[BioC] Illumina Methylation. Normalization and statistics

[Mark Dunning]

Hi all,

Has anyone tried doing analysis on log-ratios of the red and green
channels rather than beta? I would imagine they would suit existing
tools such as limma and the results can always be converted back to
betas afterwards. Just a thought.

Mark

On Thu, 2008-11-20 at 10:38 -0500, Sean Davis wrote:
> On Thu, Nov 20, 2008 at 8:57 AM, Michael Walter <
> michael.walter at med.uni-tuebingen.de> wrote:
> 
> > Dear Sean,
> >
> > Thanks for your answers. I have one array probed with fully methylated DNA
> > purchased by ZYMO. Here all beta values should be 1, which they aren't, of
> > course. Can I use these values to normalize the rest of my arrays? Let
> > assume my fully methylated value is 0.7 and my actual value is 0.5 then I
> > would correct my beta to 0.5/0.7?
> >
> 
> Hi, Mike.  That might work, but the approach we have taken is to correct the
> intensities which show a significant dye bias, so a significant bias in
> beta.
> 
> Sean
> 
> 
> 
> >
> >
> > > -----Ursprüngliche Nachricht-----
> > > Von: "Sean Davis" <sdavis2 at mail.nih.gov>
> > > Gesendet: 19.11.08 16:30:15
> > > An: "Michael Walter" <michael.walter at med.uni-tuebingen.de>
> > > CC: bioconductor at stat.math.ethz.ch
> > > Betreff: Re: [BioC] Illumina Methylation. Normalization and statistics
> >
> >
> > > On Wed, Nov 19, 2008 at 10:18 AM, Michael Walter <
> > > michael.walter at med.uni-tuebingen.de> wrote:
> > >
> > > > Dear List,
> > > >
> > > > We run our first slide of illumina's infinium methylation arrays. After
> > > > searching the archive, I still have some general questions how to best
> > > > analyze the data.
> > > >
> > > > First of all, I'm would like to know some opinion on normalization. In
> > my
> > > > personal and probably simplistic view I'd think that normalization is
> > not
> > > > necessary since the value you get from the array is a ratio which is
> > sample
> > > > inherent (unlike a classical two-color expression array where you mix
> > two
> > > > samples to generate the expression ratio). Is this assumption correct
> > or am
> > > > I missing some important aspect?
> > > >
> > >
> > > Unfortunately, there is a significant dye-bias issue.  That is, there is
> > a
> > > propensity for one dye to be brighter than the other and it appears that
> > > Illumina does not adequately correct for this bias.
> > >
> > >
> > > >
> > > > Anyway, I'd like to perform background normalization which results as
> > usual
> > > > with illumina arrays in some negative values. Does anyone one have a
> > neat
> > > > solution for this problem or shall I just skip the probes?
> > > >
> > >
> > > I have been just ignoring those probes.
> > >
> > >
> > > >
> > > > Do I have to correct for some dye effect like for the golden gate
> > > > methylation assay? Since the probes for methylated and unmethylated DNA
> > > > incorporate the same dye this shouldn't be an issue?
> > > >
> > >
> > > See above.
> > >
> > >
> > > >
> > > > My final question is basically the most pressing: What kind of
> > statistic
> > > > test should I use? Since all the values are ratios between 0 and 1 I
> > have a
> > > > real bad feeling by simply running some t-tests. And if a t-test is the
> > > > proper choice, shall I log-transform the data?
> > > >
> > >
> > > The t-statistic should still be valid, I think.  The assumptions that go
> > > into statistics like the t-stat are not based on the distribution of the
> > > data, but on differences between values.  I think these assumption
> > probably
> > > still holds in practice for these data.  However, I have not tried to
> > prove
> > > things one way or the other.  Of course, if you are concerned about,
> > > non-parametric testing will alleviate these concerns.
> > >
> > > Sean
> > >
> > >
> > > >
> > > > Any input and shared experience with this type of array is highly
> > > > appreciated.
> > > >
> > > >
> > > > Best Regards,
> > > >
> > > >
> > > > Mike
> > > > --
> > > > Dr. Michael Walter
> > > >
> > > > The Microarray Facility
> > > > University of Tuebingen
> > > > Calwerstr. 7
> > > > 72076  TÃŒbingen/GERMANY
> > > >
> > > > Tel.: +49 (0) 7071 29 83210
> > > > Fax. + 49 (0) 7071 29 5228
> > > >
> > > > Confidentiality Note:\ This message is intended only
> > for...{{dropped:9}}
> > > >
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> > >
> > >       [[alternative HTML version deleted]]
> > >
> > >
> > > <hr>
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> >
> > --
> > Dr. Michael Walter
> >
> > The Microarray Facility
> > University of Tuebingen
> > Calwerstr. 7
> > 72076  Tübingen/GERMANY
> >
> > Tel.: +49 (0) 7071 29 83210
> > Fax. + 49 (0) 7071 29 5228
> >
> > Confidentiality Note:\ This message is intended only for...{{dropped:9}}
> >
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