Gene coexpression analysis constitutes a widely used practice for gene partner identification and gene function prediction, consisting of many intricate procedures. The analysis begins with the collection of primary transcriptomic data and their preprocessing, continues with the calculation of the similarity between genes based on their expression values in the selected sample dataset and results in the construction and visualisation of a gene coexpression network (GCN) and its evaluation using biological term enrichment analysis. As gene coexpression analysis has been studied extensively, we present most parts of the methodology in a clear manner and the reasoning behind the selection of some of the techniques. In this review, we offer a comprehensive and comprehensible account of the steps required for performing a complete gene coexpression analysis in eukaryotic organisms. We comment on the use of RNA-Seq vs. microarrays, as well as the best practices for GCN construction. Furthermore, we recount the most popular webtools and standalone applications performing gene coexpression analysis, with details on their methods, features and outputs.

Background/Objectives: Genes with similar expression patterns across multiple samples are considered coexpressed, and they may participate in similar biological processes or pathways. Gene coexpression networks depict the degree of similarity between the expression profiles of all genes in a set of samples. Gene coexpression tools allow for the prediction of functional gene partners or the assignment of roles to genes of unknown function. Weighted Gene Correlation Network Analysis (WGCNA) is an R package that provides a multitude of functions for constructing and analyzing a weighted or unweighted gene coexpression network. Methods: Previously preprocessed, high-quality gene expression data of 3500 samples of Affymetrix microarray technology from various tissues of the Arabidopsis thaliana plant model species were used to construct a weighted gene coexpression network, using WGCNA. Results: The gene dendrogram was used as the basis for the creation of a new Arabidopsis coexpression tool (ACT) version (ACT2.6). The dendrogram contains 21,273 leaves, each one corresponding to a single gene. Genes that are clustered in the same clade are coexpressed. WGCNA grouped the genes into 27 functional modules, all of which were positively or negatively correlated with specific tissues. Discussion: Genes known to be involved in common metabolic pathways were discovered in the same module. By comparing the current ACT version with the previous one, it was shown that the new version outperforms the old one in discovering the functional connections between gene partners. ACT2.6 is a major upgrade over the previous version and a significant addition to the collection of public gene coexpression tools.