21 research outputs found

    Development of a read mapping analysis software and computational pan genome analysis of 20 Pseudomonas aeruginosa strains

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    Hilker R. Development of a read mapping analysis software and computational pan genome analysis of 20 Pseudomonas aeruginosa strains. Bielefeld: Bielefeld University; 2015.In times of multi-resistant pathogenic bacteria, their detailed study is of utmost importance. Their comparative analysis can even aid the emerging field of personalized medicine by enabling optimized treatment depending on the presence of virulence factors and antibiotic resistances in the infection concerned. The weaknesses and functionality of these pathogenic bacteria can be investigated using modern computer science and novel sequencing technologies. One of these methods is the bioinformatics evaluation of high-throughput sequencing data. A pathogenic bacterium posing severe health care issues is the ubiquitous Pseudomonas aeruginosa. It is involved in a wide range of infections mainly affecting the pulmonary or urinary tract, open wounds and burns. The prevalence of chronic obstructive pulmonary disease cases with P. aeruginosa in Germany alone is ~600,000 per year. Within the framework of this dissertation, computational comparative genomics experiments were conducted with a panel of 20 of the most abundant Pseudomonas aeruginosa strains. 15 of these strains were isolated from clinical cases, while the remaining 5 were strains without a known infection history isolated from the environment. This division was chosen to enable direct comparison of the pathogenic potential of clinical and environmental strains and identification of their possible characteristic differences. When designing the bioinformatics experiments and searching for an efficient visualization and automatic analysis platform for read alignment (mapping) data, it became evident that no adequate solution was available that included all required functionalities. On these grounds, the decision was made to define two main subjects for this dissertation. Besides the P. aeruginosa pan genome analysis, a novel read mapping visualization and analysis software was developed and published in the journal Bioinformatics. This software - ReadXplorer - is partly based upon a prototype, which was developed during a preceding master's thesis at the Center for Biotechnology of the Bielefeld University under the name VAMP. The software was developed into a comprehensive user-friendly platform augmented with several newly developed and implemented automatic bioinformatics read mapping analyses. Two examples of these are the transcription start site detection and the single nucleotide polymorphism detection. Moreover, new intuitive visualizations were added to the existent ones and existing visualizations were greatly enhanced. ReadXplorer is designed to support not only DNA-seq data as accrued in the P. aeruginosa experiments, but also any kind of standard read mapping data as obtained from RNA-seq or ChIP-seq experiments. The data management was designed to comply with the latest performance and efficiency needs emerging from the large next generation sequencing data sets. Finally, ReadXplorer was empowered to deal with eukaryotic read mapping data as well. Amongst other software, ReadXplorer was then used to analyze different comparative genomics aspects of P. aeruginosa and to draw conclusions regarding the development of their pathogenicity. The list of conducted experiments includes phylogeny and gene set determination, analysis of regions of genomic plasticity and identification of single nucleotide polymorphisms. The achieved results were published in the journal Environmental Biology

    ReadXplorer 2 - detailed read mapping analysis and visualization from one single source

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    Hilker R, Stadermann KB, Schwengers O, et al. ReadXplorer 2 - detailed read mapping analysis and visualization from one single source. Bioinformatics. 2016;32(24):3702-3708.Motivation: The vast amount of already available and currently generated read mapping data re-quires comprehensive visualization, and should benefit from bioinformatics tools offering a wide spec-trum of analysis functionality from just one source. Appropriate handling of multiple mapped reads during mapping analyses remains an issue that demands improvement. Results: The capabilities of the read mapping analysis and visualization tool ReadXplorer were vastly enhanced. Here, we present an even finer granulated read mapping classification, improving the level of detail for analyses and visualizations. The spectrum of automatic analysis functions has been broadened to include genome rearrangement detection as well as correlation analysis between two mapping data sets. Existing functions were refined and enhanced, namely the computation of differ-entially expressed genes, the read count and normalization analysis and the transcription start site (TSS) detection. Additionally, ReadXplorer 2 features a highly improved support for large eukaryotic data sets and a command line version, enabling its integration into workflows. Finally, the new version is now able to display any kind of tabular results from other bioinformatics tools. Availability: http://www.readxplorer.or

