Question: differentially expressed genes without contrasts
0
gravatar for hoe
2.3 years ago by
hoe0
hoe0 wrote:

Hi

I'm confused about how to identify differentially expressed genes (DEGs) in a single-channel expt.

Is it possible to identify DEGs without making a contrast.matrix?

I saw examples just did:

fit <- lmFit(...)

fit <- eBayes(fit)

tt <- topTable(fit, coef=2)

All DEGs are stored in tt

However, when I looked up the limma user guide (version 2016) in section 9.6, the example used contrast.matrix.

Can someone help?

Thanks Eric.

 

ADD COMMENTlink modified 2.3 years ago by Ryan C. Thompson7.4k • written 2.3 years ago by hoe0
Answer: differentially expressed genes without contrasts
1
gravatar for Ryan C. Thompson
2.3 years ago by
The Scripps Research Institute, La Jolla, CA
Ryan C. Thompson7.4k wrote:

If one of your model coefficients already corresponds to the hypothesis you want to test, then you do not need a contrast. Otherwise, the purpose of contrasts is to express the linear combination of coefficients that represents your hypothesis.

ADD COMMENTlink written 2.3 years ago by Ryan C. Thompson7.4k

I think my answer can be found in chapter 9 (2016 edition) on p.40-41. For a fixed reference group, there is no need to construct a contrast.matrix because eBayes and topTable always use 'intercept' or coef=1 as the reference for comparison. For this scenario, design <- model.matrix(~conditions). conditions is a factor variable defining the experimental conditions including the reference.

However, if a fixed reference group is not applicable for your problem. More flexibility can be accommodated by building a contrast.matrix. In such case, you can contrast different experimental conditions. The design must be defined as:

design <- model.matrix(~0+conditions)

In such case, you can compare e.g. condition3-condition2, condition2-condition1, by

cont.matrix <- makeContrasts(C3vsC2=condition3-condition2, C2vsC1=condition2-condition1, levels=design)

ADD REPLYlink written 2.3 years ago by hoe0
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