Identifying Significantly Impacted Pathways and Putative Mechanisms with iPathwayGuide.

iPathwayGuide is a gene expression analysis tool that provides biological context and inferences from data generated by high-throughput sequencing. iPathwayGuide utilizes a systems biology approach to identify significantly impacted signaling pathways, Gene Ontology terms, disease processes, predicted microRNAs, and putative mechanisms based on the given differential expression signature. By using a novel analytical approach called Impact Analysis, iPathwayGuide considers the role, position, and relationships of each gene within a pathway, which results in a significant reduction in false positives, as well as a better ability to identify the truly impacted pathways and putative mechanisms that can explain all measured gene expression changes. It is a Web-based, user-friendly, interactive tool that does not require prior training in bioinformatics. The protocols in this unit describe how to use iPathwayGuide to analyze a single contrast between two phenotypes (any number of samples), and provide guidance on how to interpret the results obtained from iPathwayGuide. Even though iPathwayGuide has powerful meta-analysis capabilities, these are not covered in this unit. © 2017 by John Wiley & Sons, Inc.