[BioC] Limma with multiple samples but only 2 reps

[Matthew Hannah]

Lots of posts today....

Actually this may also be of interest to others with similar designs.

I remember reading on a previous thread that even with low (<3 reps) you
could still use a linear model to estimate significant changes. Rather
than just ask if this is really possible I decided to do it myself
before seeing what others have to say. I know limma shrinks sample
variance towards a pooled mean so in principle it should work.

I used 7 genotypes, 2 treatments first with 3 replicates and then with
just 2 replicates. For contrasts I compared each genotype untreated vs.
treated (ebfit p<0.01). On average 3 replica detected 2500 changes, 2
reps detected 60% of these + 15% false +ves. I haven't looked at the
effect of fdr correction or including fold change yet, but looking for
other opinions. (actual #'s for the 7 comparisons are below).

Obviously more reps are better, but the planned analysis isn't my data,
just a suggestion for a collegue. Hopefully with a view to lowering the
arbitary cutoff from 2x, to include some consistent changes that just
miss the cutoff at present. Their design is slightly different - 10-15
treatments + 1 control x 2 reps.

Thanks,
Matt

3rep	Both	2rep
1146	1386	161
1004	1509	248
1103	2006	404
899	1013	185
772	1258	197
958	1887	402
1165	1795	298

Average
1007	1551	271