A new approach for the analysis of bacterial microarray-based Comparative Genomic Hybridization: insights from an empirical study

BACKGROUND: Microarray-based Comparative Genomic Hybridization (M-CGH) has been used to characterize the extensive intraspecies genetic diversity found in bacteria at the whole-genome level. Although conventional microarray analytical procedures have proved adequate in handling M-CGH data, data interpretation using these methods is based on a continuous character model in which gene divergence and gene absence form a spectrum of decreasing gene conservation levels. However, whereas gene divergence may yet be accompanied by retention in gene function, gene absence invariably leads to loss of function. This distinction, if ignored, leads to a loss in the information to be gained from M-CGH data. We present here results from experiments in which two genome-sequenced strains of C. jejuni were compared against each other using M-CGH. Because the gene content of both strains was known a priori, we were able to closely examine the effects of sequence divergence and gene absence on M-CGH data in order to define analytical parameters for M-CGH data interpretation. This would facilitate the examination of the relative effects of sequence divergence or gene absence in comparative genomics analyses of multiple strains of any species for which genome sequence data and a DNA microarray are available. RESULTS: As a first step towards improving the analysis of M-CGH data, we estimated the degree of experimental error in a series of experiments in which identical samples were compared against each other by M-CGH. This variance estimate was used to validate a Log Ratio-based methodology for identification of outliers in M-CGH data. We compared two genome strains by M-CGH to examine the effect of probe/target identity on the Log Ratios of signal intensities using prior knowledge of gene divergence and gene absence to establish Log Ratio thresholds for the identification of absent and conserved genes. CONCLUSION: The results from this empirical study validate the Log Ratio thresholds that have been used in other studies to establish gene divergence/absence. Moreover, the analytical framework presented here enhances the information content derived from M-CGH data by shifting the focus from divergent/absent gene detection to accurate detection of conserved and absent genes. This approach closely aligns the technical limitations of M-CGH analysis with practical limitations on the biological interpretation of comparative genomics data

Comparative genomic assessment of Multi-Locus Sequence Typing: rapid accumulation of genomic heterogeneity among clonal isolates of Campylobacter jejuni

<p>Abstract</p> <p>Background</p> <p>Multi-Locus Sequence Typing (MLST) has emerged as a leading molecular typing method owing to its high ability to discriminate among bacterial isolates, the relative ease with which data acquisition and analysis can be standardized, and the high portability of the resulting sequence data. While MLST has been successfully applied to the study of the population structure for a number of different bacterial species, it has also provided compelling evidence for high rates of recombination in some species. We have analyzed a set of <it>Campylobacter jejuni </it>strains using MLST and Comparative Genomic Hybridization (CGH) on a full-genome microarray in order to determine whether recombination and high levels of genomic mosaicism adversely affect the inference of strain relationships based on the analysis of a restricted number of genetic loci.</p> <p>Results</p> <p>Our results indicate that, in general, there is significant concordance between strain relationships established by MLST and those based on shared gene content as established by CGH. While MLST has significant predictive power with respect to overall genome similarity of isolates, we also found evidence for significant differences in genomic content among strains that would otherwise appear to be highly related based on their MLST profiles.</p> <p>Conclusion</p> <p>The extensive genomic mosaicism between closely related strains has important implications in the context of establishing strain to strain relationships because it suggests that the exact gene content of strains, and by extension their phenotype, is less likely to be "predicted" based on a small number of typing loci. This in turn suggests that a greater emphasis should be placed on analyzing genes of clinical interest as we forge ahead with the next generation of molecular typing methods.</p

Comparative genomics profiling of clinical isolates of Aeromonas salmonicida using DNA microarrays

BACKGROUND: Aeromonas salmonicida has been isolated from numerous fish species and shows wide variation in virulence and pathogenicity. As part of a larger research program to identify virulence genes and candidates for vaccine development, a DNA microarray was constructed using a subset of 2024 genes from the draft genome sequence of A. salmonicida subsp. salmonicida strain A449. The microarray included genes encoding known virulence-associated factors in A. salmonicida and homologs of virulence genes of other pathogens. We used microarray-based comparative genomic hybridizations (M-CGH) to compare selected A. salmonicida sub-species and other Aeromonas species from different hosts and geographic locations. RESULTS: Results showed variable carriage of virulence-associated genes and generally increased variation in gene content across sub-species and species boundaries. The greatest variation was observed among genes associated with plasmids and transposons. There was little correlation between geographic region and degree of variation for all isolates tested. CONCLUSION: We have used the M-CGH technique to identify subsets of conserved genes from amongst this set of A. salmonicida virulence genes for further investigation as potential vaccine candidates. Unlike other bacterial characterization methods that use a small number of gene or DNA-based functions, M-CGH examines thousands of genes and/or whole genomes and thus is a more comprehensive analytical tool for veterinary or even human health research

Getting the Grip on Nonspecific Treatment Effects: Emesis in Patients Randomized to Acupuncture or Sham Compared to Patients Receiving Standard Care

Background: It is not known whether or not delivering acupuncture triggers mechanisms cited as placebo and if acupuncture or sham reduces radiotherapy-induced emesis more than standard care. Methodology/Principal Findings: Cancer patients receiving radiotherapy over abdominal/pelvic regions were randomized to verum (penetrating) acupuncture (n = 109; 99 provided data) in the alleged antiemetic acupuncture point PC6 or sham acupuncture (n = 106; 101 provided data) performed with a telescopic non-penetrating needle at a sham point 2-3 times/week during the whole radiotherapy period. The acupuncture cohort was compared to a reference cohort receiving standard care (n = 62; 62 provided data). The occurrence of emesis in each group was compared after a mean dose of 27 Gray. Nausea and vomiting were experienced during the preceding week by 37 and 8% in the verum acupuncture group, 38 and 7% in the sham acupuncture group and 63 and 15% in the standard care group, respectively. The lower occurrence of nausea in the acupuncture cohort (verum and sham) compared to patients receiving standard care (37% versus 63%, relative risk (RR) 0.6, 95 % confidence interval (CI) 0.5-0.8) was also true after adjustment for potential confounding factors for nausea (RR 0.8, CI 0.6 to 0.9). Nausea intensity was lower in the acupuncture cohort (78% no nausea, 13% a little, 8% moderate, 1% much) compared to the standard care cohort (52% no nausea, 32% a little, 15% moderate, 2% much) (p = 0.002). The acupuncture cohort expected antiemetic effects from their treatment (95%). Patients who expected nausea had increased risk for nausea compared to patients who expected low risk for nausea (RR 1.6; Cl 1.2-2.4). Conclusions/Significance: Patients treated with verum or sham acupuncture experienced less nausea and vomiting compared to patients receiving standard care, possibly through a general care effect or due to the high level of patient expectancy.Original Publication:Anna Enblom, Mats Lekander, Mats Hammar, Anna Johnsson, Erik Onelov, Martin Ingvar, Gunnar Steineck and Sussanne Börjeson, Getting the Grip on Nonspecific Treatment Effects: Emesis in Patients Randomized to Acupuncture or Sham Compared to Patients Receiving Standard Care, 2011, PLOS ONE, (6), 3, .http://dx.doi.org/10.1371/journal.pone.0014766Licensee: Public Library of Science (PLoS)http://www.plos.org