Dear Friends,

First of all let me tell that I am not an expert bioinformatician.  I would
like to do some basic microarray analysis using R & Bioconductor with CEL
files obtained using Affymetrix HuGene-1.0-ST-v1 platform. I have so many
questions and I tried to search and read several threads in the
Bioconductor Help List and other webpages. My questions are related to or
the same as many of the previous threads but after reading several of those
answers, questions remain almost the same.

The main question is regarding the number of genes probed in this platform.
According to Affymetrix Data sheet on this platform there are 764,885
distinct probes and 28,869 estimated genes. When I use ‘affy’ and use the
function ‘ReadAffy()’ and ‘rma’ I get an expression set with 32321
features. Very different from 28,869!

I read in most of the replies to previous threads that ‘affy’ should not be
used for the analysis of this platform.  (It will be great if somebody can
explain or point to relevant literature on the reasons for these
differences). However, with ‘affy’ it automatically identifies the correct
annotation file (at least the name ‘*hugene10stv1’*) and processes the CEL
file without giving any error message or warning.

As suggested in many threads and in Bioconductor website I used the package
‘oligo’ for processing my HuGene10STv1 based CEL file. After summarizing at
the core level using ‘rma’ function, I obtained an expression set object
with 33297 features, and of course it is neither 28,869 nor 32321. Here the
annotation used is ‘pd.hugene.1.0.st.v1’ instead of the ‘hugene10stv1’ in
the previous case.

I am fine with using ‘oligo’. [See, I am ‘blindly’ using a software, like
most of the people! I found papers, even in prestigious journals, using
‘affy’ to process CEL files obtained using ‘hugene10stv1’ chip. Please help
me to open my eyes or enlighten me (and many others)!] However, when I want
to get gene Symbols corresponding to the transcripts, again there is a
‘number mismatch’. For example when I used the package
'hugene10sttranscriptcluster.db' , I found that there are 21995 keys out of
33295 (not 33297) can be mapped to gene symbols. What happened to two of
them? Or, with ‘oligo’ I have to use something else to convert ‘transcript
ids’ to SYMBOLS or ENTREZIDs, than 'hugene10sttranscriptcluster.db'?

I read that ‘affy’  can be used with * "hugene10stv1.r3cdf" *but there is
no such thing available in bioconductor website among the annotation
packages. May be that was applicable to an older Bioconductor release as
those threads were 2-3 years old. Doesn’t it imply that the currently
available ‘*hugene10stv1’ *is the correct one to use with ‘affy’? On the
other hand, if it cannot be used why is it there in Bioconductor? Where do
we use the annotation ‘*hugene10stv1’*?

I read there are other packages such as ‘aroma-affymetrix’, xps, etc, but I
am trying to do some simple things with standard, official, ‘bioconductor’
packages. Any suggestions and helpful hints are highly appreciated.

Here are the commands that used in Bioconductor version 2.8 (with R 2.13)
[Yes, I will update to most recent version soon!].

As an example, I used the CEL file, 'GSM857535.CEL.gz', down loaded  from
GEO.

> library(‘affy’)

> as <- ReadAffy('GSM857535.CEL.gz')

> as

> aset <- rma(as)

> aset

> library('hugene10sttranscriptcluster.db')

x <- hugene10sttranscriptclusterSYMBOL

xx <- x[mappedkeys(x)]

> length(x)

[1] 33295

> length(xx)

[1] 21995

library(‘oligo’)

bs <- read.celfiles('GSM857535.CEL.gz')

> bs

> bset <- rma(bs,target='core')
> bset

Thanks,
Asha

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