Entering edit mode
Natasha
▴
440
@natasha-4640
Last seen 10.2 years ago
Dear List,
I do know that this question has been approached before, but am not
sure about
the reliability of the list of genes.
I have data from an experiment that was run in two batches (3 groups
per batch,
4 samples per group, 2 array-chips per bacth), these need to be
combined and
analysed.
Thus there are 6 groups, and I know from the QC that there is a strong
batch
effect, which I need to add to the model. However when I run the
analysis it
says batch is not estimable.
I am unsure now whether the limma list of differentially expressed
genes is
valid or not, as it might have not accounted for batch variation. (I
do
appreciate that it would also be a confounding factor, so one would
not be
certain of true differential effects without further experimental
verification,
but I am still uncertain of my list of genes).
Code below:
design2 = model.matrix(~0+group+batch)
design2
WT KO H1KO H2KO TripleKO P3 batch2
1 0 1 0 0 0 0 0
2 0 1 0 0 0 0 0
3 0 1 0 0 0 0 0
4 0 1 0 0 0 0 0
5 0 0 1 0 0 0 0
6 0 0 1 0 0 0 0
7 0 0 1 0 0 0 0
8 0 0 1 0 0 0 0
9 1 0 0 0 0 0 0
10 1 0 0 0 0 0 0
11 1 0 0 0 0 0 0
12 1 0 0 0 0 0 0
13 0 0 0 0 1 0 1
14 0 0 0 0 1 0 1
15 0 0 0 0 1 0 1
16 0 0 0 0 1 0 1
17 0 0 0 1 0 0 1
18 0 0 0 1 0 0 1
19 0 0 0 1 0 0 1
20 0 0 0 1 0 0 1
21 0 0 0 0 0 1 1
22 0 0 0 0 0 1 1
23 0 0 0 0 0 1 1
24 0 0 0 0 0 1 1
nonEstimable(design2)
[1] "batch2"
fit2 <- lmFit(sig.norm, design2)
Coefficients not estimable: batch2
Warning message:
Partial NA coefficients for 26084 probe(s)
con.mat2 = makeContrasts(KO-WT, H1KO-WT, H2KO-WT, TripleKO-WT, P3-WT,
P3-KO,
TripleKO-KO, levels=design2)
fit2b <- contrasts.fit(fit2, con.mat2)
fit2b <- eBayes(fit2b)
Any help and suggestions would be much appreciated.
Many Thanks,
Natasha
