edgeR: very low p-value and very high variance within the group of replicates. What's

[valei]

I'm using `edgeR` in order to perform differential expression analysis from RNA-seq experiment.

I have 6 samples of tumor cell, same tumor and same treatment: 3 patient with good prognosis and 3 patient with bad prognosis. I want to compare the gene expression among the two groups.

I ran the `edgeR` pakage like follow:

x <- read.delim("my_reads_count.txt", row.names="GENE")
group <- factor(c(1,1,1,2,2,2))
y <- DGEList(counts=x,group=group)
y <- calcNormFactors(y)
y <- estimateCommonDisp(y)
y <- estimateTagwiseDisp(y)
et <- exactTest(y)

I obtained a very odd results: in some cases I had a very low *p*-value and FDR but looking at the raw data it is obvious that the difference between the two groups can't be significant.
This is an example for `my_reads_count.txt`:

GENE sample1_1 sample1_2 sample1_3 sample2_1 sample2_2 sample2_3
ENSG00000198842 0 3 2 2 6666 3
ENSG00000257017 3 3 25 2002 29080 4

And for `my_edgeR_resulta.txt`:

GENE logFC logCPM PValue FDR
ENSG00000198842 9.863211e+00 5.4879462930 5.368843e-07 1.953612e-04
ENSG00000257017 9.500927e+00 7.7139869397 8.072384e-10 7.171947e-07

I would like that the variance within the group is considered. Does anyone may help me? Some suggestion?

[Gordon Smyth]

The edgeR developers answered this question on the Bioconductor mailing list.

Please don't ask the same question on multiple forums at the same time -- it forces us to either answer the same question twice or to leave a question looking as if it wasn't answered.

Gordon