Hi,

We are working on a glm analysis in EdgeR and voom() and I am hoping we can help with our design.  Currently, we are getting errors.

We have 18 RNA-Seq biological replicates (samples), each sample has two columns of counts for each tag, one for the maternal allele and one for the paternal allele.  Nine samples have mother A and father B, and nine samples have mother B and father A.  

we wish to test the following models in edgeR and voom()

(~sample+allele+parent) vs (~sample+allele)
(~sample+allele+allele:parent) vs (~sample+allele)

Our design matrix is built as follows.  Once concern is that the two columns for each sample are paired, so I give them the same sample ID.

sample<-factor(c(1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,12,12,13,13,14,14,15,15,16,16,17,17,18,18))  #provides sample labels
title<-c("A", "B")   #provides allele labels
allele<-factor(c(rep(title, 18)))
parent1<-c("father", "mother")   #parent labels for the first nine replicates
parent2<-c("mother", "father")   #parent labels for the second nine replicates
parent<-factor(c(c(rep(parent1, 9)), c(rep(parent2, 9)))) 

model.matrix(~sample+allele+allele:parent)->design

The errors we get with this approach are as follows:

EdgeR
> d <-estimateGLMCommonDisp(d, design)
Error in solve.default(R, t(beta)) : 
  system is computationally singular: reciprocal condition number = 3.227e-17


voom()
> design<-model.matrix(~sample+allele+allele:parent)
> y<-voom(ctx,design, plot=TRUE, lib.size=colSums(ctx)*nf)
Coefficients not estimable: alleleA:parentmother 
Warning message:
Partial NA coefficients for 159907 probe(s) 

Thank you very much for any help!

best wishes,

Chris


> sessionInfo()
R version 2.15.0 (2012-03-30)
Platform: x86_64-apple-darwin9.8.0/x86_64 (64-bit)

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

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

other attached packages:
[1] edgeR_2.6.3  limma_3.12.0

loaded via a namespace (and not attached):
 [1] annotate_1.34.0      AnnotationDbi_1.18.0 Biobase_2.16.0       BiocGenerics_0.2.0   DBI_0.2-5            DESeq_1.9.7          genefilter_1.38.0    geneplotter_1.34.0   grid_2.15.0         
[10] IRanges_1.14.0       RColorBrewer_1.0-5   RSQLite_0.11.1       splines_2.15.0       stats4_2.15.0        survival_2.36-12     xtable_1.7-0      
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