[BioC] Increase in CV of replicated spots after normalization?

[Wolfgang Huber]

Hi Jakob,

it can be misleading to look solely at the CV of replicates to assess 
normalization. Because if you did that, a normalization method that 
simply divided all your log-ratios by 2 would be twice as good, and one 
that sets everything to zero would be even better.

What I usually do is look at the distribution of F- or t-statistics per 
gene across arrays for some meaningful biological grouping of the 
samples. There need to be enough replicate arrays within each group for 
this.

Still, if you used a "reasonable" normalization method, it sounds it 
didn't work well on your data. It is hard to say more without more 
details on what you did and diagnostic plots etc.

Best regards
  Wolfgang


-------------------------------------
Wolfgang Huber
European Bioinformatics Institute
European Molecular Biology Laboratory
Cambridge CB10 1SD
England
Phone: +44 1223 494642
Fax:   +44 1223 494486
Http:  www.ebi.ac.uk/huber
-------------------------------------


Jakob Hedegaard wrote:
> Hi list
> 
>  
> 
> I am working on a data set from 24 arrays, where each array consist of
> 6.912 spots replicated pair wise at two different spatial locations.
> 
> For quality evaluation, I have calculated the CV of "raw" log-ratios for
> each pair wise replicated spot (13.824 points per array) and have
> observed the expected tendency of decreasing CV by increasing average
> spot intensity.
> 
> When calculating the CV for normalized data, I have observed that the CV
> has increased compared to CV for raw data. This essentially means that
> normalization is making data worse in terms of variance among replicated
> spots!
> 
>  
> 
> Has anybody observed something similar?
> 
> Is this what should be expected or does it indicate that the
> normalization is not optimally performed?
> 
>  
> 
> Looking forward hearing from you!
> 
> Jakob
> 
> -------------------------------------------------------------------
> 
> Jakob Hedegaard
> 
> Danish Institute of Agricultural Sciences
> 
> Department of Genetics and Biotechnology
> 
> Molecular Genetics and System Biology
> 
> Building K25
> 
> Research Centre Foulum
> 
> P.O. box 50
> 
> DK-8830 Tjele
> 
> Denmark
> 
> Tel: (+45)89991363
> 
> Fax: (+45)89991300
> 
>  
>