Question

edgeR glmLRT and interaction effect: an example

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David R ▴ 90

@david-rengel-6321

Last seen 10 months ago

European Union

Hi,

I am analyzing some RNA-Seq data involving three genotypes and 5 time points.

I am particularly interested in those genes for whom time does not present the same effect in all genotypes, that is, genes with a significant interaction genotype:time.

Please find here below a small code summarizing what I have attempted (for info, genotypes are "Col", "KO" and "TAP")

I find the results rather puzzling cause:

1-Only a few genes are significant for the interaction Col:KO or Col:TAP

2-IF I have a closer look at certain genes, I cannot see (a) why either the interaction effect has not been called as significant (e.g. the upper gene in the attached file) or (b) why it is significant in Col (blue line) vs KO (red), but not vs TAP (green) on the bottom gene.

I would be very grateful if you could provide me with any feedback on this matter.

Best,

David Rengel

library(edgeR)
dge <- DGEList(counts=counts.F, group=design.factor)
dge <- calcNormFactors(dge)

design=as.data.frame(cbind(rep(c("Col","KO","TAP"),each=15),rep(rep(c("0h","1h","2h","4h","6h"),each=3),3)))
rownames(design)=colnames(counts.F)
colnames(design)=c("gtype","time")
design=model.matrix(~gtype*time,data=design)
rownames(design)=colnames(counts.F)

dge <- estimateGLMCommonDisp(dge,design,verbose=TRUE)
dge <- estimateGLMTrendedDisp(dge,design)
dge <- estimateGLMTagwiseDisp(dge,design)

fit <- glmFit (dge,design)

my.contrasts=as.list(c(1:5))
names(my.contrasts) <- c("KO","TAP","t","it.KO","it.TAP")
my.contrasts[[1]] <- 2 # KO genotype vs Col genotype at t=0
my.contrasts[[2]] <- 3 # TAP genotype vs Col genotype at t=0
my.contrasts[[3]] <- c(4:7) # Time effect, at any time point, on Col genotype
my.contrasts[[4]] <- c(8,10,12,14) # The interaction effect of time in KO gtype, as compared to time in Col genotype.
my.contrasts[[5]] <- c(9,11,13,15) # The interaction effect of time in TAP gtype, as compared to time in Col genotype.

lrt=my.contrasts
for (i in 1:length(my.contrasts))
lrt[[i]] <- glmLRT(fit, coef=my.contrasts[[i]])

rna-seq edger glmlrt interactions • 4.6k views

ADD COMMENT • link updated 8.6 years ago by Gordon Smyth 51k • written 8.6 years ago by David R ▴ 90

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Hi again,

I guess I did not properly attach the image. Here is the link: http://imgur.com/HIv3sbH

ADD REPLY • link 8.6 years ago David R ▴ 90

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For your first question, I don't understand what you mean by a Col:KO or Col:TAP interaction. Col, KO and TAP are levels of the same factor, so you shouldn't get any interaction terms between them, because each sample should only be one or the other. For your second question, you didn't specify which contrast was significant or not significant using for each picture, e.g., for the first picture, is it Col, TAP or KO that isn't significant? Or all of them? What is the "Col vs KO" you refer to in the second picture?

ADD REPLY • link 8.6 years ago Aaron Lun ★ 28k

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Hi Aaron,

Thanks for your post. I was not clear enoguh: I did not mean interaction between two levels of the same factor, I meant the interaction of time: genotype: i.e. does time play any different role in KO or in TAP, as compared to the role played in Col? Concerning the first picture, none of the effects are significant. What I meant in the second picture is that my test considered the blu and the curves significantly different, but not the blue and the green.

ADD REPLY • link 8.6 years ago David R ▴ 90

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Also, if you want to add to your original post, try to use "add comment" rather than adding an answer.

ADD REPLY • link 8.6 years ago Aaron Lun ★ 28k

score 3 · Answer 1 · 2016-03-31

Well, I can see a few issues.

First, you are plotting cpm values on the unlogged scale, whereas the linear model is being fitted on the log-scale. The interaction for the the top gene AT1G01120 would be far less apparent on the log-scale.

Second, it is impossible to detect an interaction between two treatments if the gene is not at all expressed in one of the treatments. This is because the time effects are indeterminate for that treatment.

Third, I think you may be attributing interpretations to parameters in the linear model that they don't have. In particular, you should not ascribe any meaning to the "main effects" in an interaction model.

As I have explained many times on this support site, I think that classical factorial models are actively misleading and pretty much useless for analyzing gene expression experiments. I think it would be more meaningful to treat your experiment as having 15 groups. Setup a single factor to distinguish the 15 groups, then form contrasts between the groups to test the hypotheses that are of interest to you. This approach is statistically equivalent to the factorial model, but has the advantage that each contrast you form has an unambiguous meaning. For example, if you want to test expression is higher in Col vs KO overall, then you would form the contrast:

(Col0+Col1+Col2+Col4+Col6-KO0-KO1-KO2-KO4-KO6)/5

This would give the average log2FC for Col vs KO, averaged over the 5 times. See also Section 3.3.1 of the edgeR User's Guide.