[BioC] Another question about normalization of data

[Gustavo Fernández Bayón]

Hi everybody. 

Not so long ago, I asked in this list about some normalization issues. The question and its very interesting replies, from which I have learned a lot, can be found here:

http://comments.gmane.org/gmane.science.biology.informatics.conductor/41812

It seems to me that, the more I am getting into Bioinformatics, the less I know about everything. I usually doubt about everything, and I am always asking, step by step, if I am doing things correctly.

Now, I want to test some ideas on a 450K methylation array data base. Main idea is to try to classify probesets in families according to their behavior with respect to some phenotype variables. I have several ideas I would like to try on this data, and the first step has been to import, review, visualize and try to understand the global structure of the beta values I have at my hand. 

Once loaded, I have made two box plots. One shows the distribution of beta values among the 40 samples, and the other shows the distribution among the first 100 probesets. 

I have shared the plots at my Google Docs account:

https://docs.google.com/open?id=0Bw-_OWjrT9U4cTlZblR0UkVhWG8
https://docs.google.com/open?id=0Bw-_OWjrT9U4d3FTZTQtNWJFUVE



My question might sound stupid, but I want to deeply understand what is going on with these plots. 

For the beta vs. probeset:

- I guess the variability is normal. Some probes are methylated most of the time, some not, and there are a lot of differences in their behavior. This is the common behavior, isn't it? 
- Boxplot might not be the best solution here, because the distribution need not to be unimodal, I think. Am I right? 
- My intuition is that these values should be normalized in case we were going to use something like SVM-RFE to do probeset selection. Again, is my intuition right?

For the beta vs. sample:

- Data distribution seems more regular than in the other plot. Is that an effect of the underlying normalization that GenomeStudio does? Or is the way beta values across samples are supposed to behave? 
- Although they seem regular, there are still small differences among medians, which made me think. Would a normalization of this data benefit following experiments? 

In general, I would like to know if the plots show a normal behavior, if it is the expected one, or if I should kind of normalize them using a predefined or standard method.

Any help or hint will be extremely appreciated.

Regards,
Gustavo



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