Dear list,
 
I am analyzing my first 2-color array experiment (Agilent arrays). After
reading the manual of LIMMA and the book BioC case studies, I decided to
use LIMMA applying VSN normalization. However, to me it is not
completely clear which arguments are passed to VSN when executed from
LIMMA.
 
>From ?normalizeBetweenArrays i learn:
"method="vsn" uses the vsn function from the vsn package. For this
option the input object should contain raw intensities, i.e., prior to
background correction, log-transformation or any normalization. Note
that the normalized intensities are on the log-2 scale, not the log-e
scale output by the vsn function in the vsn package."
 
The code I ran:
targets <- readTargets("targets.txt", row.names="Name")
RG <- read.maimages(targets$FileName, source="agilent")
MA.vsn <- normalizeBetweenArrays(RG, method="vsn")
 
 
Questions:
-  in the 3rd line code above, is VSN run with the argument
'backgroundsubstract=TRUE'? I am asking because by default VSN does NOT
background subtraction, and when I explicitly state to subtract
background an error occurs:
> MA.vsn <- normalizeBetweenArrays(RG, method="vsn",
backgroundsubstract=TRUE)
Error in vsnMatrix(x = y, ...) : 
  unused argument(s) (backgroundsubstract = TRUE)
Error in exprs(vsnMatrix(x = y, ...)) : 
  error in evaluating the argument 'object' in selecting a method for
function 'exprs'
> 

- Regarding this statement from ?normalizeBetweenArrays ("Note that the
normalized intensities are on the log-2 scale, not the log-e scale
output by the vsn function in the vsn package"): does this mean that the
raw array data is processed fully as per VSN methodology, and that only
in the end it is converted into log2 scale? Thus that the raw
intensities present in RG are glog-transformed, which are then used for
background correction (?; see above) and variance stabilization
normalization, and finally the VSN intensities are converted from glog
to log2 scale? Or does this mean the VSN methodology is used on log2,
and NOT glog transformed data? I assume the former, but plz correct me
if I am wrong.
 
TIA,
Guido
 
 
------------------------------------------------ 
Guido Hooiveld, PhD 
Nutrition, Metabolism & Genomics Group 
Division of Human Nutrition 
Wageningen University 
Biotechnion, Bomenweg 2 
NL-6703 HD Wageningen 
the Netherlands 
tel: (+)31 317 485788 
fax: (+)31 317 483342 
internet:   http://nutrigene.4t.com <http://nutrigene.4t.com/>  
email:      guido.hooiveld@wur.nl 


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