High-throughput sequencing of 16S rRNA gene amplicons : effects of extraction procedure, primer length and annealing temperature

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background: Homologous recombination mediated by the lambda-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the lambda-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these lambda-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. \ud \ud Results: Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the lambda-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6xHis, 3xFLAG, 4xProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the lambda-Red system, which can lead to unwanted secondary alterations to the chromosome. \ud \ud Conclusion: We have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in E. coli. We have demonstrated the versatility of the technique by modifying the chromosome of the enterohaemorrhagic O157:H7 (EHEC), uropathogenic CFT073 (UPEC), enteroaggregative O42 (EAEC) and enterotoxigenic H10407 (ETEC) E. coli strains as well as in K-12 laboratory strains

Guest Editorial

A novel blood test for tuberculosis prevention and treatmen

Analysis of host response to bacterial infection using error model based gene expression microarray experiments

A key step in the analysis of microarray data is the selection of genes that are differentially expressed. Ideally, such experiments should be properly replicated in order to infer both technical and biological variability, and the data should be subjected to rigorous hypothesis tests to identify the differentially expressed genes. However, in microarray experiments involving the analysis of very large numbers of biological samples, replication is not always practical. Therefore, there is a need for a method to select differentially expressed genes in a rational way from insufficiently replicated data. In this paper, we describe a simple method that uses bootstrapping to generate an error model from a replicated pilot study that can be used to identify differentially expressed genes in subsequent large-scale studies on the same platform, but in which there may be no replicated arrays. The method builds a stratified error model that includes array-to-array variability, feature-to-feature variability and the dependence of error on signal intensity. We apply this model to the characterization of the host response in a model of bacterial infection of human intestinal epithelial cells. We demonstrate the effectiveness of error model based microarray experiments and propose this as a general strategy for a microarray-based screening of large collections of biological samples

Draft Genome Sequences of Six Novel Bacterial Isolates from Chicken Ceca

The chicken is the most common domesticated animal and the most abundant bird in the world. However, the chicken gut is home to many previously uncharacterized bacterial taxa. Here, we report draft genome sequences from six bacterial isolates from chicken ceca, all of which fall outside any named species

Magnetic phase diagram of the Hubbard model

The competition between commensurate and incommensurate spin-density-wave phases in the infinite-dimensional single-band Hubbard model is examined with quantum Monte Carlo simulation and strong and weak coupling approximations. Quantum fluctuations modify the weak-coupling phase diagram by factors of order unity and produce remarkable agreement with the quantum Monte Carlo data, but strong-coupling theories (that map onto effective Falicov-Kimball models) display pathological behavior. The single-band model can be used to describe much of the experimental data in Cr and its dilute alloys with V and Mn.Comment: 12 pages plus 3 uuencoded postscript figures, ReVTe

Potential population level impact on tuberculosis incidence of using an mRNA expression signature correlate-of-risk test to target tuberculosis preventive therapy.

Achieving the WHO End-Tuberculosis (TB) targets requires approaches to prevent progression to TB among individuals with Mycobacterium tuberculosis (M.tb) infection. Effective preventive therapy (PT) exists, but current tests have low specificity for identifying who, among those infected, is at risk of developing TB. Using mathematical models, we assessed the potential population-level impact on TB incidence of using a new more specific mRNA expression signature (COR) to target PT among HIV-uninfected adults in South Africa. We compared the results to the use of the existing interferon-γ release assay (IGRA). With annual screening coverage of 30% COR-targeted PT could reduce TB incidence in 2035 by 20% (95% CI 15-27). With the same coverage, IGRA-targeted PT could reduce TB incidence by 39% (31-48) but would require greater use of PT resulting in a higher number needed to treat per TB case averted (COR: 49 (29-77); IGRA: 84 (59-123)). The relative differences between COR and IGRA were not sensitive to screening coverage. COR-targeted PT could contribute to reducing total TB burden in high incidence countries like South Africa by allowing more efficient targeting of treatment. To maximise impact, COR-like tests may be best utilised in the highest burden regions, or sub-populations, within these countries

Geographic Information System Data Analysis

Data was collected in order to further NASA Langley Research Center's Geographic Information System(GIS). Information on LaRC's communication, electrical, and facility configurations was collected. Existing data was corrected through verification, resulting in more accurate databases. In addition, Global Positioning System(GPS) points were used in order to accurately impose buildings on digitized images. Overall, this project will help the Imaging and CADD Technology Team (ICTT) prove GIS to be a valuable resource for LaRC

The ecological and geographic context of morphological and genetic divergence in an understorey-dwelling bird

Advances in understanding the process of species formation require an integrated perspective that includes the evaluation of spatial, ecological and genetic components. One approach is to focus on multiple stages of divergence within the same species. Species that comprise phenotypically different populations segregated in apparently distinct habitats, in which range is presently continuous but was putatively geographically isolated provide an interesting system to study the mechanisms of population divergence. Here, we attempt to elucidate the role of ecology and geography in explaining observed morphological and genetic variation in an understorey-dwelling bird endemic to southeastern Africa, where two subspecies are recognized according to phenotype and habitat affinity. We carried out a range-wide analysis of climatic requirements, morphological and genetic variation across southeast Africa to test the hypothesis that the extent of gene flow among populations of the brown scrub-robin are influenced by their distinct climatic niches. We recovered two distinct trends depending on whether our analyses were hierarchically structured at the subspecies or at the within subspecies level. Between subspecies we found pronounced morphological differentiation associated with strong reproductive isolation (no gene flow) between populations occupying divergent climatic niches characterized by changes in the temperature of the warmest and wettest month. In contrast, within subspecies, we recovered continuous morphological variation with extensive gene flow among populations inhabiting the temperate and sub-tropical forests of southern Africa, despite divergence along the climate axis that is mainly determined by minimum temperature and precipitation of the coldest months. Our results highlight the role of niche divergence as a diversifying force that can promote reproductive isolation in vertebrates

The Wicked Machinery of Government: Malta and the Problems of Continuity under the New Model Administration

This is a study focused on the early years of British rule in Malta (1800-1813). It explores the application to the island of the “new model” of colonial government, one based on direct rule from London mediated by the continuation of existing laws and institutions. Systemic deficiencies are identified. These tended to undermine the effectiveness of direct British rule. This study also reveals, in the context of legal and constitutional continuity, unresolved tensions between modernity and tradition. The political stability of the island was damaged and the possibility of continued British possession was threatened