    ReadXplorer - Visualization and Analysis of Mapped Sequences

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    Hilker R, Stadermann KB, Doppmeier D, et al. ReadXplorer - Visualization and Analysis of Mapped Sequences. Bioinformatics. 2014;30(16):2247-2254.MOTIVATION: Fast algorithms and well-arranged visualizations are required for the comprehensive analysis of the ever-growing size of genomic and transcriptomic next-generation sequencing data. RESULTS: ReadXplorer is a software offering straightforward visualization and extensive analysis functions for genomic and transcriptomic DNA sequences mapped on a reference. A unique specialty of ReadXplorer is the quality classification of the read mappings. It is incorporated in all analysis functions and displayed in ReadXplorer's various synchronized data viewers for (i) the reference sequence, its base coverage as (ii) normalizable plot and (iii) histogram, (iv) read alignments and (v) read pairs. ReadXplorer's analysis capability covers RNA secondary structure prediction, single nucleotide polymorphism and deletion-insertion polymorphism detection, genomic feature and general coverage analysis. Especially for RNA-Seq data, it offers differential gene expression analysis, transcription start site and operon detection as well as RPKM value and read count calculations. Furthermore, ReadXplorer can combine or superimpose coverage of different datasets. AVAILABILITY AND IMPLEMENTATION: ReadXplorer is available as open-source software at http://www.readxplorer.org along with a detailed manual. CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. The Author 2014. Published by Oxford University Press

    Deletion of the Sm1 encoding motif in the lsm gene results in distinct changes in the transcriptome and enhanced swarming activity of Haloferax cells

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    Members of the Sm protein family are important for the cellular RNA metabolism in all three domains of life. The family includes archaeal and eukaryotic Lsm proteins, eukaryotic Sm proteins and archaeal and bacterial Hfq proteins. While several studies concerning the bacterial and eukaryotic family members have been published, little is known about the archaeal Lsm proteins. Although structures for several archaeal Lsm proteins have been solved already more than ten years ago, we still do not know much about their biological function, however one can confidently propose that the archaeal Lsm proteins will also be involved in RNA metabolism. Therefore, we investigated this protein in the halophilic archaeon Haloferax volcanii. The Haloferax genome encodes a single Lsm protein, the lsm gene overlaps and is co-transcribed with the gene for the ribosomal L37.eR protein. Here, we show that the reading frame of the lsm gene contains a promoter which regulates expression of the overlapping rpl37R gene. This rpl37R specific promoter ensures high expression of the rpl37R gene in exponential growth phase. To investigate the biological function of the Lsm protein we generated a lsm deletion mutant that had the coding sequence for the Sm1 motif removed but still contained the internal promoter for the downstream rpl37R gene. The transcriptome of this deletion mutant was compared to the wild type transcriptome, revealing that several genes are down-regulated and many genes are up-regulated in the deletion strain. Northern blot analyses confirmed down-regulation of two genes. In addition, the deletion strain showed a gain of function in swarming, in congruence with the up-regulation of transcripts encoding proteins required for motility

    Genome-wide identification of transcriptional start sites in the haloarchaeon Haloferax volcanii based on differential RNA-Seq (dRNA-Seq)

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    Background: Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii. Results: Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5′-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs). Conclusion: This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated

    Additional file 2: Table S2. of Genome-wide identification of transcriptional start sites in the haloarchaeon Haloferax volcanii based on differential RNA-Seq (dRNA-Seq)

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    Containing the dRNA-Seq results. Summary of all of the TSSs identified by the dRNA-Seq approach and the additional selected parameters, e.g., replicon, localization, strand, identification in the three datasets, TSS class, associated gene, and promoter score. The first Excel sheet contains the results, and the second Excel sheet contains a list of columns and definitions. (XLSX 572 kb

    Serotype 1 and 8 Pneumococci Evade Sensing by Inflammasomes in Human Lung Tissue.

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    Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. The pore-forming toxin pneumolysin is a key virulence factor of S. pneumoniae, which can be sensed by the NLRP3 inflammasome. Among the over 90 serotypes, serotype 1 pneumococci (particularly MLST306) have emerged across the globe as a major cause of invasive disease. The cause for its particularity is, however, incompletely understood. We therefore examined pneumococcal infection in human cells and a human lung organ culture system mimicking infection of the lower respiratory tract. We demonstrate that different pneumococcal serotypes differentially activate inflammasome-dependent IL-1β production in human lung tissue and cells. Whereas serotype 2, 3, 6B, 9N pneumococci expressing fully haemolytic pneumolysins activate NLRP3 inflammasome-dependent responses, serotype 1 and 8 strains expressing non-haemolytic toxins are poor activators of IL-1β production. Accordingly, purified haemolytic pneumolysin but not serotype 1-associated non-haemolytic toxin activates strong IL-1β production in human lungs. Our data suggest that the evasion of inflammasome-dependent innate immune responses by serotype 1 pneumococci might contribute to their ability to cause invasive diseases in humans
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