Hi Gustavo,

Normalization, at least when you do it the "lumi" way is done at the probe
level, and as each locus is assigned to a fixed color, the normalization is
actually done over the M and U values. So if you have real raw data you can
pull it through your pipeline: color adjustment, background subtraction,
normalization. The color adjustment takes the red and green signals into
account and from my understanding this is done to make the loci more
comparable.

Q3) Sure that is alright. If you suspect low quality, you can use
HumMeth27QCReport (a CRAN package) to plot some of the control probe values
or you can ask if they have some control plots from GenomeStudio. Ofcourse
it could also be possible that the sample population is not suitable for
finding DMR's (like very heterogeneous disease vs control)

Disclaimer: Im still a bachelor student, so dont make me accountable for
any mistakes ;-) I'm sure there is someone around there with more wisdom,
who can answer your questions more thoroughly.

Cheers,

Djie


2012/7/19 Gustavo Fernández Bayón <gbayon@gmail.com>

> Hi everybody.
>
> Imagine I have my ideal, conceptual, pipeline for working with my Illumina
> 27k data:
>
> A) Bead level intensities
>
> ---> (color adjustment, background subtraction, normalization, …)
>
> ---> B) Meth and Unmeth intensities
>
> ---> (get ratios)
>
> ----> C) Beta values or M-values
>
> As I understand, proper normalization, if needed, should be done over the
> Red and Green signals (bead level signals, I know there are two types of
> beads, M and U, for each locus, and that each locus is assigned to a fixed
> color channel. I have studied the code in the minfi package, although it is
> for 450k, just to understand how these values get converted).
>
> Q1) Am I right? Or, is there any way we can normalize by using the M and U
> signals?
>
> Q2) As an example, I have downloaded raw data from a given GEO dataset,
> and found only the M and U (Signal A and B, in their nomenclature) data. At
> that, point, I think I cannot do neither normalization nor QC, can I? What
> happens if somebody tries to normalize these signals and, for example,
> color balance was not adjusted? Or does GenomeStudio care about that?
>
> Q3) People from another lab want us to take a look to some methylation
> datasets they have, and I am planning (having seen the facts above) to ask
> them for the .txt or .idat files, in order to do QC and normalization with
> lumi. Is that ok? Or is there any alternative? They claim they are having a
> lot of problems for finding DMR's, and I suspect they have low-quality
> data, and maybe that they are not following a correct pipeline.
>
> Regards,
> Gustavo
>
> ---------------------------
> Enviado con Sparrow (http://www.sparrowmailapp.com/?sig)
>
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