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  • 1
    In: ISME Communications, Springer Science and Business Media LLC, Vol. 3, No. 1 ( 2023-07-20)
    Abstract: Preterm birth is accompanied with many complications and requires severe therapeutic regimens at the neonatal intensive care unit. The influence of the above-mentioned factors on the premature-born infants’ respiratory metagenome or more generally its maturation is unknown. We therefore applied shotgun metagenome sequencing of oropharyngeal swabs to analyze the airway metagenome development of 24 preterm infants from one week postpartum to 15 months of age. Beta diversity analysis revealed a distinct clustering of airway microbial communities from hospitalized preterms and samples after hospital discharge. At nine and 15 months of age, the preterm infants lost their hospital-acquired individual metagenome signatures towards a common taxonomic structure. However, ecological network analysis and Random Forest classification of cross-sectional data revealed that by this age the preterm infants did not succeed in establishing the uniform and stable bacterial community structures that are characteristic for healthy full-term infants.
    Type of Medium: Online Resource
    ISSN: 2730-6151
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2023
    detail.hit.zdb_id: 3041786-7
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  • 2
    In: Frontiers in Microbiology, Frontiers Media SA, Vol. 14 ( 2023-3-13)
    Abstract: Plasticity of Pseudomonas aeruginosa chromosomes is mainly driven by an extended accessory genome that is shaped by insertion and deletion events. Further modification of the genome composition can be induced by chromosomal inversion events which lead to relocation of genes in the affected genomic DNA segments, modify the otherwise highly conserved core genome synteny and could even alter the location of the replication terminus. Although the genome of the first sequenced strain, PAO1, displayed such a large genomic inversion, knowledge on such recombination events in the P. aeruginosa population is limited. Several large inversions had been discovered in the late 1990s in cystic fibrosis isolates of the major clonal lineage C by physical genome mapping, and subsequent work on these examples led to the characterization of the DNA at the recombination breakpoints and a presumed recombination mechanism. Since then, the topic was barely addressed in spite of the compilation of thousands of P. aeruginosa genome sequences that are deposited in databases. Due to the use of second-generation sequencing, genome contig assembly had usually followed synteny blueprints provided by the existing reference genome sequences. Inversion detection was not feasible by these approaches, as the respective read lengths did not allow reliable resolution of sequence repeats that are typically found at the borders of inverted segments. In this study, we applied PacBio and MinION long-read sequencing to isolates of the mentioned clone C collection. Confirmation of inversions predicted from the physical mapping data demonstrated that unbiased sequence assembly of such read datasets allows the detection of genomic inversions and the resolution of the recombination breakpoint regions. Additional long-read sequencing of representatives of the other major clonal lineage, PA14, revealed large inversions in several isolates, from cystic fibrosis origin as well as from other sources. These findings indicated that inversion events are not restricted to strains from chronic infection background, but could be widespread in the P. aeruginosa population and contribute to genome plasticity. Moreover, the monitored examples emphasized the role of small mobile DNA units, such as IS elements or transposons, and accessory DNA elements in the inversion-related recombination processes.
    Type of Medium: Online Resource
    ISSN: 1664-302X
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2023
    detail.hit.zdb_id: 2587354-4
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  • 3
    In: BMC Genomics, Springer Science and Business Media LLC, Vol. 23, No. 1 ( 2022-11-11)
    Abstract: Shotgun metagenome analysis provides a robust and verifiable method for comprehensive microbiome analysis of fungal, viral, archaeal and bacterial taxonomy, particularly with regard to visualization of read mapping location, normalization options, growth dynamics and functional gene repertoires. Current read classification tools use non-standard output formats, or do not fully show information on mapping location. As reference datasets are not perfect, portrayal of mapping information is critical for judging results effectively. Results Our alignment-based pipeline, Wochenende, incorporates flexible quality control, trimming, mapping, various filters and normalization. Results are completely transparent and filters can be adjusted by the user. We observe stringent filtering of mismatches and use of mapping quality sharply reduces the number of false positives. Further modules allow genomic visualization and the calculation of growth rates, as well as integration and subsequent plotting of pipeline results as heatmaps or heat trees. Our novel normalization approach additionally allows calculation of absolute abundance profiles by comparison with reads assigned to the human host genome. Conclusion Wochenende has the ability to find and filter alignments to all kingdoms of life using both short and long reads, and requires only good quality reference genomes. Wochenende automatically combines multiple available modules ranging from quality control and normalization to taxonomic visualization. Wochenende is available at https://github.com/MHH-RCUG/nf_wochenende .
