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Last seen 10.0 years ago
Hi,
I am trying to figure out how to do comparisons using edgeR but have
to admit that I am on unsure ice.
My experiment is abiotic stress on two genotypes (E and J) and these
have been sampled for RNA-seq at five time points (00, 01, 05, 48, and
96).
I would like to analyze this experiment as a factorial (which I am
sure could be argued), and when I set up the following design:
> design<-model.matrix(~0 + Group + Time + Group*Time),
I get the following design matrix (I've taken out the 3 three
replicates at experimental cell.):
> design
GroupE GroupJ Time01 Time05 Time48 Time96 GroupJ:Time01
GroupJ:Time05 GroupJ:Time48 GroupJ:Time96
E00 1 0 0 0 0 0 0
0 0 0
J00 0 1 0 0 0 0 0
0 0 0
E01 1 0 1 0 0 0 0
0 0 0
J01 0 1 1 0 0 0 1
0 0 0
E05 1 0 0 1 0 0 0
0 0 0
J05 0 1 0 1 0 0 0
1 0 0
E48 1 0 0 0 1 0 0
0 0 0
J48 0 1 0 0 1 0 0
0 1 0
E96 1 0 0 0 0 1 0
0 0 0
J96 0 1 0 0 0 1 0
0 0 1
attr(,"assign")
[1] 1 1 2 2 2 2 3 3 3 3
attr(,"contrasts")
attr(,"contrasts")$Group
[1] "contr.treatment"
attr(,"contrasts")$Time
[1] "contr.treatment"
I have some questions regarding testing contrasts:
First though, I realize that I can pull out the data for E00 and J00,
say, and compare these using exactTest, but then I guess that you are
disregarding all information that lies in the remaining data set? That
does not seem to me to be a good way to do it. But I may be wrong
here?
Second, if I want to do the comparison E00 vs J00 using the design
indicated above, how should that be set up? Would
lrt <- glmLRT(fit,contrast=c(1,-1,0,0,0,0,0,0,0,0)) do it?
Third, if I want to compare (E00 + E01 + E05) vs (E48 + E96), how
would I set up that contrast?
For my own pedagogic reasons I can show how I was thinking in this
case:
E00 = L1
E01 = L1 + L4 x 2 --> 6L1 + 2L3 + 2L4
E05 = L1 + L4
versus --> 2L3 + 2L4 - 3L5 -3L6
E48 = L1 + L5 x 3 --> 6L1 + 3L5 + 3L6
E96 = L1 + L6
resulting in the contrast:
lrt <- glmLRT(fit,contrast=c(0,0,2,2,-3,-3,0,0,0,0))
Then I seem to remember that contrasts should sum up to zero? If
anybody could tell me where I am loosing it, I would be very obliged.
Thank you
jd
-- output of sessionInfo():
R version 3.1.0 (2014-04-10)
Platform: x86_64-w64-mingw32/x64 (64-bit)
locale:
[1] LC_COLLATE=Norwegian (Bokm??l)_Norway.1252 LC_CTYPE=Norwegian
(Bokm??l)_Norway.1252
[3] LC_MONETARY=Norwegian (Bokm??l)_Norway.1252 LC_NUMERIC=C
[5] LC_TIME=Norwegian (Bokm??l)_Norway.1252
attached base packages:
[1] splines stats graphics grDevices utils datasets
methods base
other attached packages:
[1] edgeR_3.6.8 limma_3.20.9 BiocInstaller_1.14.2
loaded via a namespace (and not attached):
[1] tools_3.1.0
>
--
Sent via the guest posting facility at bioconductor.org.
"On thin ice", would be the more idiomatic expression.