Deleted:edgeR - glm DE analysis for paired samples - errors

Question

[bio]

Hi All,

I had been trying to do DE analysis of my RNAseq experiment using edgeR and am having some isssues. The details of the Experiment and the R code I tried below:

(a) Paired experimental design with 45 pairs
(b) Treatment: "Before" and "After"
(c) Phenotype: 1 & 2

Aim: Look for DE genes between Phenotype 1 and 2 upon treatment taking into account the paired design

The R code tried:

library(edgeR)
counts<-read.delim(file="counts.dat",header=T)
pair=factor(pdata$pair)
Treat=factor( pdata$treat)
Phenotype=factor(pdata$pheno) 
group<-paste(Treat,Phenotype,sep=".")
design=model.matrix(~pair+Treat:Phenotype, data=counts)
counts.DGEList<-DGEList(counts, group=group)
y<-calcNormFactors(counts.DGEList)
y<-estimateCommonDisp(y, design)
y<-estimateGLMTrendedDisp(y, design)

Error message I get:

Error in glmFit.default(y, design = design, dispersion = dispersion, offset = offset,  : 
  Design matrix not of full rank.  The following coefficients not estimable:
 TreatBefore:Phenotype1 TreatBefore:Phenotype2

Any idea to solve this out?

> sessionInfo()
R version 3.1.0 (2014-04-10)
Platform: x86_64-pc-linux-gnu (64-bit)

locale:
 [1] LC_CTYPE=fi_FI.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_GB.UTF-8        LC_COLLATE=en_GB.UTF-8    
 [5] LC_MONETARY=fi_FI.UTF-8    LC_MESSAGES=en_GB.UTF-8   
 [7] LC_PAPER=en_GB.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_GB.UTF-8 LC_IDENTIFICATION=C       

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

other attached packages:
[1] edgeR_3.4.2   limma_3.18.13

Thanks a lot,
Preethy