[BioC] RNAseq expression analysis using DESeq: technical replicates, paired samples

[Simon Anders]

Dear Michael

On 2011-11-07 22:56, Michael Muratet wrote:
> I would like to verify that 18 months later, adding counts for technical
> replicates is still the best approach for combining technical replicates.
>
> We are constrained to single biological replicates, but we're interested
> in using the technical replicates if we can. I recall that limma had
> ways to set up the model matrix.

Your question sounds as if you consider the issue as a technical 
limitation of the available methods. Unfortunately, it is not, and I am 
afraid, 18 months are not enough time for fundamental principles of 
statistics to change.

If you want to know whether a difference between control and treatment 
may be attributed to the treatment, you need to verify that this 
difference is large compared to what you expect from random biological 
variation, and without biological replicates you cannot know the extent 
of biological variation.

Of course, you can perform the analysis on the level of technical 
replicates, as you can with microarrays in limma. Then, you will get 
lots of results, but any call of significant differences may only be 
interpreted to mean that your two biological samples differ in this 
gene. You may not infer that the difference is more likely to be the 
result of your treatment than just due to chance differences between the 
samples.

Best regards
   Simon