[BioC] aroma.affymetrix and affy - different results
schmidi
schmidberger at gmx.at
Mon Jul 7 09:43:59 CEST 2008
Hi Ken,
you are right. I removed the normalised signals in the probeData
directory and re-run:
Now I have these results:
> all.equal(exprs(affyBatch_bgc),exprs(affyBatch_aroma_bgc))
[1] "Attributes: < Component 2: Component 2: 10 string mismatches >"
[2] "Mean relative difference: 2.300415e-08"
> all.equal(exprs(affyBatch_norm),exprs(affyBatch_aroma_norm))
[1] "Attributes: < Component 2: Component 2: 10 string mismatches >"
[2] "Mean relative difference: 0.000647665"
But there is still an error of dimension 10^(-4). This is very much
for two methods, which should create the same results. But the raw
data only hava an accuracy of 10^(-1), so I think this could be OK.
I also had a look to some quality plots (hist and boxplot), the plots
look very well.
Best
Markus
On Jul 4, 2:58 pm, "Ken Simpson" <ken.m.simp... at gmail.com> wrote:
> Hi Markus,
>
> Please make sure that process() is actually updating the signals in the
> normalized CEL files. I strongly suspect that the cached signals are being
> returned. If in doubt, remove the normalised signals in the probeData
> directory and re-run. I am almost 100% sure that you will get consistent
> results after the re-run.
>
> Cheers,
>
> Ken
>
> On Fri, Jul 4, 2008 at 10:54 PM, schmidi <schmidber... at gmx.at> wrote:
>
> > Hi,
>
> > this was only a testcode, therefore I did quantile normalization at
> > the unadjusted signals.
>
> > Now I used
> > qn <- QuantileNormalization(cs, typesToUpdate="pm")
> > and still get a BIG DIFFERENCES:
> > all.equal(exprs(affyBatch_norm), exprs(affyBatch_aroma_norm))
> > [1] "Attributes: < Component 2: Component 2: 10 string mismatches >"
> > [2] "Mean relative difference: 0.2979911"
>
> > The differences in the background corrected sig
>
> > Best
> > Markus
>
> > On Jul 4, 2:16 pm, "Ken Simpson" <ken.m.simp... at gmail.com> wrote:
> > > Further to Mark's email, it is not the probe ordering that is the problem
> > > (note that the difference in the background corrected signals is ~ 1e-8),
> > > but the following line:
>
> > > > qn <- QuantileNormalization(cs, typesToAvg="pm")
>
> > > This is telling aroma.affymetrix to average the quantiles from the PM
> > only
> > > to get the target distribution. However, by default, the normalisation
> > will
> > > adjust *all* the signals using the target distribution, not just the PM -
> > > hence the difference Markus is seeing. If this line is changed to
>
> > > > qn <- QuantileNormalization(cs, typesToUpdate="pm")
>
> > > then you should see very little difference between the two
> > implementations
> > > (~ 1e-5 in the test run I did). By default, typesToAvg = typesToUpdate
> > and
> > > so this call will average the PM quantiles (typesToAvg="pm"), and update
> > > *only* the PM signals (typesToUpdate="pm").
>
> > > (Markus - I'm not sure why you performed background correction but then
> > > quantile normalised the unadjusted signals - was this just for testing
> > > purposes?)
>
> > > Cheers,
>
> > > Ken
>
> > > On Fri, Jul 4, 2008 at 10:07 AM, Mark Robinson <mrobin... at wehi.edu.au>
> > > wrote:
>
> > > > Markus.
>
> > > > The aroma.affymetrix processing is based on the defaults of affyPLM's
> > > > 'fitPLM', which is: background.method = "RMA.2" and normalize.method =
> > > > "quantile".
>
> > > > Here is a test script that I wrote about a year ago (so take it with a
> > > > grain of salt):
> > > >http://bioinf.wehi.edu.au/folders/mrobinson/for_hb/u133.test.mark.R
>
> > > > My test was based on the chip effects and they matched up, so
> > > > presumably the BG adjustment and normalization also matched. I
> > > > believe a variation of this test is used everytime aroma.affymetrix is
> > > > built. Note I haven't used the 'extractAffyBatch' and I'm not sure if
> > > > that introduces any differences in the order, etc.
>
> > > > Sorry, I haven't answered your question as I don't have access to run
> > > > my code at the moment ... but hopefully this give you an avenue to
> > > > check.
>
> > > > Mark
>
> > > > On 03/07/2008, at 10:49 PM, Markus Schmidberger wrote:
>
> > > > > Hi,
>
> > > > > I compared results from aroma.affymetrix and affy. Especially for
> > > > > rma background correction and quantil normalization. But there are
> > > > > DIFFERENCES!
>
> > > > > Any ideas for these differences?
> > > > > Attached my test code.
