Hi Henrik,
thanks for your quick reply.
Our goal is to reproduce the results of Affymetrix Powertools by using
our own implementation (written in Java) for normalization and median
polish:
Until now we can reproduce the normalization and we did it as follows:
(Affymetrix 500k)
Normalization:
1) We executed the Affymetrix Powertools program "apt-cel-
transformer":
apt-cel-transformer -d Mapping250K_Nsp.cdf -c quant-norm.sketch=0 --
out-dir outputFolder inputFiles (all values are used for
normalization, not only pm)
2) Then, we executed the normalization step as well using
aroma.affymetrix:
cdf <- AffymetrixCdfFile$byChipType("Mapping250K_Nsp");
cs <- AffymetrixCelSet$byName("Project1", cdf=cdf);
qn <- QuantileNormalization(cs);
csN <- process(qn);
3) After that we extracted several, random chosen SNPs from the binary
CEL files and compared the results, which were identical for all three
approaches (ours, affymetrix powertools and aroma.affymetrix).
So far so good.
---------------------
The next step is the implementation of median polish where we run into
trouble:
Median Polish: (apt-cel-transformer is included in this script "apt-
probeset-summarize")
1) apt-probeset-summarize --cdf-file Mapping250K_Nsp.cdf --out-dir
outDirectory --cel-files inputFiles -a quant-norm.sketch=0,med-
polish,expr.genotype=true
2)aroma:
plm <- RmaPlm(csN, flavor="oligo");
fit(plm);
ces <- getChipEffectSet(plm);
unitNames <- getUnitNames(cdf);
theta <- extractTheta(ces,groups=1:2);
theta <- log2(theta);
dimnames(theta)[[1]] <- unitNames;
3)
Unfortunately, we could not reproduce neither the values from the
powertools nor from aroma.affymetrix (using oligo) with our
implementation. All are going in the same direction but we didn't get
mathematical exact results (Some SNPs are pretty close)
So the point where we stuck at the moment is if (1) we are missing a
step out what has to be done between normalization and median polish
(for example the log2 transformation is done at the powertools before
median polish and in aroma after median polish) or (2) we maybe have
an error in reasoning while reading out the values (extractTheta).
So thanks again for aroma.affymetrix, it helps a lot to interpret our
results.
Cheers Sebastian
On Dec 4, 3:32 am, Henrik Bengtsson <henrik.bengts...@aroma-
project.org> wrote:
> Hi Sebastian.
>
> 2010/12/3 Sebastian Schönherr <ratsc...@gmail.com>:
>
> > Hi there,
> > I've a question regarding to RMA in aroma:
> > In a first step we've compared the normalization method used in aroma
> > with the method used in the Affymetrix Powertools which gave us
> > identical results. (500K chip) ("QuantileNormalization(cs)" vs "apt-
> > cel-transformer")
>
> Thanks for this information. It is really useful. If you are not
> aware of it already, we're collecting results of this kind at:
>
> http://aroma-project.org/replication/
>
> Would you mind writing down/sharing the details on exactly how you did
> the comparison, e.g. one R/aroma script and one APT script? How do
> you conclude they give identical results, e.g. by correlation,
> graphical, ...?
>
> > So in a next step we wanted to test median polish as well (standard
> > flavor) on this data and compared it again to Affymetrix (using "apt-
> > probeset-summarize" with med-polish,expr.genotype=true). We tried hard
> > and tested a lot of configurations but we've never got the same
> > results. (Same trend, but slightly different in the digits after the
> > comma).
> > R-script, quite straight forward:
> > cdf <- AffymetrixCdfFile$byChipType("Mapping250K_Nsp");
> > cs <- AffymetrixCelSet$byName("testset", cdf=cdf);
> > qn <- QuantileNormalization(cs);
> > csN <- process(qn);
> > plm <- RmaPlm(csN);
> > fit(plm);
>
> Atually, the default for the RmaPlm class is actually
> flavor="affyPLM", which uses the robust linear model M-estimator as
> implemented by preprocessCore/affyPLM.
> Inhttp://aroma-project.org/replication/RMAwe show that aroma.affymetrix
> can reproduce the affyPLM estimates quiet well. To use the median
> polish estimator, use flavor="oligo".
