[BioC] edgeR: very low p-value and very high variance within the group of replicates

[valentina [guest]]

I have a question about you edgeR, in order  to fix a problem that may be very simple for you.

I'm using `edgeR`  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_results.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

It seems very odd that "0 3 2" versus "2 6666 3" is significant....
I would like that the variance within the group is considered. Can you help me? Some suggestion?
Are there some alternative function in edgeR that is capable to fix my problem??

 -- output of sessionInfo(): 

> sessionInfo()
R version 2.15.0 (2012-03-30)
Platform: x86_64-redhat-linux-gnu (64-bit)

locale:
[1] C

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
[1] edgeR_3.0.8              limma_3.12.3             tweeDEseqCountData_1.0.8 Biobase_2.16.0           BiocGenerics_0.2.0       BiocInstaller_1.8.3     

loaded via a namespace (and not attached):
[1] tools_2.15.0

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