[BioC] Limma, SAM and Fold Change Calculation

[Peter White]

I wondered if anyone could comment on what is generally considered the most
appropriate method of calculating fold change values from Affy data. I have
a data set from a test vs. control experiment (n=3 in each group) performed
on the MOE430_2 array. I used the Affy package to read in the CEL file data
and GCRMA to normalize. Finally I used limma to analyze (lmFit and eBayes).
Now the FC coefficients I get back from limma appear to be log2(2^(mean(test
replicates in log2))/(2^mean(control replicates in log2)). For example:

> exprs(eset)[18731,1:6]
 TA2.CEL  TA5.CEL  TA7.CEL  TA1.CEL  TA8.CEL  TA9.CEL 
3.294215 4.851795 4.403851 4.782934 7.716893 9.284909

> fit$coefficients[18731,1:2] #returns the mean of the above log2 values
  Fed_MT   Fed_WT 
4.183287 7.261578 

> fit2$coefficients[18731,1]
[1] -3.078291

> -1/2^fit2$coefficients[18731,1]
[1] -8.446136

So we have a probe that is reporting a -8.4 fold downregulation.

The problem is that SAM does not calculate the fold change values in the
same manner. It appears to take the average of the unlogged data and then
use those values to calculate fold changes. Thus:

> 2^exprs(eset)[18731,1:6]
   TA2.CEL    TA5.CEL    TA7.CEL    TA1.CEL    TA8.CEL    TA9.CEL 
  9.809738  28.875927  21.168555  27.530016 210.385700 623.786694

> tmp <- c(mean(2^exprs(eset)[18731,1:3]),mean(2^exprs(eset)[18731,4:6]))
> tmp
[1]  19.95141 287.23414

> tmp[1]/tmp[2]
[1] 0.06946043

> -1/(tmp[1]/tmp[2])
[1] -14.39669

So now we have -14.4 fold downregulation.

The example given is of course an extreme, but using the limma method there
are 282 probes with a fold change >2, while using the sam method there are
only 159, with 141 probes in common. The probes that were called uniquely by
limma were almost all downregulated (-2 to -5 fold).

MY QUESTION IS WHICH METHOD IS THE CORRECT WAY?

Thanks,

Peter 

Peter White, Ph.D.
Technical Director
Functional Genomics Core
Department of Genetics
University of Pennsylvania
570 Clinical Research Building
415 Curie Boulevard
Philadelphia, PA 19104-6145
 
Tel: +1 (215) 898-0773
Fax: +1 (215) 573-2326
E-mail: pwhite at mail.med.upenn.edu
 
http://www.med.upenn.edu/pdc/cores_fgc.html
http://www.med.upenn.edu/kaestnerlab/
http://www.betacell.org/microarrays
http://www.cbil.upenn.edu/EPConDB/