602 research outputs found
The 13 high-quality draft genomes
No full text<p>The 13 high-quality draft genomes from: </p>
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<p>Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes</p>
<p>Mads Albertsen, Philip Hugenholtz, Adam Skarshewski, Gene W. Tyson, Kåre L. Nielsen and Per .H. Nielsen</p>
<p>Nature Biotechnology 2013</p
Mapping DNA Methylation to Methyltransferases in Microbial Communities
No full textAcross all domains of life, the genetic code is overlaid with epigenetic modifications that ex-tend beyond the primary nucleotide sequence. The most common and nearly universal mech-anism of epigenetic signaling is DNA methylation. In bacteria, it modulates a range of biologi-cal processes, including host defense mechanisms, cell cycle regulation, gene expression, and virulence. This modification is facilitated by DNA methyltransferases, which dictate the methylation patterns of bacterial genomes in a motif-specific manner, often differing among species and strains. Recent technological advances in Nanopore sequencing now enable the direct detection of DNA methylation from a standard sequencing run. Despite this, only a few efforts have been made to utilize ONT methylation calls for methylation motif discovery in bac-teria, but none which scales or extends motif discovery to metagenome sequencing of micro-bial communities. To address this, we developed Nanomotif, a fast, scalable, bioinformatic tool for identification and utilization of methylation motifs in metagenomic samples. The MTa-se-linker submodule of Nanomotif replaces existing manual and non-scalable methods with a modern, user-friendly bioinformatics tool that pairs methylation motifs to their cognate DNA methyltransferases. In the era of metagenomics, tools like this are essential for faster epige-netic profiling across entire microbial communities. Motif-methyltransferase pairs not only help circumvent restriction-modification barriers but also open new avenues to explore the func-tional roles of methylation and its implications for microbial physiology and ecology
Modification af moose (Alces alces) microbiome after af translocation from Sweden to Lille Vildmose in Denmark
No full textMetagenomes obtained by "deep sequencing" - what do they tell about the EBPR communities
No full textMetagenomes obtained by "deep sequencing" - what do they tell about the EBPR communities?Mads Albertsen1, Aaron M. Saunders1, Kåre L. Nielsen1 and Per H. Nielsen11 Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Aalborg, Denmark Presenting Author: Mads AlbertsenKeywords: Metagenomics; Accumulibacter; Micro-diversity; Enhanced Biological Phosphorus RemovalIntroductionMetagenomics, or environmental genomics, provides comprehensive information about the entire microbial community of a certain ecosystem, e.g. a wastewater treatment plant. So far, metagenomic analyses have been hampered by high costs and high level of expertise needed to conduct the investigations, but it is changing now with development of new technologies allowing analyses of billions of DNA sequences (deep-sequencing) and user-friendly pipelines for analyses of the huge data sets. Analyses of metagenomes can give extensive information about the identity of microbes in the community as well as their functional potential (Kuczynski et al., 2011). Metagenomics can also be regarded as the blueprint needed to conduct the next tier of studies of gene and protein (transcriptomics and proteomics) providing information about the function of the microbes in the system. The combination of metagenomics, transcriptomics and proteomics will bring us closer to a systems microbiology perspective of a certain microbial community and will aid in the goal of establishing predictive models. Recently we presented the first metagenome of a full-scale wastewater treatment plant carrying out nitrification/denitrification and enhanced biological phosphorus removal (EBPR) (Albertsen et al., 2011). Such studies on full-scale plants are needed to improve the understanding of key microorganisms in the EBPR process (e.g., the polyphosphate accumulating bacteria, PAO) and the entire community in order to improve the process for efficient P-removal and P-recovery (Nielsen et al., 2010; 2011). The aim of this study was to compare metagenomes from two full-scale EBPR plants to other environments and to investigate in detail similarities and differences between the two EBPR communities. Material and MethodsDNA extraction from activated sludge from the EBPR wastewater treatment plants Aalborg East and West and the further metagenomic sequencing, assembly and annotation were