    Type of Medium: Online Resource
    ISSN: 1471-2164
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2022
    detail.hit.zdb_id: 2041499-7
    SSG: 12
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  • 4
    In: Microbiology Spectrum, American Society for Microbiology, Vol. 10, No. 5 ( 2022-10-26)
    Abstract: The introduction of mutation-specific combination therapy with the cystic fibrosis transmembrane conductance regulator (CFTR) modulators elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) has substantially improved lung function and quality of life of people with cystic fibrosis (CF). Collecting deep cough swabs and induced sputum, this postapproval study examined the effect of 14- and 50-week treatment with ELX/TEZ/IVA on the airway microbial metagenome of pancreatic- insufficient CF patients aged 12 years and older. Compared to pretreatment, the total bacterial load decreased, the individual species were more evenly distributed in the community, and the individual microbial metagenomes became more similar in their composition. However, the microbial network remained vulnerable to fragmentation. The initial shift of the CF metagenome was attributable to the ELX/TEZ/IVA-mediated gain of CFTR activity followed by a diversification driven by a group of commensals at the 1-year time point that are typical for healthy airways. IMPORTANCE Shotgun metagenome sequencing of respiratory secretions with spike-in controls for normalization demonstrated that 1 year of high-efficient CFTR modulation with elexacaftor/tezacaftor/ivacaftor extensively reduced the bacterial load. Longer observation periods will be necessary to resolve whether the partial reversion of the basic defect that is achieved with ELX/TEZ/IVA is sufficient in the long run to render the CF lungs robust against the recolonization with common opportunistic pathogens.
    Type of Medium: Online Resource
    ISSN: 2165-0497
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 2807133-5
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  • 5
    In: Microbiology Spectrum, American Society for Microbiology, Vol. 11, No. 2 ( 2023-04-13)
    Abstract: The microbial metagenome in cystic fibrosis (CF) airways was investigated by whole-genome shotgun sequencing of total DNA isolated from nasal lavage samples, oropharyngeal swabs, and induced sputum samples collected from 65 individuals with CF aged 7 to 50 years. Each patient harbored a personalized microbial metagenome unique in microbial load and composition, the exception being monocultures of the most common CF pathogens Staphylococcus aureus and Pseudomonas aeruginosa from patients with advanced lung disease. The sampling of the upper airways by nasal lavage uncovered the fungus Malassezia restricta and the bacterium Staphylococcus epidermidis as prominent species. Healthy and CF donors harbored qualitatively and quantitatively different spectra of commensal bacteria in their sputa, even in the absence of any typical CF pathogen. If P. aeruginosa , S. aureus , or Stenotrophomonas maltophilia belonged to the trio of the most abundant species in the CF sputum metagenome, common inhabitants of the respiratory tract of healthy subjects, i.e., Eubacterium sulci , Fusobacterium periodonticum , and Neisseria subflava , were present only in low numbers or not detectable. Random forest analysis identified the numerical ecological parameters of the bacterial community, such as Shannon and Simpson diversity, as the key parameters that globally distinguish sputum samples from CF and healthy donors. IMPORTANCE Cystic fibrosis (CF) is the most common life-limiting monogenetic disease in European populations and is caused by mutations in the CFTR gene. Chronic airway infections with opportunistic pathogens are the major morbidity that determines prognosis and quality of life in most people with CF. We examined the composition of the microbial communities of the oral cavity and upper and lower airways in CF patients across all age groups. From early on, the spectrum of commensals is different in health and CF. Later on, when the common CF pathogens take up residence in the lungs, we observed differential modes of depletion of the commensal microbiota in the presence of S. aureus , P. aeruginosa , S. maltophilia , or combinations thereof. It remains to be seen whether the implementation of lifelong CFTR (cystic fibrosis transmembrane conductance regulator) modulation will change the temporal evolution of the CF airway metagenome.
    Type of Medium: Online Resource
    ISSN: 2165-0497
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 2807133-5
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  • 6
    Online Resource
    Online Resource
    Springer Science and Business Media LLC ; 2021
    In:  ISME Communications Vol. 1, No. 1 ( 2021-03-24)
    In: ISME Communications, Springer Science and Business Media LLC, Vol. 1, No. 1 ( 2021-03-24)
    Abstract: In shotgun metagenomic sequencing applications, low signal-to-noise ratios may complicate species-level differentiation of genetically similar core species and impede high-confidence detection of rare species. However, core and rare species can take pivotal roles in their habitats and should hence be studied as one entity to gain insights into the total potential of microbial communities in terms of taxonomy and functionality. Here, we offer a solution towards increased species-level specificity, decreased false discovery and omission rates of core and rare species in complex metagenomic samples by introducing the rare species identifier (raspir) tool. The python software is based on discrete Fourier transforms and spectral comparisons of biological and reference frequency signals obtained from real and ideal distributions of short DNA reads mapping towards circular reference genomes. Simulation-based testing of raspir enabled the detection of rare species with genome coverages of less than 0.2%. Species-level differentiation of rare Escherichia coli and Shigella spp., as well as the clear delineation between human Streptococcus spp. was feasible with low false discovery (1.3%) and omission rates (13%). Publicly available human placenta sequencing data were reanalysed with raspir. Raspir was unable to identify placental microbial communities, reinforcing the sterile womb paradigm.
