Is limma suitable for identifying differentially expressed genes across species?

Question

[EvoGen]

For microarray gene expression data for

5 species: sp1, sp2, sp3, ...

7 time-points: t1, t2, t3, ...

3 replicates: r1, r2, r3, ...

And the aim is to identify genes differentially expressed genes w.r.t to sp1 (assume sp1 is reference species with whom I want compare expression values).

Using limma I compared created a contrast matrix to compare sp2-sp1, sp3-sp1, sp3-sp1.

Design matrix looks like:

design <- model.matrix(~0+ targets$Organism)
colnames(design) <- c("sp1","sp2","sp3","sp4","sp5","sp6")
head(design)
  sp1 sp2 sp3 sp4 sp5 sp6
1   0   1   0   0   0   0
2   0   1   0   0   0   0
3   0   1   0   0   0   0
4   0   1   0   0   0   0
5   0   1   0   0   0   0
6   0   1   0   0   0   0
tail(design)
    sp1 sp2 sp3 sp4 sp5 sp6
139   0   0   0   0   0   1
140   0   0   0   0   0   1
141   0   0   0   0   0   1
142   0   0   0   0   0   1
143   0   0   0   0   0   1
144   0   0   0   0   0   1

contrast.matrix <- makeContrasts(sp2-sp1,sp3-sp1,sp4-sp1,sp5-sp1,sp6-sp1,levels=design)
contrast.matrix
      Contrasts
Levels sp2 - sp1 sp3 - sp1 sp4 - sp1 sp5 - sp1 sp6 - sp1
   sp1        -1        -1        -1        -1        -1
   sp2         1         0         0         0         0
   sp3         0         1         0         0         0
   sp4         0         0         1         0         0
   sp5         0         0         0         1         0
   sp6         0         0         0         0         1

But for example for sp2-sp1 set, not only genes differentially expressed in sp2 but also differentially expressed in some other species e.g., sp4 is given as output.

So, I am not sure if limma package is suitable for such time-point and species specific data?

Can anyone suggest any other approach or package to identify genes differentially expressed across species?

[1234anjalianjali1234]

will limma work for samples without replicates?

Answer 1

[Devon Ryan]

Limma is a completely appropriate tool for this sort of experiment (in fact, it'd probably be the most commonly used tool). Just because a particular gene is differentially expressed between sp2 and sp1 doesn't mean it can't also be DE between sp4 and sp1 (or even between sp4 and sp2).

[EvoGen]

OK I completely agree with you that a gene can be DE in multiple species.

But would there be any means to identify or pin-point genes whose expression is conserved in sp1,sp3,sp4 but DE only for sp2?

What if I group sp1,sp2,sp4 together say into allsps and compare sp2-allsps, to get DE in sp2 against all the remaining species?

[Devon Ryan]

For that sort of thing it's often best to use something like WGCNA. I say "better" because I suspect there are a bunch of patterns like this that you'd like to look at and this is a convenient^* method to do that.

^*This does not imply that WGCNA is easy to use and/or understand/debug.