largely conducted as described in Albertsen et al., (2011).Results and DiscussionWe sequenced two metagenomes from Aalborg East and West EBPR wastewater treatment plants at a depth of 12 and 8 Gb using Illumina short read sequencing. The EBPR plants form a distinct group when compared to metagenomes from a wide range of environments, both on phylogenetic and functional level (Fig. 1). Even though the samples were taken at different times of the year (August vs. December) and from different EBPR plants, they cluster tightly, which may be attributed to the wide range of selection pressures acting on the EBPR communities. These results confirm the findings of a core microbial community using quantitative fluorescence in situ hybridization (qFISH) and other techniques (Nielsen et al., 2010; 2011). Through the use of qFISH probes we investigated the micro-diversity of the key PAO Accumulibacter (clade I and II) in the two EBPR plants. In Aalborg East clade I was estimated to 1.6% and II to 1.3% of the total population, and 1.5% and 1.1% in West. As a reference genome exists for clade IIA we used the raw metagenome reads to estimate the Accumulibacter micro-diversity in the metagenomes. This revealed a much greater micro-diversity than observed through qFISH - also between the two metagenomes. Despite the large diversity the PAO core functions seemed conserved between the different Accumulibacter species. In addition, there seemed to be a high selection pressure from viruses (phages) acting on the different Accumulibacter populations. ConclusionsThe improved resolution of deep metagenomics enables insights of both community function and micro-diversity. We showed that the complex selection pressures in EBPR plants seem to create a tightly controlled core gene pool, although significant micro-diversity exists. The dynamics of micro-diversity at genome level and the implications for stable plant operation and P-removal will be an interesting question to investigate further. One current limitation for application of metagenomics and metatranscriptomics on a systems level is the need of more reference genomes that are closely related to the species in the EBPR plants. Currently only a handful of relevant genomes are available, however the limited core species in the EBPR process and the rapid improvement of single cell genomics makes us confident that we soon will be able to conduct comprehensive systems microbiology investigations in EBPR plants.ReferencesAlbertsen M., Hansen L.B.S., Saunders A.M., Nielsen P.H., and Nielsen K.L. (2011). A metagenome of a full-scale microbial community carrying out enhanced biological phosphorus removal. ISME journal, ePub ahead of print, doi :10.1038/ismej.2011.176.Nielsen P.H., Mielczarek A.T., Kragelund C., Nielsen J.L., Saunders A.M., Kong Y., et al. (2010). A conceptual ecosystem model of microbial communities in enhanced biological phosphorus removal plants. Water Res 44, 5070-5088.Nielsen P.H., Saunders A.M, Hansen A.A, Larsen P., Nielsen J.L (2011) Microbial communities involved in enhanced biological phosphorus removal from wastewater - a model system in environmental biotechnology. Current Opinion in Biotechnology 23, 1-8.Kuczynski J., Lauber C.L., Walters W.A.., Parfrey L.W., Clemente J.C., Gevers D., et al. (2011). Experimental and analytical tools for studying the human microbiome. Nature Reviews Genetics, 13, 47-58.<br/
FERTILIZATION-INDEPENDENT SEED-Polycomb Repressive Complex 2 Plays a Dual Role in Regulating Type I MADS-Box Genes in Early Endosperm Development
Get PDFEarly endosperm development presents a unique system in which to uncover epigenetic regulatory mechanisms because the contributing maternal and paternal genomes possess differential epigenetic modi?cations. In Arabidopsis (Arabidopsis thaliana), the initiation of endosperm coenocytic growth upon fertilization and the transition to endosperm cellularization are regulated by the FERTILIZATION-INDEPENDENT SEED (FIS)-Polycomb Repressive Complex 2 (PRC2), a putative H3K27 methyltransferase. Here, we address the possible role of the FIS-PRC2 complex in regulating the type I MADS-box gene family, which has been shown previously to regulate early endosperm development. We show that a subclass of type I MADS-box genes (C2 genes) was expressed in distinct domains of the coenocytic endosperm in wild-type seeds. Furthermore, the C2 genes were mostly up-regulated biallelically during the extended coenocytic phase of endosperm development in the FIS-PRC2 mutant background. Using allele-speci?c expression analysis, we also identi?ed a small subset of C2 genes subjected to FIS-PRC2-dependent maternal or FIS-PRC2-independent paternal imprinting. Our data support a dual role for the FIS-PRC2 complex in the regulation of C2 type I MADS-box genes, as evidenced by a generalized role in the repression of gene expression at both alleles associated with endosperm cellularization and a specialized role in silencing the maternal allele of imprinted genes
