Bacterial 16S rDNA gene fragement
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PROVIDER: PRJNA358454 | ENA |
REPOSITORIES: ENA
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Project description:The MinION sequencer has made in situ sequencing feasible in remote locations. Following our initial demonstration of its high performance off planet with Earth-prepared samples, we developed and tested an end-to-end, sample-to-sequencer process that could be conducted entirely aboard the International Space Station (ISS). Initial experiments demonstrated the process with a microbial mock community standard. The DNA was successfully amplified, primers were degraded, and libraries prepared and sequenced. The median percent identities for both datasets were 84%, as assessed from alignment of the mock community. The ability to correctly identify the organisms in the mock community standard was comparable for the sequencing data obtained in flight and on the ground. To validate the process on microbes collected from and cultured aboard the ISS, bacterial cells were selected from a NASA Environmental Health Systems Surface Sample Kit contact slide. The locations of bacterial colonies chosen for identification were labeled, and a small number of cells were directly added as input into the sequencing workflow. Prepared DNA was sequenced, and the data were downlinked to Earth. Return of the contact slide to the ground allowed for standard laboratory processing for bacterial identification. The identifications obtained aboard the ISS, Staphylococcus hominis and Staphylococcus capitis, matched those determined on the ground down to the species level. This marks the first ever identification of microbes entirely off Earth, and this validated process could be used for in-flight microbial identification, diagnosis of infectious disease in a crewmember, and as a research platform for investigators around the world.
| S-EPMC7016637 | biostudies-literature
Project description:Repeated quantitative measurement of bacterial DNA on whole blood has been shown to be a promising method for monitoring bloodstream infection (BSI) with selected bacterial species. To enable broad use of this method, we developed a quantitative droplet digital PCR (ddPCR) method for 16S rDNA. It was validated with species-specific ddPCRs for Staphylococcus aureus (nuc), Streptococcus pneumoniae (lytA), and Escherichia coli (uidA) on spiked whole blood samples and on repeated whole blood samples (days 0, 1-2, 3-4, 6-8, and 13-15) from 83 patients with BSI with these pathogens. In these patients, 16S rDNA and species-specific DNA were detected in 60% and 61%, respectively, at least at one time-point. The highest positivity rates were seen in S. aureus BSI, where 92% of the patients were 16S rDNA-positive and 85% nuc-positive. Quantitative 16S rDNA and species-specific DNA showed strong correlations in spiked samples (r = 0.98; p < 0.0001) and clinical samples (r = 0.84; p < 0.0001). Positivity for 16S rDNA was rapidly cleared in patients with S. pneumoniae and E. coli BSI, but more slowly and sometimes persisted, in those with S. aureus BSI. The initial 16S rDNA load was higher in BSI patients with sepsis (Sepsis-3 definition) than without sepsis (median 2.38 vs. 0 lg10 copies/mL; p = 0.031) and in non-survivors than in survivors (median 2.83 vs. 0 lg10 copies/mL; p = 0.006). 16S rDNA ddPCR appears to be a promising method for bacterial DNA monitoring during BSI. The clinical value of such monitoring should be further studied.
| S-EPMC6853374 | biostudies-literature
Project description:Soil contamination with heavy metals is a widespread problem, especially prominent on grounds lying in the vicinity of mines, smelters, and other industrial facilities. Many such areas are located in Southern Poland; they are polluted mainly with Pb, Zn, Cd, or Cu, and locally also with Cr. As for now, little is known about most bacterial species thriving in such soils and even less about a core bacterial community--a set of taxa common to polluted soils. Therefore, we wanted to answer the question if such a set could be found in samples differing physicochemically and phytosociologically. To answer the question, we analyzed bacterial communities in three soil samples contaminated with Pb and Zn and two contaminated with Cr and lower levels of Pb and Zn. The communities were assessed with 16S rRNA gene fragments pyrosequencing. It was found that the samples differed significantly and Zn decreased both diversity and species richness at species and family levels, while plant species richness did not correlate with bacterial diversity. In spite of the differences between the samples, they shared many operational taxonomic units (OTUs) and it was possible to delineate the core microbiome of our sample set. The core set of OTUs comprised members of such taxa as Sphingomonas, Candidatus Solibacter, or Flexibacter showing that particular genera might be shared among sites ~40 km distant.
| S-EPMC3962847 | biostudies-literature
Project description:Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods.
| S-EPMC4286631 | biostudies-literature
Project description:The bacterial communities present in smokeless tobacco (ST) products have not previously reported. In this study, we used Next Generation Sequencing to study the bacteria present in U.S.-made dry snuff, moist snuff and Sudanese toombak. Sample diversity and taxonomic abundances were investigated in these products. A total of 33 bacterial families from four phyla, Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes, were identified. U.S.-produced dry snuff products contained a diverse distribution of all four phyla. Moist snuff products were dominated by Firmicutes. Toombak samples contained mainly Actinobacteria and Firmicutes (Aerococcaceae, Enterococcaceae, and Staphylococcaceae). The program PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to impute the prevalence of genes encoding selected bacterial toxins, antibiotic resistance genes and other pro-inflammatory molecules. PICRUSt also predicted the presence of specific nitrate reductase genes, whose products can contribute to the formation of carcinogenic nitrosamines. Characterization of microbial community abundances and their associated genomes gives us an indication of the presence or absence of pathways of interest and can be used as a foundation for further investigation into the unique microbiological and chemical environments of smokeless tobacco products.
