Question: limma voom analysis with multiple comparisons
1
gravatar for ea1402
3.2 years ago by
ea140210
ea140210 wrote:

Hi,

I have an experiment where I have treated a cell line with two drugs A and B, their combination AB, as well as control C. So I have 4 treatments but also 3 time point time=3,9 and 24 hours. At each condition and time point i.e. A_3 I have 3 treatments. My goal is a dynamic analysis as such at each time point (3,9,24) and treatment (A,B,AB) I want to find DE genes wrt to control C at that time point.

I have two options:

(1) Run all of the voom-limma (including calcnorm factors and mean variance trend) pipeline separately for each comparision. i.e (A_3 -C_3 etc.) and get DE genes.

(2) Combine all the data into one matrix and run voom-limma pipeline on the whole matrix once then run contrast.fit for each comparison.

When I try to run both ways and compare the results the correlation between t_score (column 3 of toptable output) is 0.98. However, the magnitude varies significantly same gene has a tscore of 39 in (2) vs 25 (1), such are the p.values. Rank wise they look ok but effect wise they are different. I am not quite sure whether I gain power by applying (2) or lose power. Or which way is preferable. Any help is appreciated.

Thanks

voom limma • 1.6k views
ADD COMMENTlink modified 3.2 years ago • written 3.2 years ago by ea140210

You have mentioned some important points. The rank does not change much but the effective size has changed. That's because by pooling all of the data you increase the degree of freedoms for fitting linear model and make variance estimation for shrinkage more accurate.

ADD REPLYlink modified 3.2 years ago • written 3.2 years ago by GZ1995350

Thanks for the reply. My intuition was the same but what consufed me more is the t-statistics in case (2) are much higher than in (1) when I expected the opposite. Whats your intuition on that?

ADD REPLYlink written 3.2 years ago by ea140210

It is hard to say because increasing n does not necessarily lead to decrease of moderate t-statistics.

ADD REPLYlink written 3.2 years ago by GZ1995350

Hello ea1402!

It appears that your post has been cross-posted to another site: https://support.bioconductor.org/p/88905/

This is typically not recommended as it runs the risk of annoying people in both communities.

ADD REPLYlink written 3.2 years ago by WouterDeCoster42k
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