Project description:BACKGROUND: Many microarray experiments search for genes with differential expression between a common "reference" group and multiple "test" groups. In such cases currently employed statistical approaches based on t-tests or close derivatives have limited efficacy, mainly because estimation of the standard error is done on only two groups at a time. Alternative approaches based on ANOVA correctly capture within-group variance from all the groups, but then do not confront single test groups with the reference. Ideally, a t-test better suited for this type of data would compare each test group with the reference, but use within-group variance calculated from all the groups. RESULTS: We implemented an R-Bioconductor package named Mulcom, with a statistical test derived from the Dunnett's t-test, designed to compare multiple test groups individually against a common reference. Interestingly, the Dunnett's test uses for the denominator of each comparison a within-group standard error aggregated from all the experimental groups. In addition to the basic Dunnett's t value, the package includes an optional minimal fold-change threshold, m. Due to the automated, permutation-based estimation of False Discovery Rate (FDR), the package also permits fast optimization of the test, to obtain the maximum number of significant genes at a given FDR value. When applied to a time-course experiment profiled in parallel on two microarray platforms, and compared with two commonly used tests, Mulcom displayed better concordance of significant genes in the two array platforms (39% vs. 26% or 15%), and higher enrichment in functional annotation to categories related to the biology of the experiment (p value < 0.001 in 4 categories vs. 3). CONCLUSIONS: The Mulcom package provides a powerful tool for the identification of differentially expressed genes when several experimental conditions are compared against a common reference. The results of the practical example presented here show that lists of differentially expressed genes generated by Mulcom are particularly consistent across microarray platforms and enriched in genes belonging to functionally significant groups.

Project description:Background Many microarray experiments search for genes with differential expression between a common “reference” group and multiple test groups, like in the case of time-course designs or of various treatments versus a control condition. In such cases, currently employed statistical approaches based on t-test or close derivatives have limited efficacy, mostly because estimation of noise is done on only two groups at time. Alternative approaches based on ANOVA correctly capture noise from all the groups, but then do not confront single test groups with the reference. We therefore conceived a statistical test for pairwise comparisons between the reference group and each test group that uses within-group variance calculated from all the groups. Results We implemented an R-Bioconductor package named Mulcom, with a statistical test derived from the Dunnett’s test, designed to compare multiple experimental groups against a common reference. In addition to the basic Dunnett’s t value, the package includes an optional minimal fold-change threshold, m. Thanks to automated, permutation-based estimation of False Discovery Rate (FDR), the package also permits fast optimization of the test, to obtain the maximum number of significant genes at a given FDR value. When applied on a time-course experiment profiled in parallel on two microarray platforms, and compared with currently used tests, Mulcom displayed higher concordance of significant genes in the two array platforms, and higher enrichment in functional annotation to categories related to the biology of the experiment. Conclusions The Mulcom package provides a fast and powerful tool for the identification of differentially expressed genes when several experimental conditions are compared with a common reference. We found that Mulcom leads to lists of differentially expressed genes that are particularly consistent across microarray platforms and enriched in significant classes of genes. In our opinion, the main reasons for these good performances are three: (i) within-group variability is estimated from all experimental groups even if only two of them are compared each time; (ii) the optional fold-change threshold m avoids false positives due to aberrantly low within-group variability; (iii) automated test optimization allows maximizing sensitivity without compromising specificity.

Project description:Background Many microarray experiments search for genes with differential expression between a common “reference” group and multiple test groups, like in the case of time-course designs or of various treatments versus a control condition. In such cases, currently employed statistical approaches based on t-test or close derivatives have limited efficacy, mostly because estimation of noise is done on only two groups at time. Alternative approaches based on ANOVA correctly capture noise from all the groups, but then do not confront single test groups with the reference. We therefore conceived a statistical test for pairwise comparisons between the reference group and each test group that uses within-group variance calculated from all the groups. Results We implemented an R-Bioconductor package named Mulcom, with a statistical test derived from the Dunnett’s test, designed to compare multiple experimental groups against a common reference. In addition to the basic Dunnett’s t value, the package includes an optional minimal fold-change threshold, m. Thanks to automated, permutation-based estimation of False Discovery Rate (FDR), the package also permits fast optimization of the test, to obtain the maximum number of significant genes at a given FDR value. When applied on a time-course experiment profiled in parallel on two microarray platforms, and compared with currently used tests, Mulcom displayed higher concordance of significant genes in the two array platforms, and higher enrichment in functional annotation to categories related to the biology of the experiment. Conclusions The Mulcom package provides a fast and powerful tool for the identification of differentially expressed genes when several experimental conditions are compared with a common reference. We found that Mulcom leads to lists of differentially expressed genes that are particularly consistent across microarray platforms and enriched in significant classes of genes. In our opinion, the main reasons for these good performances are three: (i) within-group variability is estimated from all experimental groups even if only two of them are compared each time; (ii) the optional fold-change threshold m avoids false positives due to aberrantly low within-group variability; (iii) automated test optimization allows maximizing sensitivity without compromising specificity. Ten MDA-MB-435 samples, biological duplicates of each condition (untreated, integrin Beta4 treatment, hepatocyte growth factor treatment for 1 hr, 6 hrs, or 24 hrs).

Project description:BackgroundThis paper describes the results of a Gene Ontology (GO) term enrichment analysis of chicken microarray data using the Bioconductor packages. By checking the enriched GO terms in three contrasts, MM8-PM8, MM8-MA8, and MM8-MM24, of the provided microarray data during this workshop, this analysis aimed to investigate the host reactions in chickens occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria. The results of GO enrichment analysis using GO terms annotated to chicken genes and GO terms annotated to chicken-human orthologous genes were also compared. Furthermore, a locally adaptive statistical procedure (LAP) was performed to test differentially expressed chromosomal regions, rather than individual genes, in the chicken genome after Eimeria challenge.ResultsGO enrichment analysis identified significant (raw p-value < 0.05) GO terms for all three contrasts included in the analysis. Some of the GO terms linked to, generally, primary immune responses or secondary immune responses indicating the GO enrichment analysis is a useful approach to analyze microarray data. The comparisons of GO enrichment results using chicken gene information and chicken-human orthologous gene information showed more refined GO terms related to immune responses when using chicken-human orthologous gene information, this suggests that using chicken-human orthologous gene information has higher power to detect significant GO terms with more refined functionality. Furthermore, three chromosome regions were identified to be significantly up-regulated in contrast MM8-PM8 (q-value < 0.01).ConclusionOverall, this paper describes a practical approach to analyze microarray data in farm animals where the genome information is still incomplete. For farm animals, such as chicken, with currently limited gene annotation, borrowing gene annotation information from orthologous genes in well-annotated species, such as human, will help improve the pathway analysis results substantially. Furthermore, LAP analysis approach is a relatively new and very useful way to be applied in microarray analysis.

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Mulcom: a multiple comparison statistical test for microarray data in Bioconductor

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