Hi, I have DESeqDataSet object 'ddsHTSeq' which contain multiple sample types. But i want to perform differential gene expression analysis only at a subset of this data. I have subset the data and ran DESeq, but the result is not what i was expected, since i knew some genes which were among the most differentially expressed genes from microarray data. if i could get any comments or suggest where i could go wrong, it would be much appreciated. Thank you in advance.
##Extract coldata >coldata_1 = colData(ddsHTSeq) ## subset the coldata >coldata = coldata_1[coldata_1$Subgroup %in% c("MB.WNT","MB.SHH","MB.Grp3","MB.Grp4"),] ## Extract and subset count data >countdata = assay(ddsHTSeq) >countdata = countdata[, coldata_1$Subgroup %in% c("MB.WNT","MB.SHH","MB.Grp3","MB.Grp4")] ## Make variables for design, to perform differential expression of MB.Grp4 compared to all others combined >Subgroup = as.character(coldata$Subgroup) >Subgroup[Subgroup !="MB.Grp4"] = "other" >Subgroup = factor(Subgroup, levels = c("other","MB.Grp4")) ## Re-assign the coldata Subgroup >coldata$Subgroup = Subgroup ## Create a new DESeq object >dds = DESeqDataSetFromMatrix(countdata = countdata, coldata = coldata, design =~Subgroup) >dds = DESeq(dds) >res = results(dds, alpha = 0.01) ## Check the top differentially expressed genes >rownames(res) [1:50]