Effects of vertebrate-associated microbiota on physiology and health are of significant interest in current biological research. Most previous studies have focused on host-microbiota interactions in captive-bred mammalian models. These interactions and their outcomes are still relatively understudied, however, in wild populations and non-mammalian taxa. Using deep pyrosequencing, we described the cloacal microbiome (CM) composition in free living barn swallows Hirundo rustica, a long-distance migratory passerine bird. Barn swallow CM was dominated by bacteria of the Actinobacteria, Proteobacteria and Firmicutes phyla. Bacteroidetes, which represent an important proportion of the digestive tract microbiome in many vertebrate species, was relatively rare in barn swallow CM (< 5%). CM composition did not differ between males and females. A significant correlation of CM within breeding pair members is consistent with the hypothesis that cloacal contact during within-pair copulation may promote transfer of bacterial assemblages. This effect on CM composition had a relatively low effect size, however, possibly due to the species' high level of sexual promiscuity.

• MeSH
• bakteriální RNA MeSH
• biodiverzita MeSH
• chov MeSH
• kloaka mikrobiologie   MeSH
• metagenom MeSH
• migrace zvířat * MeSH
• mikrobiota * MeSH
• ptáci mikrobiologie   MeSH
• RNA ribozomální 16S genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Bacterial and fungal biodiversity throughout different biostimulation and bioaugmentation treatments applied to an industrial creosote-polluted soil were analyzed by means of polyphasic approach in order to gain insight into the microbial community structure and dynamics. Pyrosequencing data obtained from initial creosote polluted soil (after a biopiling step) revealed that Alpha and Gammaproteobacteria were the most abundant bacterial groups, whereas Fusarium and Scedosporium were the main fungal genera in the contaminated soil. At the end of 60-days laboratory scale bioremediation assays, pyrosequencing and DGGE data showed that (i) major bacterial community shifts were caused by the type of mobilizing agent added to the soil and, to a lesser extent, by the addition of lignocellulosic substrate; and (ii) the presence of the non-ionic surfactant (Brij 30) hampered the proliferation of Actinobacteria (Mycobacteriaceae) and Bacteroidetes (Chitinophagaceae) and, in the absence of lignocellulosic substrate, also impeded polycyclic aromatic hydrocarbons (PAHs) degradation. The results show the importance of implementing bioremediation experiments combined with microbiome assessment to gain insight on the effect of crucial parameters (e.g. use of additives) over the potential functions of complex microbial communities harbored in polluted soils, essential for bioremediation success.

• MeSH
• Bacteria klasifikace   MeSH
• biodegradace MeSH
• biodiverzita MeSH
• denaturační gradientová gelová elektroforéza MeSH
• houby klasifikace   MeSH
• kreosot analýza   MeSH
• látky znečišťující půdu analýza   MeSH
• mezerníky ribozomální DNA genetika   MeSH
• polycyklické aromatické uhlovodíky analýza   MeSH
• povrchově aktivní látky chemie   MeSH
• průmysl MeSH
• půda chemie   MeSH
• půdní mikrobiologie * MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Current technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors. Although pyro- and ion semiconductor sequencing both have the advantage of delivering long and high quality reads, problems might occur when sequencing homopolymer-containing regions, since the repeating identical bases are going to incorporate during the same synthesis cycle, which leads to uncertainty in base calling. The aim of this study was to evaluate the analytical performance of a pyrosequencing-based next-generation sequencing system in detecting homopolymer sequences using homopolymer-preintegrated plasmid constructs and human DNA samples originating from patients with cystic fibrosis. RESULTS: In the plasmid system average correct genotyping was 95.8% in 4-mers, 87.4% in 5-mers and 72.1% in 6-mers. Despite the experienced low genotyping accuracy in 5- and 6-mers, it was possible to generate amplicons with more than a 90% adequate detection rate in every homopolymer tract. When homopolymers in the CFTR gene were sequenced average accuracy was 89.3%, but varied in a wide range (52.2 - 99.1%). In all but one case, an optimal amplicon-sequencing primer combination could be identified. In that single case (7A tract in exon 14 (c.2046_2052)), none of the tested primer sets produced the required analytical performance. CONCLUSIONS: Our results show that pyrosequencing is the most reliable in case of 4-mers and as homopolymer length gradually increases, accuracy deteriorates. With careful primer selection, the NGS system was able to correctly genotype all but one of the homopolymers in the CFTR gene. In conclusion, we configured a plasmid test system that can be used to assess genotyping accuracy of NGS devices and developed an accurate NGS assay for the molecular diagnosis of CF using self-designed primers for amplification and sequencing.

