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

[Claus Mayer]

Hi Wolfgang and Jakob

I think there is some confusion here. The CV is (at least as far as I 
know) standard deviation divided by mean, so it is scale-invariant, i.e 
dividing all log-ratios by 2 shouldn't make a difference. It is not 
location-invariant though, which could be the explanation for the 
increased CV. The normalisation centers the log-ratio distribution, so 
for most genes the mean should be closer to 0 than before, which will 
result in an increased CV.
For that reason the CV is not an appropriate tool here to assess the 
effect of the normalisation. As Wolfgang points out, the distribution 
of  F- or t-statistics (or the corresponding p-values)  should be a 
reasonable (and scale-invariant!) exploratory tool to assess the sucess 
of the normalisation.

Best Wishes

Claus


Wolfgang Huber wrote:

>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
>
>
>
>
>
>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
>>
>>    
>>


-- 
***********************************************************************************
 Claus-D. Mayer                       | http://www.bioss.ac.uk
 Biomathematics & Statistics Scotland | email: claus at bioss.ac.uk
 Rowett Research Institute            | Telephone: +44 (0) 1224 716652
 Aberdeen AB21 9SB, Scotland, UK.     | Fax: +44 (0) 1224 715349