Visual annotation display (VLAD): a tool for finding functional themes in lists of genes

Mamm Genome (2015) 26:567–573 DOI 10.1007/s00335-015-9570-2 Visual annotation display (VLAD): a tool for finding functional themes in lists of genes Joel E. Richardson1 • Carol J. Bult1 Received: 6 March 2015 / Accepted: 19 May 2015 / Published online: 6 June 2015  The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Experiments that employ genome scale technology platforms frequently result in lists of tens to thousands of genes with potential significance to a specific biological process or disease. Searching for biologically relevant connections among the genes or gene products in these lists is a common data analysis task. We have implemented a software application for uncovering functional themes in sets of genes based on their annotations to bio-ontologies, such as the gene ontology and the mammalian phenotype ontology. The application, called VisuaL Annotation Display (VLAD), performs a statistical analysis to test for the enrichment of ontology terms in a set of genes submitted by a researcher. The results for each analysis using VLAD includes a table of ontology terms, sorted in decreasing order of significance. Each row contains the term, statistics such as the number of annotated terms, the p value, etc., and the symbols of annotated genes. An accompanying graphical display shows portions of the ontology hierarchy, where node sizes are scaled based on p values. Although numerous ontology term enrichment programs already exist, VLAD is unique in that it allows users to upload their own annotation files and ontologies for customized term enrichment analyses, Electronic supplementary material The online version of this article (doi:10.1007/s00335-015-9570-2) contains supplementary material, which is available to authorized users. & Carol J. Bult Joel E. Richardson 1 Mouse Genome Informatics (MGI) Database Consortium, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA supports the analysis of multiple gene sets at once, provides interfaces to customize graphical output, and is tightly integrated with functional and biological details about mouse genes in the Mouse Genome Informatics (MGI) database. VLAD is available as a web-based application from the MGI web site (http://proto.informatics.jax.org/ prototypes/vlad/). Introduction One of the challenges facing biologists in the era of genome scale science is to glean biological meaning from large experimental datasets such as those generated by microarray, RNA Seq, ChIP (chromatin immunoprecipitation) Seq, genome wide copy number variation (CNV) analysis, and exome sequencing. The development of biomedical ontologies such as the Gene Ontology (GO) (Ashburner et al. 2000; Gene Ontology 2015) and annotated gene sets (Subramanian et al. 2005) have been essential for mining functional properties of genes from large-scale datasets. Numerous software tools that use curated annotations and ontologies for extracting functional information from gene sets have been developed over the years including GO::TermFinder (Boyle et al. 2004), DAVID (da Huang et al. 2009), BiNGO (Maere et al. 2005), AmiGO (Carbon et al. 2009), GoMiner (Zeeberg et al. 2003), and WebGestalt (Wang et al. 2013). In general, these programs are designed to analyze gene sets that show statistically significant patterns of gene expression, variation, etc. Other gene set analysis methods such as gene set enrichment analysis (GSEA) (Subramanian et al. 2005), parametric analysis of gene set enrichment (PAGE) (Kim and Volsky 123 568 2005), and generally applicable gene set enrichment (GAGE) (Luo et al. 2009) allow for the analysis of all genes in global transcriptomics studies. These methods were developed to address the issue that not all meaningful gene expression changes rise to the level of statistical significance. Both the ‘‘cutoff -based’’ and ‘‘cutoff-free’’ methods (Luo et al. 2009) rely on comparisons of experimental gene sets to annotated gene sets and ontologies to facilitate data interpretation. We describe here a web-based application called VLAD (VisuaL Annotation Display) for finding functional themes in sets of genes based on their ontology term annotations. VLAD uses the hypergeometric test for determining significance and is appropriate for the analysis of gene sets that are generated by ‘‘cutoff-based’’ statistical analyses methods. VLAD is highly configurable; there are many parameters that can be set by users that control input, data processing, and output. A unique feature of the software relative to existing term enrichment tools is that it is not limited to the two native ontologies in the system: the Gene Ontology (Ashburner et al. 2000) and the Mammalian Phenotype Ontology (Smith and Eppig 2012); rather, VLAD can compare lists of genes to any structured vocabulary that is in the standard open biological and biomedical ontologies (OBO) format (http://www.obo foundry.org) (Smith et al. 2007) and for which there is a file of gene-to-annotation-term associations in the GO Annotation Format (GAF; http://geneontology.org/page/ go-annotation-file-gaf-format-10). VLAD also provides users with a level of control over the graphical display of results that is not available in other similar analysis sites. VLAD is available as a web-based application from the Mouse Genome Informatics (MGI) web site (http://proto. informatics.jax.org/prototypes/vlad/). Materials and methods Data sources To illustrate the functionality of VLAD, we analyzed genes from a previously published study that described the genome wide gene expression patterns across key developmental stages of normal mouse diaphragm (Russell et al. 2012). In this study, the investigators used time-series analysis (Ernst and Bar-Joseph 2006) of microarray-based expression data to identify over 650 genes whose expression levels increased significantly between embryonic day 11.5 and embryonic day 16.5 and over 360 genes whose expression levels decreased significantly over this same time period. To demonstrate the extensibility of VLAD to user-provided ontologies, an OBO ontology of mouse biochemical 123 J. E. Richardson, C. J. Bult: Visual annotation display (VLAD)… pathways (mousecyc_obo.txt) and a corresponding set of annotations in GAF format (mousecyc_gaf.txt) from the curated MouseCyc database (Evsikov et al. 2009) were downloaded from the MouseCyc project ftp site (ftp:// informatics.jax.org/pub/curatorwork/MouseCycDB/) and chosen as the basis for a custom term enrichment analysis using the Annotation Data Set options on the VLAD homepage. The mouse diaphragm gene lists, OBO ontology, and GAF files are available as supplemental data and from the following ftp site: ftp://informatics.jax.org/pub/ supplemental/MammGenome2015. Running VLAD VLAD is preconfigured to work with either gene-function annotations from MGI using the GO and/or gene-phenotype annotations from MGI using the Mammalian Phenotype (MP) Ontology. The (...truncated)