• MeSH
• cystická fibróza genetika   MeSH
• lidé MeSH
• plazmidy MeSH
• protein CFTR genetika   MeSH
• sekvenční analýza DNA metody   MeSH
• tandemové repetitivní sekvence * MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH

BACKGROUND: It is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters. METHODS: We compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits. RESULTS AND CONCLUSIONS: Our results demonstrate that TheraScreen DxS and the StripAssay, in that order, were most effective at diagnosing mutations in KRAS. However, there were still unsatisfactory disagreements between them for 6.1% of all samples tested. Despite this, our findings are likely to assist molecular biologists in making rational decisions when selecting a reliable, efficient, and cost-effective method for detecting KRAS mutations in heterogeneous clinical tumor samples.

• MeSH
• analýza nákladů a výnosů MeSH
• DNA nádorová analýza   genetika   MeSH
• kolorektální nádory * genetika   sekundární   MeSH
• lidé MeSH
• mutace MeSH
• nemalobuněčný karcinom plic genetika   MeSH
• protoonkogenní proteiny genetika   MeSH
• ras proteiny genetika   MeSH
• sekvenční analýza DNA * ekonomika   metody   MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• financování organizované MeSH
• Publikační typ
• abstrakty MeSH

The molecular mechanisms underlying fluconazole resistance in C. albicans involve mutations and the overexpression of the ERG11 gene and membrane transport proteins. We examined the relationship between the reduced fluconazole susceptibility of C. albicans and mutations of V404I and V509M in the ERG11 gene in 182 C. albicans clinical isolates using the Pyrosequencing method. DNAs from these clinical isolates with different levels of in-vitro fluconazole susceptibility--one resistant, five susceptible dose-dependent (SDD), four trailer, and 172 susceptible--were analyzed. None of the fluconazole-susceptible, SDD, trailer or resistant isolates had mutations of V404I or V509M. Our results showed that no correlation can be found between the V404I or V509M mutation and fluconazole susceptibility in C. albicans.

• MeSH
• antifungální látky farmakologie   MeSH
• Candida albicans enzymologie   genetika   účinky léků   MeSH
• DNA fungální genetika   chemie   MeSH
• financování organizované MeSH
• flukonazol farmakologie   MeSH
• fungální léková rezistence MeSH
• fungální proteiny genetika   metabolismus   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• substituce aminokyselin genetika   MeSH
• systém (enzymů) cytochromů P-450 genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH

Symbioses are increasingly seen as dynamic ecosystems with multiple associates and varying fidelity. Symbiont specificity remains elusive in one of the most ecologically successful and economically damaging eukaryotic symbioses: the ambrosia symbiosis of wood-boring beetles and fungi. We used multiplexed pyrosequencing of amplified internal transcribed spacer II (ITS2) ribosomal DNA (rDNA) libraries to document the communities of fungal associates and symbionts inside the mycangia (fungus transfer organ) of three ambrosia beetle species, Xyleborus affinis, Xyleborus ferrugineus and Xylosandrus crassiusculus. We processed 93 beetle samples from 5 locations across Florida, including reference communities. Fungal communities within mycangia included 14-20 fungus species, many more than reported by culture-based studies. We recovered previously known nutritional symbionts as members of the core community. We also detected several other fungal taxa that are equally frequent but whose function is unknown and many other transient species. The composition of fungal assemblages was significantly correlated with beetle species but not with locality. The type of mycangium appears to determine specificity: two Xyleborus with mandibular mycangia had multiple dominant associates with even abundances; Xylosandrus crassiusculus (mesonotal mycangium) communities were dominated by a single symbiont, Ambrosiella sp. Beetle mycangia also carried many fungi from the environment, including plant pathogens and endophytes. The ITS2 marker proved useful for ecological analyses, but the taxonomic resolution was limited to fungal genus or family, particularly in Ophiostomatales, which are under-represented in our amplicons as well as in public databases. This initial analysis of three beetle species suggests that each clade of ambrosia beetles and each mycangium type may support a functionally and taxonomically distinct symbiosis.

• MeSH
• biodiverzita MeSH
• brouci mikrobiologie   MeSH
• DNA fungální genetika   izolace a purifikace   MeSH
• DNA primery MeSH
• genetické markery genetika   MeSH
• houby klasifikace   genetika   MeSH
• mezerníky ribozomální DNA genetika   MeSH
• polymerázová řetězová reakce MeSH
• symbióza * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Geografické názvy
• Florida MeSH

