On Wed, Nov 19, 2008 at 10:18 AM, Michael Walter <
michael.walter@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  Tübingen/GERMANY
>
> Tel.: +49 (0) 7071 29 83210
> Fax. + 49 (0) 7071 29 5228
>
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