library(topGO)

library(limma)

library(edgeR)

library(org.Hs.eg.db)

data = read.table("lung.txt", header = T, sep = "\t")

data2 = data[data$X != "SLC35E2",]

rownames(data2) = data2[,1]

data = data2[,-1]

condition = factor(rep(c("T1", "T2"), c(576, 552)))

design = model.matrix(~condition)

dge = DGEList(counts=as.matrix(data), group = condition)

dge = calcNormFactors(dge, method = "TMM") #webtool'da var. RLE dediği deseq, TMM dediği TMM, none dediği none

v = voom(dge,design,plot=F) 

fit = lmFit(v,design)

fit = eBayes(fit)

res = topTable(fit,coef=ncol(design),number = dim(data)[1])

geneList = res$adj.P.Val #voomNSC tarafından seçilen genler

names(geneList) = rownames(res) #voomNSC tarafından seçilen genler

biomarkers = rownames(res[10:50,]) #voomNSC tarafından seçilen genler

#truefalse = is.element(names(geneList),biomarkers)

#selection = function(x) TRUE 

#allGO2genes = annFUN.org(whichOnto='BP', feasibleGenes = NULL,

#                         mapping="org.Hs.eg.db", ID = "genename")

#GOdata = new("topGOdata", description = "Simple session", ontology = "BP",

# allGenes = geneList, geneSel = topDiffGenes, nodeSize = 10,

# annot = annFUN.db, affyLib = affyLib)

truefalse = function(allScore) {  

  truefalse = is.element(names(geneList),biomarkers)

  return(truefalse)

}

allGO2genes = annFUN.org(whichOnto='BP', feasibleGenes = NULL,

                         mapping="org.Hs.eg.db", ID = "symbol")

GOdata =  new('topGOdata', ontology = 'BP', allGenes = geneList, 

              annot = annFUN.GO2genes, GO2genes = allGO2genes, 

              geneSel = truefalse, nodeSize=10)

results.ks = runTest(GOdata, algorithm = "classic", statistic = "ks")

results.ks

allRes = GenTable(GOdata, KS = results.ks, orderBy = "KS", topNodes = 20)

allRes[,c('GO.ID','Term','KS')]

showSigOfNodes(GOdata, score(results.ks), firstSigNodes = 5, useInfo = 'all')