> Inhttp://aroma-project.org/replication/gcRMAwe use that to show that
> aroma.affymetrix can reproduce gcrrma too. Likewise, justSNPRMA() for
> AffymetrixCelSet uses flavor="oligo" to reproduce SNPRMA pipeline of
> the oligo package, cf.http://aroma-project.org/replication/SNPRMA
>
> Hope this helps
>
> /Henrik
>
> > The background correction was omitted in both scripts
> > Any ideas?
> > Thanks for your help
> > Sebastian
>
> > --
> > When reporting problems on aroma.affymetrix, make sure 1) to run the latest
> > version of the package, 2) to report the output of sessionInfo() and
> > traceback(), and 3) to post a complete code example.
>
> > You received this message because you are subscribed to the Google Groups
> > "aroma.affymetrix" group with websitehttp://www.aroma-project.org/.
> > To post to this group, send email to aroma-affymetrix@googlegroups.com
> > To unsubscribe and other options, go tohttp://www.aroma-project.org/forum/
On Dec 4, 3:32 am, Henrik Bengtsson <henrik.bengts...@aroma-
project.org> wrote:
> Hi Sebastian.
>
> 2010/12/3 Sebastian Schönherr <ratsc...@gmail.com>:
>
> > Hi there,
> > I've a question regarding to RMA in aroma:
> > In a first step we've compared the normalization method used in aroma
> > with the method used in the Affymetrix Powertools which gave us
> > identical results. (500K chip) ("QuantileNormalization(cs)" vs "apt-
> > cel-transformer")
>
> Thanks for this information. It is really useful. If you are not
> aware of it already, we're collecting results of this kind at:
>
> http://aroma-project.org/replication/
>
> Would you mind writing down/sharing the details on exactly how you did
> the comparison, e.g. one R/aroma script and one APT script? How do
> you conclude they give identical results, e.g. by correlation,
> graphical, ...?
>
> > So in a next step we wanted to test median polish as well (standard
> > flavor) on this data and compared it again to Affymetrix (using "apt-
> > probeset-summarize" with med-polish,expr.genotype=true). We tried hard
> > and tested a lot of configurations but we've never got the same
> > results. (Same trend, but slightly different in the digits after the
> > comma).
> > R-script, quite straight forward:
> > cdf <- AffymetrixCdfFile$byChipType("Mapping250K_Nsp");
> > cs <- AffymetrixCelSet$byName("testset", cdf=cdf);
> > qn <- QuantileNormalization(cs);
> > csN <- process(qn);
> > plm <- RmaPlm(csN);
> > fit(plm);
>
> Atually, the default for the RmaPlm class is actually
> flavor="affyPLM", which uses the robust linear model M-estimator as
> implemented by preprocessCore/affyPLM.
> Inhttp://aroma-project.org/replication/RMAwe show that aroma.affymetrix
> can reproduce the affyPLM estimates quiet well. To use the median
> polish estimator, use flavor="oligo".
> Inhttp://aroma-project.org/replication/gcRMAwe use that to show that
> aroma.affymetrix can reproduce gcrrma too. Likewise, justSNPRMA() for
> AffymetrixCelSet uses flavor="oligo" to reproduce SNPRMA pipeline of
> the oligo package, cf.http://aroma-project.org/replication/SNPRMA
>
> Hope this helps
>
> /Henrik
>
> > The background correction was omitted in both scripts
> > Any ideas?
> > Thanks for your help
> > Sebastian
>
> > --
> > When reporting problems on aroma.affymetrix, make sure 1) to run the latest
> > version of the package, 2) to report the output of sessionInfo() and
> > traceback(), and 3) to post a complete code example.
>
> > You received this message because you are subscribed to the Google Groups
> > "aroma.affymetrix" group with websitehttp://www.aroma-project.org/.
> > To post to this group, send email to aroma-affymetrix@googlegroups.com
> > To unsubscribe and other options, go tohttp://www.aroma-project.org/forum/
--
When reporting problems on aroma.affymetrix, make sure 1) to run the latest
version of the package, 2) to report the output of sessionInfo() and
traceback(), and 3) to post a complete code example.
You received this message because you are subscribed to the Google Groups
"aroma.affymetrix" group with website http://www.aroma-project.org/.
To post to this group, send email to aroma-affymetrix@googlegroups.com
To unsubscribe and other options, go to http://www.aroma-project.org/forum/
