[BioC] how deal with multiplicate affy probes?

[Ron Ophir]

Hi All,
There is a project called GeneAnnot from the people of GeneCards that
implement this idea by "blatting" each probe to many RNA annotation
resources and integrate the results into two scores that define the
specificity and the sensitivity of the whole probe set. It was done on
U95 sets and please encourage that to do it on other chips.
You can fine the papers at:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14725348
and at
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14962929
and please have a look at
http://genecards.weizmann.ac.il/geneannot/
regards,
Ron

>>> <Arne.Muller at aventis.com> 03/26/04 11:26 AM >>>
Hi,

you may be able to automate this by blasting all the target sequences
(as
Lawrence suggested) against the ENSEMBLE confirmed or predicted genes
(i.e.
not the complete genome but just the genes). Then only look at matches
with
>95% sequence identity (not sure about this cut off). In your analysis
ignore
all probe sets that do not have a confident match (<95% sequence id).
One
could say this is the actual informative subset of probe sets on the
chip.

Note that you can still get >1 match with >95% id per probe set! In my
opinion the correspnding *single* expression measure is meaningless,
since
you cannot measure the >1 exprssion measures with the same probe set ...

For cases with >1 gene per probe set (as determined by blast using ther
target sequence) you may need to fall back to the single probe level
where
you may find that one of the genes has >95% sequence id in many probes
whereas the other doesn't.

As I said above you could automate this, but I it's not an easy task ..
:-(

    regards,

    Arne

--
Arne Muller, Ph.D.
Toxicogenomics, Aventis Pharma
arne dot muller domain=aventis com

> -----Original Message-----
> From: bioconductor-bounces at stat.math.ethz.ch
> [mailto:bioconductor-bounces at stat.math.ethz.ch]On Behalf Of Johnnidis,
> Jonathan
> Sent: 26 March 2004 00:53
> To: Lawrence Paul Petalidis; Michael Seewald
> Cc: bioconductor at stat.math.ethz.ch
> Subject: RE: [BioC] how deal with multiplicate affy probes?
> 
> 
> thank you for your suggestions.  However, in this instance 
> I'm not interested in particular transcripts but rather an 
> entire range of transcripts (several hundred)--so I'm not 
> sure it would be feasible to individually lookup and verify 
> every single probe set...
> Jonathan
> 
> -----Original Message-----
> From: Lawrence Paul Petalidis [mailto:lpp22 at cam.ac.uk]
> Sent: Thursday, March 25, 2004 6:35 PM
> To: Michael Seewald; Johnnidis, Jonathan
> Cc: bioconductor at stat.math.ethz.ch
> Subject: RE: [BioC] how deal with multiplicate affy probes?
> 
> 
> Hello,
> As a note following on from Michael Seewald's message, I 
> totally agree that
> there is a STRONG need to BLAST probe set sequences. I tend 
> to use the probe
> set target sequence instead of the indicidual probe sequences 
> however. You
> will be surprised to see the inconsistency of the Affy 
> annotation, in many
> cases _at probes are really not unique at all. So if you are really
> interested in a transcript, BLAST it to make sure you are 
> actually seeing
> what you think you are.
> 
> Best regards to all, Lawrence
> 
> ______________________________
> Lawrence Paul Petalidis
> Ph.D. Candidate
> 
> University of Cambridge
> Department of Pathology
> ______________________________
> 
> -----Original Message-----
> From: bioconductor-bounces at stat.math.ethz.ch
> [mailto:bioconductor-bounces at stat.math.ethz.ch]On Behalf Of Michael
> Seewald
> Sent: 25 March 2004 20:48
> To: Johnnidis, Jonathan
> Cc: bioconductor at stat.math.ethz.ch
> Subject: Re: [BioC] how deal with multiplicate affy probes?
> 
> 
> 
> As a rule of thumb: If statistics based on a given probe set 
> data tells you,
> that a transcript is significantly deregulated, you can 
> usually trust it and
> discard every other probe set for that transcript!
> 
> The thing to look at is the probe design itself: Download the 
> probe set from
> NetAffx and blast the single probes agains the genome (e.g. 
> in ensembl). You
> will be surprised, how many probes match up with introns or 
> genomic regions
> that do not correspond to any cDNA!
> 
> 2 examples: There are 4 probe sets for human Wnt6 
> (HG-U133AB), 2 match with
> the sense (!) strand and have to be discarded. Out of >12 
> probe sets for
> human
> CD44, only 4 have probes that are completely matching the 
> transcripts. >8
> can
> be discarded.
> 
> Best,
> Michael
> 
> PS: www.ensembl.org is always a good place to check probe sets. Their
> mapping
> of probe sets does not show the location of single probes, though...
> 
> PPS: In affymetrix.com you can check out the "Details" view 
> for a probe set.
> There you can discover, that 2 probe sets of Wnt 6 map to the 
> (-) strand,
> which is bad. It doesn't tell you, however, that many probe sets match
> intron
> regions.
> 
> 
> On Sat, 20 Mar 2004, Johnnidis, Jonathan wrote:
> > I'm a new list member and am not quite sure if this question is
> appropriate
> > for the list, but will shoot anyway. I'm analyzing a bunch 
> of data from
> Affy
> > MgU74Av2 chips and am a bit perplexed as to how to treat conflicting
> > expression data from multiplicate probe sets (that is a 
> gene that has >1
> > probe set designed against it (for example, 97569_r_at and 
> 97658_r_at are
> > both probes for the Insulin gene).
> 
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