Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape.

Cytoscape is a general network visualization, data integration, and analysis software package. Its development and use has been focused on the modeling requirements of systems biology, though it has been used in other fields. Cytoscape's flexibility has encouraged many users to adopt it and adapt it to their own research by using the plugin framework offered to specialize data analysis, data integration, or visualization. Plugins represent collections of community-contributed functionality and can be used to dynamically extend Cytoscape functionality. This community of users and developers has worked together since Cytoscape's initial release to improve the basic project through contributions to the core code and public offerings of plugin modules. This chapter discusses what Cytoscape does, why it was developed, and the extensions numerous groups have made available to the public. It also describes the development of a plugin used to investigate a particular research question in systems biology and walks through an example analysis using Cytoscape.

Data availability. The WGS and RNA expression data can be found at the European Genome-phenome Archive (EGA) under accessions EGAD00001004417 and EGAD00001004423, respectively. Code availability. Code associated with the analysis is available upon request. Ethics. The study was registered (UKCRNID 8880), approved by the Institutional Ethics Committees (REC 07/H0305/52 and 10/ H0305/1), and all subjects gave individual informed consent. Reporting summary. Additional information is included in the Life Sciences Reporting Summary, which details exact software and biological materials used and efforts made to ensure reproducibility of results. Author contributions RCF and AMF conceived the overall study. AMF and SJ analyzed the genomic data and performed statistical analyses. RCF, AMF and XL designed the experiments. AMF, XL and JM performed the experiments. GC contributed to the structural variant analysis and data visualization. SK helped compile the clinical data and aided statistical analyses. JP and SA produced and QC'ed the RNA-seq data. EO aided the whole genome sequencing of EAC cell lines. SM and NG coordinated the clinical centres and were responsible for sample collections. ME benchmarked our mutation calling pipelines. MO led the pathological sample QC for sequencing. LB and GD constructed and managed the sequencing alignment and variant calling pipelines. RCF and ST supervised the research. RCF and ST obtained funding. AMF and RCF wrote the manuscript. All authors approved the manuscript.