Figure 1.

Pathway analysis methodologies from gene expression: (A) Over-representation analysis first performs a statistical test for each gene by comparing expression values in phenotypes to identify a set of significantly DE genes, obtaining a gene count N_D. The procedure then counts the number of DE genes that are also annotated to a specified pathway (N_D,P) and calculates a P-value for enrichment of that pathway by testing if N_D,P is unusually high relative to N_D and N_P (the number of genes in the pathway). (B) Enrichment analysis first assigns an individual DE score to each of the genes annotated to a pathway, and aggregates these into a pathway score Z_P. A similar score is computed for a null distribution, Z_N. For example, this null distribution may be defined empirically from the DE score for alternative sets of genes or permuted sample labels. Enrichment analysis forms a pathway statistic by comparing the distribution of DE scores in Z_P to that of DE scores in Z_N. (C) Differential variability analysis defines a statistic to measure variability of the expression of pathway genes for samples from a given phenotype, denoted by V_P1 and V_P2 for phenotypes 1 and 2, respectively. If the variability between two phenotypes is significantly high (ie, |V_P1 − V_P2| >> 0), the pathway is identified as dysregulated.