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# GSEAmining Make biological sense of Gene Set Enrichment Analysis outputs Gene Set Enrichment Analysis ([GSEA]( is a very powerful and interesting computational method that allows an easy correlation between differential expressed genes and biological processes. Unfortunately, although it was designed to help researchers to interpret gene expression data it can generate huge amounts of results whose biological meaning can be difficult to interpret. Many available tools rely on the hierarchically structured Gene Ontology (GO) classification to reduce reundandcy in the results. However, due to the popularity of GSEA many more gene set collections, such as those in the Molecular Signatures Database ([MSigDB](, are emerging. Since these collections are not organized as those in GO, their usage for GSEA do not always give a straightforward answer or, in other words, getting all the meaninful information can be challenging with the currently available tools. For these reasons, GSEAmining was born to be an easy tool to create reproducible reports to help researchers make biological sense of GSEA outputs. Given the results of GSEA, GSEAmining clusters the different gene sets collections based on the presence of the same genes in the leadind edge (core) subset. Leading edge subsets are those genes that contribute most to the enrichment score of each collection of genes or gene sets. For this reason, gene sets that participate in similar biological processes should share genes in common and in turn cluster together. After that, GSEAmining is able to identify and represent for each cluster: - The most enriched terms in the names of gene sets (as wordclouds) - The most enriched genes in the leading edge subsets (as bar plots). In each case, positive and negative enrichments are shown in different colors so it is easy to distinguish biological processes or genes that may be of interest in that particular study. # Installation You can install `GSEAmining` from Bioconductor: ``` if (!requireNamespace("BiocManager", quietly = TRUE)) install.packages("BiocManager") BiocManager::install("GSEAmining") ``` Or directly from GitHub: ``` install.packages("devtools") # If you have not installed "devtools" package library(devtools) devtools::install_github("oriolarques/GSEAmining") ``` # Input data format: gm_filter By default GSEAmining is designed to accept the resuls of the GSEA function from the [clusterProfiler]( package. For more information about this function click [here]( However, it is *not mandatory to use this package*. In this example the data corresponds to GSEA analysis of differential expressed genes from treated versus control samples in [HGPalmer-PDX-P30 experiment]( Differential gene expression (tableTop) was obtained using the oligo and limma R packages. - Generation of a geneList, which is a ranked list of all the genes, for GSEA using clusterProfiler. ``` # A geneList contains three features: # 1.numeric vector: fold change or other type of numerical variable # 2.named vector: every number has a name, the corresponding gene ID # 3.sorted vector: number should be sorted in decreasing order tableTop_p30 <- geneList = tableTop_p30[,2] names(geneList) = as.character(tableTop_p30[,1]) ``` - Gmt files containing the different gene sets that will be analysed by GSEA. They can be downloaded from the [MSigDB]( ``` library(clusterProfiler) # Read the .gmt file from MSigDB gmtC2<- read.gmt("gmt files/c2.all.v7.1.symbols.gmt") gmtC5<- read.gmt('gmt files/c5.all.v7.1.symbols.gmt') gmtHALL <- read.gmt('gmt files/h.all.v7.1.symbols.gmt') # Merge all the gene sets gmt_all <- rbind(gmtC2, gmtC5, gmtHALL) ``` - Perform the GSEA. ``` GSEA_p30<-GSEA(geneList, TERM2GENE = gmt_all, nPerm = 1000, pvalueCutoff = 0.5) # Selection of gene sets with a specific thershold in terms of NES and p.adjust genesets_sel <- GSEA_p30@result ``` Data should be in a data.frame with, **at least** three columns labelled as follows: - **ID**: The name of the gene set. - **NES**: Normalized Enrichment Score of the gene set. - **core_enrichment**: Genes that appear in the leading edge subset of the gene set. ``` # Structure of the data included in the package data("genesets_sel", package = 'GSEAmining') tibble::glimpse(genesets_sel) ``` **gm_filter: Filter the input data** GSEA outputs normally presents tens or hundreds of genesets but many of them may not meet the thresholds for considering them significantly enriched. For that it is better to filter the data for a better visualization. The `gm_filter()` function of GSEAmining allows this process to be very easy, especially if the format of your data meets the aforementioned criteria. ``` library(GSEAmining) data("genesets_sel", package = 'GSEAmining') gs.filt <- gm_filter(genesets_sel, p.adj = 0.05, neg_NES = 2.6, pos_NES = 2) ``` # Clustering ## gm_clust: Creation of a gm_clust object Using the `gm_clust()` function, we can create an object that will contain the hierarchical clustering of the gene sets according to their genes in the core_enrichment column. This function accepts the data frame created with gm_filter. In the process, a distance matrix is calculated using the *binary* method (from the `dist()` function in `stats`) and then a cluster with the *complete* method (from `hclust()` function in `stats`) is created. ``` # Create an object that will contain the cluster of gene sets. <- gm_clust(gs.filt) ``` ## gm_dendplot: Plot the cluster The `gm_dendplot()` function uses (i) the filtered data frame and (ii) the gm_clust object to plot the cluster dendrogram. It shows which gene sets are positively or negatively enriched coloring them in red or blue respectively. Additionally by default the function will highlight every other cluster with a rectangle to facilitate the differentiation of all the clusters. ``` gm_dendplot(gs.filt, ``` ![dendplot](/vignettes/dendplot.png) It is possible to tune the height of the dendrogram, the colors for the enrichment and the use of rectangles (and their sizes) for highlighting clusters. ``` gm_dendplot(gs.filt,, col_pos = 'orange', col_neg = 'black', rect = TRUE, dend_len = 20, rect_len = 2) ``` ![dendplot2](/vignettes/dendplot2.png) # Evaluation of gene sets enriched terms by cluster To start getting an idea of what kind of biological processes are represented in each cluster we can use `gm_enrichterms()`. This function plots wordclouds for the most enriched terms in the names of the different gene sets in each cluster. Again it needs (i) the filtered data frame and (ii) the gm_clust object created before. By default Positive or negative enrichment are colored in red or in blue respectively and two ploting options are available: - By cluster: ``` gm_enrichterms(gs.filt, ``` ![enrichterms](/vignettes/enrichterms.png) - Without clustering: ``` gm_enrichterms(gs.filt,, clust = FALSE, col_pos = 'chocolate3', col_neg = 'skyblue3') ``` ![enrichterms2](/vignettes/enrichterms2.png) # Evaluation of gene enrichment in leading edge subsets by clusters As for the gene set terms, it is also possible to evaluate which are the most enriched genes in the leading edge (or core enrichment) subsets in the different clusters with the function `gm_enrichcores()`. In this case the representation is shown as bar plots following the same options as in `gm_enrichterms()` regarding colors and clustering the results. ``` gm_enrichcores(gs.filt, ``` ![enrichcores](/vignettes/enrichcores.png) Additionally, this function allows the possibility to decide the number of the most top genes that will be plotted using the parameter `top`. In case more than the indicated number of top genes appear the same number of times in the same cluster they all will be shown. # gm_enrichreport: Create a report Finally, the `gm_enrichreport()` is a tool that generates a pdf file where each page contains both the terms wordcloud and the leading edge bar plot for each of the different clusters in the gm_clust object. ``` gm_enrichreport(gs.filt,, output = 'gm_report') ``` The `output`parameter can be used to name the file that will be generated (by default it will be called gm_report.pdf). As in the previous functions the colors and the number of top genes are also editable parameters of the function.