    Type of Medium: Online Resource
    ISSN: 2730-6151
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2021
    detail.hit.zdb_id: 3041786-7
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  • 7
    Online Resource
    Online Resource
    Elsevier BV ; 2022
    In:  Computational and Structural Biotechnology Journal Vol. 20 ( 2022), p. 175-186
    In: Computational and Structural Biotechnology Journal, Elsevier BV, Vol. 20 ( 2022), p. 175-186
    Type of Medium: Online Resource
    ISSN: 2001-0370
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2022
    detail.hit.zdb_id: 2694435-2
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  • 8
    Online Resource
    Online Resource
    American Society for Microbiology ; 2022
    In:  Journal of Bacteriology Vol. 204, No. 1 ( 2022-01-18)
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 204, No. 1 ( 2022-01-18)
    Abstract: The transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phases of planktonic growth were evaluated by direct RNA sequencing on the nanopore platform and compared with established short-read cDNA sequencing on the Illumina platform. Fifty to ninety percent of the sense RNAs turned out to be rRNA molecules, followed by similar proportions of mRNA transcripts and noncoding RNAs. The two platforms detected similar proportions of uncharged tRNAs and 29 yet-undescribed antisense tRNAs. For example, the rarest arginine codon was paired with the most abundant tRNA Arg , and the tRNA Arg gene is missing for the most frequent arginine codon. More than 90% of the antisense RNA molecules were complementary to a coding sequence. The antisense RNAs were evenly distributed in the genomes. Direct RNA sequencing identified more than 4,000 distinct nonoverlapping antisense RNAs during exponential and stationary growth. Besides highly expressed small antisense RNAs less than 200 bases in size, a population of longer antisense RNAs was sequenced that covered a broad range (a few hundred to thousands of bases) and could be complementary to a contig of several genes. In summary, direct RNA sequencing identified yet-undescribed RNA molecules and an unexpected composition of the pools of tRNAs and sense and antisense RNAs. IMPORTANCE Genome-wide gene expression of bacteria is commonly studied by high-throughput sequencing of size-selected cDNA fragment libraries of reverse-transcribed RNA preparations. However, the depletion of rRNAs, enzymatic reverse transcription, and the fragmentation, size selection, and amplification during library preparation lead to inevitable losses of information about the initial composition of the RNA pool. We demonstrate that direct RNA sequencing on the Nanopore platform can overcome these limitations. Nanopore sequencing of total RNA yielded novel insights into the Pseudomonas aeruginosa transcriptome that—if replicated in other species—will change our view of the bacterial RNA world. The discovery of sense-antisense pairs of transfer-messenger RNA (tmRNA), tRNAs, and mRNAs indicates a further and unknown level of gene regulation in bacteria.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 9
    Online Resource
    Online Resource
    Springer Science and Business Media LLC ; 2020
    In:  npj Biofilms and Microbiomes Vol. 6, No. 1 ( 2020-12-15)
    In: npj Biofilms and Microbiomes, Springer Science and Business Media LLC, Vol. 6, No. 1 ( 2020-12-15)
    Abstract: The metagenome development of the human respiratory tract was investigated by shotgun metagenome metagenomic sequencing of cough swabs from healthy children and children with cystic fibrosis (CF) between 3 weeks and 6 years of age. A healthy microbial community signature was associated with increased absolute abundances in terms of bacterial–human cell ratios of core and rare species across all age groups, with a higher diversity of rare species and a tightly interconnected species co-occurrence network, in which individual members were found in close proximity to each other and negative correlations were absent. Even without typical CF pathogens, the CF infant co-occurrence network was found to be less stable and prone to fragmentation due to fewer connections between species, a higher number of bridging species and the presence of negative species correlations. Detection of low-abundant DNA of the CF hallmark pathogen Pseudomonas aeruginosa was neither disease- nor age-associated in our cohort. Healthy and CF children come into contact with P. aeruginosa on a regular basis and from early on.
    Type of Medium: Online Resource
    ISSN: 2055-5008
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2020
    detail.hit.zdb_id: 2817021-0
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