Introduction

The GSVA (Gene Set Variation Analysis) (Hänzelmann, S., Castelo, R. & Guinney, J. GSVA: gene set variation analysis for microarray and RNA-Seq data. BMC Bioinformatics 14, 7 (2013).) is a popular method for GSE (Gene Set Enrichment) and allows the identification of changes in pathway activity in RNA-Seq data. Overall, gene sets can be uploaded or selected from the available databases and the GSVA method can be run on the selected gene sets which can eventually be visualized using Violin or Heatmap plots.

General Worflow

The figure below describes the workflow of using GSVA through singleCellTK package:

Workflow Guide

  1. Upload or import gene sets:
#import a GMT file
gmtFilePath <- "C:/Users/HP/Desktop/gmtfile.GMT"
sce <- importGeneSetsFromGMT(inSCE = sce,
                             file = gmtFilePath,
                             by = "rownames",
                             collectionName = "Collection1")

#select from an existing database
sce <- importGeneSetsFromMSigDB(inSCE = sce,
                                categoryIDs = "C1",
                                by = "rownames")
  1. Run GSVA:
sce$gsvaRes <- gsvaSCE(inSCE = sce,
               useAssay = "logcounts",
               pathwayNames = c("C1"))
  1. Visualize the results:
#populate results
conditionFactor <- factor(colData(sce)[, "Biological_Condition"])
      if(nlevels(conditionFactor)<=1){
        stop("Condition variable must have atleast two levels!")
      }
fit <- limma::lmFit(sce$gsvaRes, stats::model.matrix(~conditionFactor))
fit <- limma::eBayes(fit)
toptableres <- limma::topTable(fit, number = nrow(sce$gsvaRes))
temptable <- cbind(rownames(toptableres), toptableres)
rownames(temptable) <- NULL
colnames(temptable)[1] <- "Pathway"
     

tempgsvares <- sce$gsvaRes
topPaths <- 10
#visualize violin plot
tempgsvares <- tempgsvares[1:min(49, topPaths, nrow(tempgsvares)), , drop = FALSE]
gsvaPlot(inSCE = sce,
         gsvaData = tempgsvares,
         plotType = "Violin",
         condition = "Biological_Condition")

#visualize heatmap plot
gsvaPlot(inSCE = sce,
         gsvaData = tempgsvares,
         plotType = "Heatmap",
         condition = "Biological_Condition")