Dear Adi and other members, I am trying to apply my microarray data to SPIA R package that is created by Adi Tarca. I only got one pathway. I am wondering if I did anything wrong. Could you help me on this? I attached my topTable of fit data that contains lists of genes that is differently expressed, LFC , p.value, adj.p.value and so on. Here is my R: > library(lattice) > m <- exprs(eset) > A <- unname(rowMeans(m)) > M <- as.vector(m) -A > Sample <- colnames(m)[col(m)] > df <- data.frame(A=A, M=M, Sample=Sample) > treatments=factor(c(1,1,1,2,2,2,3,3,3,4,4,4), labels=c("CTRL","HIF1a","HIF2a","HIF1a2a")) > design=model.matrix(~treatments) > colnames(design)=c("CTRL","HIF1a","HIF2a","HIF1a2a") > design CTRL HIF1a HIF2a HIF1a2a 1 1 0 0 0 2 1 0 0 0 3 1 0 0 0 4 1 1 0 0 5 1 1 0 0 6 1 1 0 0 7 1 0 1 0 8 1 0 1 0 9 1 0 1 0 10 1 0 0 1 11 1 0 0 1 12 1 0 0 1 attr(,"assign") [1] 0 1 1 1 attr(,"contrasts") attr(,"contrasts")$treatments [1] "contr.treatment" > fit=lmFit(eset,design) > fit=eBayes(fit) > results=classifyTestsF(fit, p.value=0.001) > summary(results) CTRL HIF1a HIF2a HIF1a2a -1 0 440 84 577 0 0 50437 53683 50649 1 54675 3798 908 3449 > options(digits=3) > colnames(topTable(fit,coef="HIF1a",n=20)) [1] "ID" "Gene.title" "Gene.symbol" [4] "Gene.ID" "UniGene.title" "UniGene.symbol" [7] "UniGene.ID" "Nucleotide.Title" "GI" [10] "GenBank.Accession" "Platform_CLONEID" "Platform_ORF" [13] "Platform_SPOTID" "Chromosome.location" "Chromosome.annotation" [16] "GO.Function" "GO.Process" "GO.Component" [19] "GO.Function.1" "GO.Process.1" "GO.Component.1" [22] "logFC" "AveExpr" "t" [25] "P.Value" "adj.P.Val" "B" > library(SPIA) > x=topTable(fit,coef="HIF1a") > library(hgu133plus2.db) Loading required package: AnnotationDbi Loading required package: org.Hs.eg.db Loading required package: DBI > y=hgu133plus2ENTREZID > x$ENTREZ=unlist(as.list(y[x$ID])) > x=x[!is.na(x$ENTREZ),] > x=x[!duplicated(x$ENTREZ),] > tg1=x[x$adj.P.Val<0.1,] > HIF1a=tg1$logFC > names(HIF1a)=as.vector(tg1$ENTREZ) > HIF1a1=x$ENTREZ > res=spia(de=HIF1a,all=HIF1a1,organism="hsa",nB=2000,plots=F,beta=NUL L,combine="fisher",verbose=F) > res Name ID pSize NDE pNDE tA pPERT pG pGFdr pGFWER Status 1 RNA transport 03013 1 1 1 0 NA 1 1 1 Inhibited KEGGLINK 1 http://www.genome.jp/dbget-bin/show_pathway?hsa03013+54913 Many Many Thanks Jing Huang PhD Oregon Health $ Sciences University Portland Oregon

On Mon, 22 Aug 2011 07:56:28 -0700, Jing Huang wrote: > Dear Adi and other members, > > I am trying to apply my microarray data to SPIA R package that is > created by Adi Tarca. I only got one pathway. I am wondering if I did > anything wrong. Could you help me on this? > > I attached my topTable of fit data that contains lists of genes that > is differently expressed, LFC , p.value, adj.p.value and so on. [...] >> library(SPIA) >> x=topTable(fit,coef="HIF1a") topTable only returns the top 10 genes by default so it looks to me like your gene universe only contains 10 genes. Check the length of HIF1a1 to be sure. If that is the case then x=topTable(fit,coef="HIF1a",number=Inf) Should do something closer to what you want. >> library(hgu133plus2.db) > Loading required package: AnnotationDbi > Loading required package: org.Hs.eg.db > Loading required package: DBI > > >> y=hgu133plus2ENTREZID >> x$ENTREZ=unlist(as.list(y[x$ID])) >> x=x[!is.na(x$ENTREZ),] >> x=x[!duplicated(x$ENTREZ),] >> tg1=x[x$adj.P.Val<0.1,] >> HIF1a=tg1$logFC >> names(HIF1a)=as.vector(tg1$ENTREZ) >> HIF1a1=x$ENTREZ >> >> res=spia(de=HIF1a,all=HIF1a1,organism="hsa",nB=2000,plots=F,beta=NU LL,combine="fisher",verbose=F) >> res > Name ID pSize NDE pNDE tA pPERT pG pGFdr pGFWER > Status > 1 RNA transport 03013 1 1 1 0 NA 1 1 1 > Inhibited > KEGGLINK > 1 http://www.genome.jp/dbget-bin/show_pathway?hsa03013+54913 > > > Many Many Thanks > > Jing Huang PhD > > Oregon Health $ Sciences University > > Portland Oregon -- Alex Gutteridge