>
> > > > > Best
> > > > > Markus
>
> > > > > ########################################################
> > > > > Code:
>
> > > > > cs <- AffymetrixCelSet$fromName("test", chipType="HG-U133_Plus_2")
> > > > > cs <- extract(cs, 1:10)
> > > > > bc <- RmaBackgroundCorrection(cs)
> > > > > csBC <- process(bc)
> > > > > affyBatch_aroma_bgc <- extractAffyBatch(csBC)
>
> > > > > qn <- QuantileNormalization(cs, typesToAvg="pm")
> > > > > csN <- process(qn)
> > > > > affyBatch_aroma_norm <- extractAffyBatch(csN)
>
> > > > > pathCELFiles <- "/home/schmidb/tmp/rawData/test/HG-U133_Plus_2"
> > > > > celFile <- list.celfiles(path=pathCELFiles,full.names=TRUE);
> > > > > affyBatch <- ReadAffy(filenames=celFile[1:10])
> > > > > affyBatch_bgc <- bg.correct(affyBatch, method="rma")
> > > > > affyBatch_norm <- normalize.AffyBatch.quantiles(affyBatch,
> > > > > type="pmonly")
>
> > > > > ###########################################################
> > > > > Comparison:
>
> > > > > all.equal(exprs(affyBatch_bgc), exprs(affyBatch_aroma_bgc))
> > > > > [1] "Attributes: < Component 2: Component 2: 10 string mismatches
> > >"
> > > > > [2] "Mean relative difference: 2.300415e-08"
> > > > > all.equal(exprs(affyBatch_norm), exprs(affyBatch_aroma_norm))
> > > > > [1] "Attributes: < Component 2: Component 2: 10 string mismatches >"
> > > > > [2] "Mean relative difference: 0.2979911"
>
> > > > > (sampleNames are the same!)
>
> > > > > ####################################################
> > > > > sessionInfo()
> > > > > R version 2.7.1 (2008-06-23)
> > > > > x86_64-unknown-linux-gnu
>
> > > > > locale:
> > > > > LC_CTYPE
> > > > > =
> > > > > de_DE
> > > > > .UTF
> > > > > -8
> > > > > ;LC_NUMERIC
> > > > > =
> > > > > C
> > > > > ;LC_TIME
> > > > > =
> > > > > de_DE
> > > > > .UTF
> > > > > -8
> > > > > ;LC_COLLATE
> > > > > =
> > > > > de_DE
> > > > > .UTF
> > > > > -8
> > > > > ;LC_MONETARY
> > > > > =
> > > > > C
> > > > > ;LC_MESSAGES
> > > > > =
> > > > > de_DE
> > > > > .UTF
> > > > > -8
> > > > > ;LC_PAPER
> > > > > =
> > > > > de_DE
> > > > > .UTF
> > > > > -8
> > > > > ;LC_NAME
> > > > > =
> > > > > C
> > > > > ;LC_ADDRESS
> > > > > =C;LC_TELEPHONE=C;LC_MEASUREMENT=de_DE.UTF-8;LC_IDENTIFICATION=C
>
> > > > > attached base packages:
> > > > > [1] tools stats graphics grDevices utils datasets
> > > > > methods [8] base
> > > > > other attached packages:
> > > > > [1] hgu133plus2cdf_2.2.0 affyPara_1.1.5
> > > > > snow_0.3-0 [4] aroma.affymetrix_0.9.3
> > > > > aroma.apd_0.1.3 R.huge_0.1.5 [7]
> > > > > affxparser_1.12.2 aroma.core_0.9.3 sfit_0.1.5
> > > > > [10] aroma.light_1.8.1 digest_0.3.1
> > > > > matrixStats_0.1.2 [13] R.rsp_0.3.4
> > > > > R.cache_0.1.7 R.utils_1.0.2 [16]
> > > > > R.oo_1.4.3 R.methodsS3_1.0.1 affy_1.18.2
> > > > > [19] preprocessCore_1.2.0 affyio_1.8.0 Biobase_2.0.1
> > > > > --
> > > > > Dipl.-Tech. Math. Markus Schmidberger
>
> > > > > Ludwig-Maximilians-Universität München
> > > > > IBE - Institut für medizinische Informationsverarbeitung,
> > > > > Biometrie und Epidemiologie
> > > > > Marchioninistr. 15, D-81377 Muenchen
> > > > > URL:http://ibe.web.med.uni-muenchen.deMail:Markus.Schmidberger
> > > > > [at] ibe.med.uni-muenchen.de
> > > > > Tel: +49 (089) 7095 - 4599
>
> > > > > _______________________________________________
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>
> > > > ------------------------------
> > > > Mark Robinson
> > > > Epigenetics Laboratory, Garvan
> > > > Bioinformatics Division, WEHI
> > > > e: m.robin... at garvan.org.au
> > > > e: mrobin... at wehi.edu.au
> > > > p: 03 9345 2628
> > > > f: 03 9347 0852
> > > > ------------------------------
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