[BioC] Re: Limma: M value in toptable

[Hua Weng]

Dear Dr. Smyth and Bioconductor members:

I have a question about M Values in toptable in LIMMA package. I know the M in toptable is not a simple average from all the replicates, it is a coefficient  value from lmFit.  I have three biological replicate array and within each array I have six technical replicates. My commands are the following:

design <- c(1,1,1)
 fit <- lmFit(MA.norm, design, ndups=6, spacing=800)
eb <- ebayes(fit)
x <- toptable(number=length(fit$coefficients), genelist=genes, fit=fit, A = fit$Amean, eb=eb, adjust="fdr")

These commands seems successful, I got what I want. For most of the genes, their M very close to simple average across 18 replicates. But one M vaule for one gene seems wired.

The result for this gene from toptable :
          Block Row Column Name           ID                     M                 A             T                P.value            B     
"58"    "1"     "6"     "8"     "P24-E1"        "P24-E1"         1.441473       12.652   4.74793        2.507e-04        0.9625

The M values I pluged in to run lmFit:
ID                     M(bio rep1)         A      weight    M(bio rep2)     A       weight     M(bio rep3)      A         weight
"P24-E1"         1.388976       14.8516    1.0      1.367237       13.1931   1.0      1.4567540      13.6927   1.0
P24-E1"         1.682181       15.0347     1.0      1.410991       13.3535   1.0      0.9180444      13.7189    1.0
"P24-E1"         1.419217       14.9294    1.0      0.978001       13.1710   1.0     -1.0304558       3.3577    0.1
"P24-E1"         0.911255       14.8090    1.0      1.007387       13.1383   1.0      0.5174605      13.2928   1.0
"P24-E1"         1.058592       14.8387    1.0      0.972540       13.1374   1.0      0.6585728      13.4881   1.0
"P24-E1"         0.343243       14.2827    1.0      0.859519       12.9058   1.0     -2.1799057       2.5352    0.1

But the simple average for these replicates exclude those with weight of 0.1 is 1.05938. I tried to reproduce 1.441473, but it seems out of my limmit. Could you help me to explain how this happens? How dose LIMMA calculate the M in toptable?

Thanks,
Hua




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