| S-EPMC4718623 | biostudies-literature
Project description:The leading cause of morbidity and mortality in cystic fibrosis (CF) patients stems from repeated bacterial respiratory infections. Many bacterial species have been cultured from CF specimens and so are associated with lung disease. Despite this, much remains to be determined. In the present study, we characterized without prior cultivation the total bacterial community present in specimens taken from adult CF patients, extracting DNA directly from 14 bronchoscopy or sputum samples. Bacterial 16S ribosomal DNA (rRNA) gene PCR products were amplified from extracted nucleic acids, with analyses by terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), and sequencing of individual cloned PCR products to characterize these communities. Using the same loading of PCR products, 12 distinct T-RFLP profiles were identified that had between 3 and 32 T-RFLP bands. Nine distinct LH-PCR profiles were identified containing between one and four bands. T-RFLP bands were detected in certain samples at positions that corresponded to pathogens cultured from CF samples, e.g., Burkholderia cepacia and Haemophilus influenzae. In every sample studied, one T-RFLP band was identified that corresponded to that produced by Pseudomonas aeruginosa. A total of 103 16S rRNA gene clones were examined from five patients. P. aeruginosa was the most commonly identified species (59% of clones). Stenotrophomonas species were also common, with eight other (typically anaerobic) bacterial species identified within the remaining 17 clones. In conclusion, T-RFLP analysis coupled with 16S rRNA gene sequencing is a powerful means of analyzing the composition and diversity of the bacterial community in specimens sampled from CF patients.
| S-EPMC179861 | biostudies-literature
Project description:hyperaccumulator rhizobiome
| PRJEB40196 | ENA
Project description:BackgroundThe high prevalence of suspected early-onset neonatal sepsis among preterm infants leads to immediate antibiotic administration upon admission. Notably, most blood cultures for suspected early-onset neonatal sepsis do not yield a causative pathogen. This study aimed to assess polymerase chain reaction (PCR) targeting the variable region V4 of the 16S ribosomal gene (16S rDNA) and Sanger sequencing for bacterial identification in preterm infants with suspected early-onset neonatal sepsis.MethodsTherefore, this prospective study was conducted. Preterm infants with suspected early-onset neonatal sepsis were included in this study. The three groups were formed based on the risk of infection and clinical sepsis. Blood samples were collected upon admission to the neonatal unit for culture and molecular analysis. PCR amplification and subsequent Sanger sequencing of the V4 region of the 16S rDNA were performed.ResultsTwenty-eight patients were included in this study. Blood cultures were negative in 100% of the patients. Amplification and sequencing of the V4 region identified bacterial genera in 19 patients across distinct groups. The predominant taxonomically identified genus was Pseudomonas.ConclusionsAmplifying the 16S rDNA variable region through PCR and subsequent Sanger sequencing in preterm neonates with suspected early-onset neonatal sepsis can enhance the identification of microbial species that cause infection, especially in negative cultures.
| S-EPMC11281265 | biostudies-literature
Project description:Traceability of seafood has become crucial with market globalization and consumer's awareness. The present study used PCR-DGGE and 454 pyrosequencing to assess if bacterial communities fingerprint associated to seabass (Dicentrarchus labrax) skin mucus can be used to discriminate the geographic origin of fishes cultured in three semi-intensive fish farms. PCR-DGGE and pyrosequencing results were congruent and suggested that this molecular approach has the potential to trace fish farms with a spatial resolution <500 m. Pyrosequencing results provided a detailed insight into the bacterial community composition of seabass skin mucus and revealed the existence of a core of bacterial communities within family Pseudomonadaceae and Rhodobacteraceae. This approach also allowed to recognized key OTUs that are potentially relevant to discriminate the geographic origin of the fish being surveyed. Overall, the present study increased our knowledge on farmed seabass microbiome and demonstrated that specific and unique bacterial taxa can act as natural signatures that allow us to trace fish to its respective geographic origin. Our study provides valuable clues that should be more investigated in future studies as a way to fulfill current traceability needs in the global trade of seafood.
| S-EPMC5605529 | biostudies-literature
Project description:BackgroundNext generation sequencing (NGS) enables a more comprehensive analysis of bacterial diversity from complex environmental samples. NGS data can be analysed using a variety of workflows. We test several simple and complex workflows, including frequently used as well as recently published tools, and report on their respective accuracy and efficiency under various conditions covering different sequence lengths, number of sequences and real world experimental data from rhizobacterial populations of glyphosate-tolerant maize treated or untreated with two different herbicides representative of differential diversity studies.ResultsAlignment and distance calculations affect OTU estimations, and multiple sequence alignment exerts a major impact on the computational time needed. Generally speaking, most of the analyses produced consistent results that may be used to assess differential diversity changes, however, dataset characteristics dictate which workflow should be preferred in each case.ConclusionsWhen estimating bacterial diversity, ESPRIT as well as the web-based workflow, RDP pyrosequencing pipeline, produced good results in all circumstances, however, its computational requirements can make method-combination workflows more attractive, depending on sequence variability, number and length.
| S-EPMC3258296 | biostudies-literature