Gene set enrichment analysis (GSEA) (also functional enrichment analysis) is a method to identify classes of genes or proteins that are over-represented in a large set of genes or proteins, and may have an association with disease phenotypes. The method uses statistical approaches to identify significantly enriched or depleted groups of genes. Microarray and proteomics results often identify thousands of genes which are used for the analysis.
Researchers performing high-throughput experiments that yield sets of genes (for example, genes that are differentially expressed under different conditions) often want to retrieve a functional profile of that gene set, in order to better understand the underlying biological processes. This can be done by comparing the input gene set to each of the bins (terms) in the gene ontology - a statistical test can be performed for each bin to see if it is enriched for the input genes. FunRich can also be used for gene ontology enrichment analysis.
Video Gene set enrichment analysis
Background
While the completion of the Human Genome Project gifted researchers with an enormous amount of new information, it also left them with the problem of how to interpret and analyze the incredible amount of data. In order to seek out genes associated with diseases, researchers utilized DNA microarrays, which measure the amount of gene expression in different cells. Researchers would perform these microarrays on thousands of different genes, and compare the results of two different cell categories, e.g. normal cells versus cancerous cells. However, this method of comparison is not sensitive enough to detect the subtle differences between the expression of individual genes, because diseases typically involve entire groups of genes. Multiple genes are linked to a single biological pathway, and so it is the additive change in expression within gene sets that leads to the difference in phenotypic expression. Gene Set Enrichment Analysis was developed to focus on the changes of expression in groups of a priori defined gene sets. By doing so, this method resolves the problem of the undetectable, small changes in the expression of single genes.
Maps Gene set enrichment analysis
Methods of GSEA
Gene set enrichment analysis uses a priori gene sets that have been grouped together by their involvement in the same biological pathway, or by proximal location on a chromosome. A database of these predefined sets can be found at the Molecular signatures database (MSigDB). In GSEA, DNA microarrays, or now RNA-Seq, are still performed and compared between two cell categories, but instead of focusing on individual genes in a long list, L, the focus is put on a gene set. Researchers analyze whether the majority of genes in the set fall in the extremes of this list: the top and bottom of the list correspond to the largest differences in expression between the two cell types. If the gene set falls at either the top (over-expressed) or bottom (under-expressed), it is thought to be related to the phenotypic differences.
In the method that is typically referred to as standard GSEA, there are three steps involved in the analytical process. The general steps are summarized below:
- Calculate the enrichment score (ES) that represents the amount to which the genes in the set are over-represented at either the top or bottom of the list. This score is a Kolmogorov-Smirnov-like statistic.
- Estimate the statistical significance of the ES. This calculation is done by a phenotypic-based permutation test in order to produce a null distribution for the ES.
- Adjust for multiple hypothesis testing for when a large number of gene sets are being analyzed at one time. The enrichment scores for each set are normalized and a false discovery rate is calculated.
Limitations and proposed alternatives to standard GSEA
SEA
When GSEA was first proposed in 2003 some immediate concerns were raised, regarding its methodology. These criticisms led to the use of the correlation-weighted Kolmogorov-Smirnov test, the normalized ES, and the false discovery rate calculation, all of which are the factors that currently define standard GSEA. However, GSEA has now also been criticized for the fact that its null distribution is superfluous, and too difficult to be worth calculating, as well as the fact that its Kolmogorov-Smirnov-like statistic is not as sensitive as the original. As an alternative, the method known as Singular Enrichment Analysis (SEA), was proposed. This method assumes gene independence and uses a simpler approach to calculate t-test. However, it is thought that these assumptions are in fact too simplifying, and gene correlation cannot be disregarded.
SGSE
One other limitation to Gene Set Enrichment Analysis is that the results are very dependent on the algorithm that clusters the genes, and the number of clusters being tested. Spectral Gene Set Enrichment (SGSE) is a proposed, unsupervised test. The method's founders claim that it is a better way to find associations between MSigDB gene sets and microarray data. The general steps include:
1. Calculating the association between principal components and gene sets.
2. Using the weighted Z-method to calculate association between the gene sets and the spectral structure of the data.
Tools for performing GSEA
GSEA uses complicated statistics, so it requires a computer program to run the calculations. However, because GSEA has become standard practice in the last decade, there are many websites and downloadable programs that will provide the data sets and run the analysis.
PlantRegMap
The gene ontology (GO) annotation for 165 plant species and GO enrichment analysis is available.
MSigDB
The Molecular Signatures Database hosts an extensive collection of annotated gene sets that can be used with most GSEA Software.
Broad Institute
The Broad Institute website is in cooperation with MSigDB and has a downloadable GSEA software, as well a general tutorial for those new to performing this analytical technique.
Enrichr
Enrichr is an easy to use gene set enrichment analysis tool for mammalian gene sets. It contains background libraries for transcription regulation, pathways and protein interactions, ontologies including GO and the human and mouse phenotype ontologies, signatures from cells treated with drugs, and expression of genes in different cells and tissues. Enrichr was developed by the Ma'ayan laboratory at Mount Sinai. The background libraries are from over 70 resources and contain over 200,000 annotated gene sets. The tool can be accessed through API and provides different ways to visualize the results.
GeneSCF
GeneSCF is a real-time based functional enrichment tool with support for multiple organisms and is designed to overcome the problems associated with using outdated resources and databases. Advantages of using GeneSCF: real-time analysis, users do not have to depend on enrichment tools to get updated, easy for computational biologists to integrate GeneSCF with their NGS pipeline, it supports multiple organisms, enrichment analysis for multiple gene list using multiple source database in single run, retrieve or download complete GO terms/Pathways/Functions with associated genes as simple table format in a plain text file.
