RFLP profiles obtained from the same samples. DNA extracts were subjected to amplification of the 16S rRNA gene pool, T-RFLP with the HaeIII restriction enzyme, 454 tag encoded FLX amplicon pyrosequencing, and PyroTRF-ID analysis. Digital T-RFLP profiles were generated from the denoised full pyrosequencing datasets, and the sequences contributing to each digital T-RF were classified to taxonomic bins using the Greengenes reference database. The method was tested both on bacterial communities found in chloroethene-contaminated groundwater samples and in aerobic granular sludge biofilms originating from wastewater treatment systems. Results: PyroTRF-ID was efficient for high-throughput mapping and digital T-RFLP profiling of pyrosequencing datasets. After denoising, a dataset comprising ca. 10'000 reads of 300 to 500 bp was typically processed within ca. 20 minutes on a high-performance computing cluster, running on a Linux-related CentOS 5.5 operating system, enabling parallel processing of multiple samples. Both digital and experimental T-RFLP profiles were aligned with maximum cross-correlation coefficients of 0.71 and 0.92 for high- and low-complexity environments, respectively. On average, 63 +/- 18% of all experimental T-RFs (30 to 93 peaks per sample) were affiliated to phylotypes. Conclusions: PyroTRF-ID profits from complementary advantages of pyrosequencing and T-RFLP and is particularly adapted for optimizing laboratory and computational efforts to describe microbial communities and their dynamics in any biological system. The high resolution of the microbial community composition is provided by pyrosequencing, which can be performed on a restricted set of selected samples, whereas T-RFLP enables simultaneous fingerprinting of numerous samples at relatively low cost and is especially adapted for routine analysis and follow-up of microbial communities on the long run.

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