A crucial step in the understanding of any phenotype is the correct identification of the signaling pathways that are significantly impacted in that phenotype. However, most current pathway analysis methods produce both false positives as well as false negatives in certain circumstances. We hypothesized that such incorrect results are due to the fact that the existing methods fail to distinguish between the primary dis-regulation of a given gene itself and the effects of signaling coming from upstream. Furthermore, a modern whole-genome experiment performed with a next-generation technology spends a great deal of effort to measure the entire set of 30,000-100,000 transcripts in the genome. This is followed by the selection of a few hundreds differentially expressed genes, step that literally discards more than 99% of the collected data. We also hypothesized that such a drastic filtering could discard many genes that play crucial roles in the phenotype. We propose a novel topology-based pathway analysis method that identifies significantly impacted pathways using the entire set of measurements, thus allowing the full use of the data provided by NGS techniques. The results obtained on 24 real data sets involving 12 different human diseases, as well as on 8 yeast knock-out data sets show that the proposed method yields significant improvements with respect to the state-of-the-art methods: SPIA, GSEA and GSA. AVAILABILITY: Primary dis-regulation analysis is implemented in R and included in ROntoTools Bioconductor package (versions ≥ 2.0.0). https://www.bioconductor.org/packages/release/bioc/html/ROntoTools.html.

Identifying the pathways and mechanisms that are significantly impacted in a given phenotype is challenging. Issues include patient heterogeneity and noise. Many experiments do not have a large enough sample size to achieve the statistical power necessary to identify significantly impacted pathways. Meta-analysis based on combining p-values from individual experiments has been used to improve power. However, all classical meta-analysis approaches work under the assumption that the p-values produced by experiment-level statistical tests follow a uniform distribution under the null hypothesis. Here we show that this assumption does not hold for three mainstream pathway analysis methods, and significant bias is likely to affect many, if not all such meta-analysis studies. We introduce DANUBE, a novel and unbiased approach to combine statistics computed from individual studies. Our framework uses control samples to construct empirical null distributions, from which empirical p-values of individual studies are calculated and combined using either a Central Limit Theorem approach or the additive method. We assess the performance of DANUBE using four different pathway analysis methods. DANUBE is compared with five meta-analysis approaches, as well as with a pathway analysis approach that employs multiple datasets (MetaPath). The 25 approaches have been tested on 16 different datasets related to two human diseases, Alzheimer's disease (7 datasets) and acute myeloid leukemia (9 datasets). We demonstrate that DANUBE overcomes bias in order to consistently identify relevant pathways. We also show how the framework improves results in more general cases, compared to classical meta-analysis performed with common experiment-level statistical tests such as Wilcoxon and t-test.

MOTIVATION: The accumulation of high-throughput data in public repositories creates a pressing need for integrative analysis of multiple datasets from independent experiments. However, study heterogeneity, study bias, outliers and the lack of power of available methods present real challenge in integrating genomic data. One practical drawback of many P-value-based meta-analysis methods, including Fisher's, Stouffer's, minP and maxP, is that they are sensitive to outliers. Another drawback is that, because they perform just one statistical test for each individual experiment, they may not fully exploit the potentially large number of samples within each study. RESULTS: We propose a novel bi-level meta-analysis approach that employs the additive method and the Central Limit Theorem within each individual experiment and also across multiple experiments. We prove that the bi-level framework is robust against bias, less sensitive to outliers than other methods, and more sensitive to small changes in signal. For comparative analysis, we demonstrate that the intra-experiment analysis has more power than the equivalent statistical test performed on a single large experiment. For pathway analysis, we compare the proposed framework versus classical meta-analysis approaches (Fisher's, Stouffer's and the additive method) as well as against a dedicated pathway meta-analysis package (MetaPath), using 1252 samples from 21 datasets related to three human diseases, acute myeloid leukemia (9 datasets), type II diabetes (5 datasets) and Alzheimer's disease (7 datasets). Our framework outperforms its competitors to correctly identify pathways relevant to the phenotypes. The framework is sufficiently general to be applied to any type of statistical meta-analysis. AVAILABILITY AND IMPLEMENTATION: The R scripts are available on demand from the authors. CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.