[BioC] coefficient not estimable (limma warning)

[Moshe Olshansky]

Hi Natasha,

In your case, H2KO + TripleKO + P3 = batch2 (column-wise), so your
matrix's columns are dependent and this causes the technical problem.
Intuitively, since you have 3 samples in batch 1 and 3 other samples in
batch 2, you can never know whether you have a batch effect or your one
set of samples is very different from the other one.

Best regards,
Moshe.

> 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
>
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-- 
Moshe Olshansky
Division of Bioinformatics
The Walter & Eliza Hall Institute of Medical Research
1G Royal Parade, Parkville, Vic 3052
e-mail: olshansky at wehi.edu.au
tel: (03) 9345 2697


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