I am trying to use DESeq2 to analyze my data. I have 2 conditions (wild type and mutant), and 3 samples for each condition. My samples are paired: wt1 with mutant1, wt2 with mutant2, wt3 with mutant3.
I am using R version 3.2.0 (2015-04-16) and DESeq2_1.8.1.The first script I wrote starts as followed:
d <- read.delim(infiles) m <- c(rep("MU",3),rep("WT",3)) cd <- data.frame(id=colnames(d), line=m, pair=c(1,2,3,1,2,3)) cd$line <- relevel(cd$line, ref="WT") dds <- DESeqDataSetFromMatrix(countData = d, colData = cd, design = ~ pair + line) dds <- DESeq(dds) res <- results(dds) sig <- res[which(res$padj<0.01),]
It ran and gave me a list of differentially expressed genes. However, reading the DESeq2 manual, I realized that "pair" should be a factor, rather than a numeric. So I reran the previous script, with a modified 3rd line:
cd <- data.frame(id=colnames(d), line=m, pair=factor(c(1,2,3,1,2,3)))
With this factor, about 150 genes which were previously DE are not DE anymore. I don't really understand what is causing this difference. Could you tell me if my design is correct?