ampvis2: an R package to analyse and visualise 16S rRNA amplicon data
No full textAbstractSummaryMicrobial community analysis using 16S rRNA gene amplicon sequencing is the backbone of many microbial ecology studies. Several approaches and pipelines exist for processing the raw data generated through DNA sequencing and convert the data into OTU-tables. Here we present ampvis2, an R package designed for analysis of microbial community data in OTU-table format with focus on simplicity, reproducibility, and sample metadata integration, with a minimal set of intuitive commands. Unique features include flexible heatmaps and simplified ordination. By generating plots using the ggplot2 package, ampvis2 produces publication-ready figures that can be easily customised. Furthermore, ampvis2 includes features for interactive visualisation, which can be convenient for larger, more complex data.Availabilityampvis2 is implemented in the R statistical language and is released under the GNU A-GPL license. Documentation website and source code is maintained at: https://github.com/MadsAlbertsen/ampvis2ContactMads Albertsen ([email protected])</jats:sec
Introduction and transmission of SARS-CoV-2 lineage B.1.1.7, Alpha variant, in Denmark
No full textBackground: In early 2021, the SARS-CoV-2 lineage B.1.1.7 (Alpha variant) became dominant across large parts of the world. In Denmark, comprehensive and real-time test, contact-tracing, and sequencing efforts were applied to sustain epidemic control. Here, we use these data to investigate the transmissibility, introduction, and onward transmission of B.1.1.7 in Denmark. Methods: We analyzed a comprehensive set of 60,178 SARS-CoV-2 genomes generated from high-throughput sequencing by the Danish COVID-19 Genome Consortium, representing 34% of all positive cases in the period 14 November 2020 to 7 February 2021. We calculated the transmissibility of B.1.1.7 relative to other lineages using Poisson regression. Including all 1976 high-quality B.1.1.7 genomes collected in the study period, we constructed a time-scaled phylogeny, which was coupled with detailed travel history and register data to outline the introduction and onward transmission of B.1.1.7 in Denmark. Results: In a period with unchanged restrictions, we estimated an increased B.1.1.7 transmissibility of 58% (95% CI: [56%, 60%]) relative to other lineages. Epidemiological and phylogenetic analyses revealed that 37% of B.1.1.7 cases were related to the initial introduction in November 2020. The relative number of cases directly linked to introductions varied between 10 and 50% throughout the study period. Conclusions: Our findings corroborate early estimates of increased transmissibility of B.1.1.7. Both substantial early expansion when B.1.1.7 was still unmonitored and continuous foreign introductions contributed considerably to case numbers. Finally, our study highlights the benefit of balanced travel restrictions and self-isolation procedures coupled with comprehensive surveillance efforts, to sustain epidemic control in the face of emerging variants.</p
Developing methods for on-site DNA sequencing
No full textEffektiviteten af spildevandsrensningsanlæg er i høj grad bestemt af deres mikrobielle sammensætning. Derfor er identifikation af det mikrobielle samfund en vigtig del af at køre et bestemt spildevandsrensningsanlæg og forstå, hvordan det fungerer. I øjeblikket gøres dette i højt specialiserede laboratorier, men det begrænser metoden til at se tilbage ved ændringer og bruges ikke til at styre operationelle beslutninger. Imidlertid gøres der løbende fremskridt med sekventeringsteknologi (Oxford Nanopore Minion) og automatiseret prøveforberedelse gør det teoretisk muligt at flytte sekventering ud af laboratoriet. Men for at gøre dette til en realitet er der et behov for en hurtig, billig, pålidelig og meget mobil DNA-ekstraktion, der fungerer i lighed med State-of-the-art ekstraktionsmetoder.I denne afhandling er der udviklet en brugervenlig, hurtig og meget mobil DNA-ekstraktionsmetode. Metoden er baseret på et kraftværktøj med en 3D-trykt adapter til bead beating baseret lysering af celler, og DNA isoleres ved anvendelse af fastfase-reversible immobiliseringsbeads. Metoden blev sammenlignet med den nyeste og anbefalede DNA-ekstraktionsmetode for feltet af aktiveret