Brown mud, as a waste product of the industrial process of aluminum production, represents a great environmental burden due to its toxicity to living organisms. However, some microorganisms are able to survive in this habitat, and they can be used in bioremediation processes. Traditional cultivation methods have a limited capacity to characterize bacterial composition in environmental samples. Recently, next-generation sequencing methods have provided new perspectives on microbial community studies. The aim of this study was to analyze the bacterial community in the drainage water of brown mud disposal site near Žiar nad Hronom (Banská Bystrica region, Slovakia) using 454 pyrosequencing. We obtained 9964 sequences assigned to 163 operational taxonomic units belonging to 10 bacterial phyla. The phylum Proteobacteria showed the highest abundance (80.39%) within the bacterial community, followed by Firmicutes (13.05%) and Bacteroidetes (5.64%). Other bacterial phyla showed an abundance lower than 1%. The classification yielded 85 genera. Sulfurospirillum spp. (45.19%) dominated the bacterial population, followed by Pseudomonas spp. (13.76%) and Exiguobacterium spp. (13.02%). These results indicate that high heavy metals content, high pH, and lack of essential nutrients are the drivers of a dramatic reduction of diversity in the bacterial population in this environment.

• MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   MeSH
• biodegradace MeSH
• biodiverzita * MeSH
• chemické látky znečišťující vodu analýza   metabolismus   MeSH
• fylogeneze MeSH
• fyziologie bakterií * MeSH
• hutnictví MeSH
• koncentrace vodíkových iontů MeSH
• mikrobiální společenstva genetika   MeSH
• odpadní voda chemie   mikrobiologie   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• těžké kovy analýza   metabolismus   MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Slovenská republika MeSH

Genes of the highly dynamic major histocompatibility complex (MHC) are directly linked to individual fitness and are of high interest in evolutionary ecology and conservation genetics. Gene duplication and positive selection usually lead to high levels of polymorphism in the MHC region, making genotyping of MHC a challenging task. Here, we compare the performance of two methods for MHC class I genotyping in a passerine with highly duplicated MHC class I genes: capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis and 454 GS FLX Titanium pyrosequencing. According to our findings, the number of MHC variants (called alleles for simplicity) detected by CE-SSCP is significantly lower than detected by 454. To resolve discrepancies between the two methods, we cloned and Sanger sequenced a MHC class I amplicon for an individual with high number of alleles. We found a perfect congruence between cloning/Sanger sequencing results and 454. Thus, in case of multi-locus amplification, CE-SSCP considerably underestimates individual MHC diversity. However, numbers of alleles detected by both methods are significantly correlated, although the correlation is weak (r = 0.32). Thus, in systems with highly duplicated MHC, 454 provides more reliable information on individual diversity than CE-SSCP.

• MeSH
• elektroforéza kapilární metody   MeSH
• fylogeneze MeSH
• genotyp MeSH
• geny MHC třídy II MeSH
• molekulární sekvence - údaje MeSH
• Passeriformes klasifikace   genetika   MeSH
• polymorfismus konformace jednovláknové DNA MeSH
• ptačí proteiny genetika   MeSH
• sekvenční analýza DNA metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

The effects of gastrointestinal tract microbiota (GTM) on host physiology and health have been the subject of considerable interest in recent years. While a variety of captive bred species have been used in experiments, the extent to which GTM of captive and/or inbred individuals resembles natural composition and variation in wild populations is poorly understood. Using 454 pyrosequencing, we performed 16S rDNA GTM barcoding for 30 wild house mice (Mus musculus) and wild-derived inbred strain mice belonging to two subspecies (M. m. musculus and M. m. domesticus). Sequenced individuals were selected according to a 2 × 2 experimental design: wild (14) vs. inbred origin (16) and M. m. musculus (15) vs. M. m. domesticus (15). We compared alpha diversity (i.e. number of operational taxonomic units - OTUs), beta diversity (i.e. interindividual variability) and microbiota composition across the four groups. We found no difference between M. m. musculus and M. m. domesticus subspecies, suggesting low effect of genetic differentiation between these two subspecies on GTM structure. Both inbred and wild populations showed the same level of microbial alpha and beta diversity; however, we found strong differentiation in microbiota composition between wild and inbred populations. Relative abundance of ~ 16% of OTUs differed significantly between wild and inbred individuals. As laboratory mice represent the most abundant model for studying the effects of gut microbiota on host metabolism, immunity and neurology, we suggest that the distinctness of laboratory-kept mouse microbiota, which differs from wild mouse microbiota, needs to be considered in future biomedical research.

• MeSH
• Bacteria klasifikace   MeSH
• divoká zvířata mikrobiologie   MeSH
• gastrointestinální trakt mikrobiologie   MeSH
• genetická variace * MeSH
• inbrední kmeny myší mikrobiologie   MeSH
• metagenom MeSH
• mikrobiota genetika   MeSH
• myši MeSH
• RNA ribozomální 16S genetika   MeSH
• taxonomické DNA čárové kódování MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH