Hi Simon,

Le 09/05/12 22:12, Simon Anders a écrit :
> Hi Marie
>
>> We are wondering what is the best procedure to prepare this dataset for
>> this analysis (steps of normalization + variance estimation):
>> 1) is it better to start with normalizing + estimating dispersion on the
>> whole dataset (5 points + 3 controls), and then to test for differential
>> expression in
>> the two by two comparisons just mentionned
>> 2) or is it better to normalize + estimate dispersion on restricted
>> datasets composed of 1 time-point + 3 controls, and then test for
>> differential expression between this time point and the controls.
>
> It makes no difference: Only replicated samples contain information 
> about variance, and hence, DESeq ignores your non-controls anyway when 
> estimating dispersion.
Thanks for your quick answer! I do agree that this experimental design 
is suboptimal. [Things will be improved for the next experiments]

In our dataset, we tried both procedure and we do see a difference in 
the DESeq output. Maybe, as you said, the  estimation of dispersion is 
the same for both procedures, but the normalization step (estimation of 
size Factors ) gives different outputs when using complete or partial 
tables (with only a subset of the samples)?

Thanks for your help!

Marie


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