DAVID
The Database for Annotation, Visualization and Integrated Discovery (DAVID) is a bioinformatics tool that pools together information from most major bioinformatics sources, with the aim of analyzing large gene lists in a high-throughput manner. DAVID goes beyond standard GSEA with additional functions like switching between gene and protein identifiers on the genome-wide scale, however, it is important to note that the annotations used by DAVID have not been updated since January 2010 which can have a considerable impact on practical interpretation of results. In October 2016, DAVID Knowledgebase version 6.8 was released with a complete rebuild.
AmiGO 2
The Gene ontology consortium has also developed their own online GO term enrichment tool, allowing species-specific enrichment analysis versus the complete database, coarser-grained GO slims, or custom references.
GREAT
In 2010, Gill Bejerano from Stanford University released the Genomic region enrichment of annotations tool (GREAT), a software which takes advantage of regulatory domains to better associate gene ontology terms to genes.
FunRich
The Functional Enrichment Analysis (FunRich) tool is mainly used for the functional enrichment and network analysis of OMICS data.
InterMine
Instances of InterMine automatically provide enrichment analysis for uploaded sets of genes and other biological entities.
ToppGene Suite
ToppGene is a one-stop portal for gene list enrichment analysis and candidate gene prioritization based on functional annotations and protein interactions network. Developed and maintained by the Division of Biomedical Informatics at Cincinnati Children's Hospital Medical Center.
QuSAGE
Quantitative Set Analysis for Gene Expression (QuSAGE) is a computational method for gene set enrichment analysis . QuSAGE improves power by accounting for inter-gene correlations and quantifies gene set activity with a complete probability density function (PDF). From this PDF, P values and confidence intervals can be easily extracted. Preserving the PDF also allows for post-hoc analysis (e.g., pair-wise comparisons of gene set activity) while maintaining statistical traceability. Turner et al. extended the applicability of QuSAGE to longitudinal studies by adding functionality for general linear mixed models. QuSAGE was used by the NIH/NIAID Human Immunology Project Consortium to identify baseline transcriptional signatures that were associated with human influenza vaccination responses. QuSAGE is available as an R/Bioconductor package, and is maintained by the Kleinstein Lab at Yale School of Medicine.
Applications and results of GSEA
GSEA and genome-wide association studies
Single-nucleotide polymorphisms, or SNPs, are single base mutations that may be associated with diseases. One base change has the potential to affect the protein that results from that gene being expressed; however, it also has the potential to have no effect at all. Genome-wide association studies are comparisons between healthy and disease genotypes to try to find SNPs that are overrepresented in the disease genomes, and might be associationed with that condition. Before GSEA, the accuracy of genome-wide SNP association studies was severely limited by a high number of false positives. The theory that the SNPs contributing to a disease tend to be grouped in a set of genes that are all involved in the same biological pathway, is what the GSEA-SNP method is based on. This application of GSEA does not only aid in the discovery of disease-associated SNPs, but helps illuminate the corresponding pathways and mechanisms of the diseases.
GSEA and Spontaneous Preterm Birth
Gene Set Enrichment methods led to the discovery of new suspect genes and biological pathways related to the Spontaneous Preterm Birth. Exome sequences from women who had experienced SPTB were compared to those from females from the 1000 Genome Project, using a tool that scored possible disease-causing variants. Genes with higher scores were then run through different programs to group them into gene sets based on pathways and ontology groups. This study found that the variants were significantly clustered in sets related to several pathways, all suspects in SPTB.
GSEA and cancer cell profiling
Gene Set Enrichment Analysis can be used to understand the changes that cells undergo during carcinogenesis and metastasis. In a study, microarrays were performed on renal cell carcinoma metastases, primary renal tumors, and normal kidney tissue, and the data was analyzed using GSEA. This analysis showed significant changes of expression in genes involved in pathways that have not been previously associated with the progression of renal cancer. From this study, GSEA has provided potential new targets for renal cell carcinoma therapy.10
GSEA and schizophrenia
GSEA can be used to help understand the molecular mechanisms of complex disorders. Schizophrenia is a largely heritable disorder, but is also very complex, and the onset of the disease involves many genes interacting within multiple pathways, as well the interaction of those genes with environmental factors. For instance, epigenetic changes, like DNA methylation, are affected by the environment, but are also inherently dependent on the DNA itself. DNA methylation is the most well-studied epigenetic change, and was recently analyzed using GSEA in relation to schizophrenia-related intermediate phenotypes. Researchers ranked genes for their correlation between methylation patterns and each of the phenotypes. They then used GSEA to look for an enrichment of genes that are predicted to be targeted by microRNAs in the progression of the disease.
GSEA and depression
GSEA can help provide molecular evidence for the association of biological pathways with diseases. Previous studies shown that long-term depression symptoms are correlated with changes in immune response and inflammatory pathways. A study by Elovainio et al. was aimed at finding genetic and molecular evidence that supports this association. The researchers took blood samples from the Young Finns Study (participants were depression patients), and used genome-wide expression data, along with GSEA to find expression differences in gene sets related to inflammatory pathways. The study found that patients who rated with the most severe depression symptoms also had significant expression differences in those gene sets, and this result supports the association hypothesis.
References
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