[BioC] coefficient not estimable (limma warning)

[Naomi Altman]

According to your design matrix, the first 3 treatments were run in 
batch 0 and the last 3 in batch 1.  So batch is confounded with 
treatment.  If so, there is no way to analyze the experiment that 
produces a valid comparison between treatments run in the different batches.

Naomi



At 10:17 AM 5/11/2011, Natasha wrote:
>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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