slam-MiDAS-feltguide. Sammenligningen af fremgangsmåderne blev lavet på flere niveauer, inklusive mængden af ekstraheret DNA, renhed og fragmentering. Desuden blev 16S rRNA amplicon-sekventering brugt til at evaluere enhver potentiel ekstraktionsforstyrrelse i det observerede mikrobielle samfund.Det blev vist, at den foreslåede DNA-ekstraktionsmetode ikke introducerede en bias i mikrobielle samfunds sammensætning og præsterede lige så godt på udbytte og renhed. Tilsvarende reduceres den samlede tid til DNA-extraction ned til ca. 10 minutter sammenlignet med 1-timers standard protokollen.Der er dog behov for yderligere optimering for at sikre, at metoden opfylder kravet om høj renhed og lang DNA-fragmentlængde for MinION. Samlet set giver den udviklede tilgang et fundament for at flytte DNA-ekstraktionen og sekventeringen ud af laboratoriet og ind i feltet. The efficiency of wastewater treatment plants is largely determined by their microbial composition. Therefore, identification of the microbial community is an important part of running a particular wastewater treatment plant and understanding how it functions. Currently, this is done in highly specialized laboratories, but this limits the method to being only able to look back at changes, and it is not being used to guide operational decisions. However, ongoing advancement of sequencing technology (Oxford Nanopore MinION) and automated sample preparation makes it theoretically possible to move sequencing out of the laboratory. However, to make this a reality, there is a need for a fast, cheap, reliable and highly mobile DNA extraction that works on par with state-of-the-art extraction methods.In this thesis, an easy to use, fast and highly mobile DNA extraction method is developed. The method is based on a power tool with a 3D printed adapter for bead-beating based lysis of cells, and DNA is isolated using solid phase reversible immobilization beads. The method was compared to the state-of-the-art and recommended DNA extraction method for the field of activated sludge: the MiDAS field guide. The comparison of the methods was made on several levels including the amount of extracted DNA, purity and fragmentation. Furthermore, 16S rRNA amplicon sequencing was used to evaluate any potential extraction bias in the observed microbial community. It was shown that the proposed DNA extraction method did not introduce a bias in microbial community composition and performed just as good on yield and purity. Correspondingly, it cut the total time for DNA extraction down to roughly 10 minutes compared to the 1-hour standard protocol.However, further optimization is needed to make sure the method fulfills the high purity and long DNA fragment length requirement for the MinION. Overall, the developed approach provides a foundation for moving the DNA extraction and sequencing out of the laboratory and into the field. <br/
Data_Sheet_1_Investigation of Detection Limits and the Influence of DNA Extraction and Primer Choice on the Observed Microbial Communities in Drinking Water Samples Using 16S rRNA Gene Amplicon Sequencing.PDF
Get PDFIn recent years, 16S rRNA gene amplicon sequencing has been widely adopted for analyzing the microbial communities in drinking water (DW). However, no comprehensive attempts have been made to illuminate the inherent method biases specifically relating to DW communities. In this study, we investigated the impact of DNA extraction and primer choice on the observed microbial community, and furthermore estimated the detection limit of the 16S rRNA gene amplicon sequencing in these experimental settings. Of the two DNA extraction kits investigated, the PowerWater DNA Isolation Kit resulted in higher yield, better reproducibility and more OTUs identified compared to the FastDNA SPIN Kit for Soil, which is also commonly used within DW microbiome research. The use of three separate primer-sets targeting the V1-3, V3-4, and V4 region of the 16S rRNA gene revealed large differences in OTU abundances, with some of the primers unable to detect entire phyla. Estimations of the detection limit were based on bacteria-free water samples (1 L) spiked with Escherichia coli cells in different concentrations [101–106 cells/ml]. E.coli could be detected in all samples, however, samples with ∼101 cells/ml had several contaminating OTUs constituting approximately 8% of the read abundances. Based on our findings, we recommend using the PowerWater DNA Isolation Kit for DNA extraction in combination with PCR amplification of the V3-4 or V4 region for DW samples if a broad overview of the microbial community is to be obtained.</p
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