Question: Determining direction of logFC in limma
0
gravatar for pt2395
3.4 years ago by
pt23950
United States
pt23950 wrote:

I'm sure this is an easy question, but I'm fairly new to using limma and R and I'd appreciate any help or guidance. I've run a paired limma analysis trying to look at differentially expressed genes in a pre-post experiment design. I'm trying to understand what direction my logFC values represent i.e. whether the gene is being up regulated or down regulated from pre to post or post to pre. My code is as follows: 

write.exprs(e,file="expressionDataHFNIH5-7-14.txt") 

d<-read.table("expressionDataHFNIH5-7-14.txt", header=T, row.names=1)
names(d)<-gsub(".CEL", "", gsub("X", "", names(d)))
d<-round(d, 5)

write.table(d, file="filepath.txt", quote=F, sep='\t')

#create a design matrix
#congestion 1=pre and 2=post

design <- cbind(ID = c(1,1,2,2,7,7,8,8,9,9,10,10,11,11,14,14,16,16,20,20,21,21,22,22,23,23,24,24,25,25,26,26,27,27,28,28,29,29,30,30,33,33,34,34,35,35,36,36,37,37,39,39,40,40,41,41), Congestion=c(1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2,1,2))

ID<-factor(design[,1])
Congestion<-factor(design[,2], levels=c("1","2"))

#create a design matrix
design<-model.matrix(~ID+Congestion)

fit <- lmFit(e, design)
fit <- eBayes(fit)

topTable(fit, coef = "Congestion2", adjust = "fdr")

topt2<-topTable(fit, coef = "Congestion2", adjust.method = "fdr", n=Inf, sort.by="p")
write.table(topt2,file="filepath.txt",quote=F,sep='\t'

Thank you! 

 

 

limma logfc • 1.4k views
ADD COMMENTlink modified 3.4 years ago • written 3.4 years ago by pt23950
Answer: Determining direction of logFC in limma
1
gravatar for Aaron Lun
3.4 years ago by
Aaron Lun23k
Cambridge, United Kingdom
Aaron Lun23k wrote:

Your Congestion2 coefficient represents the log-fold change of your "2" samples over your "1" samples (i.e., post over pre). When you drop this coefficient during your DE comparison, the reported log-fold change corresponds to the value of this coefficient. Thus, you can interpret positive values as being upregulated in the post condition compared to pre, and negative values as being downregulated.

Incidentally, if you ever get confused, it's always a good idea to look at the expression values to figure out what's going on. In this case, you can pick a couple of the top DE genes and have a look at the direction of change within each patient for those genes. This is also a good way to check that you've set up the design and contrasts correctly for more complicated experiments; it never hurts to be on the safe side.

ADD COMMENTlink modified 3.4 years ago • written 3.4 years ago by Aaron Lun23k

Thank you! One more question for my understanding, is it post over pre because of the way in which I've ordered it in my levels and design matrix? So if I decided that I wanted to determine my log-fold change of my "1" samples over my "2" samples then I would need to interchange their order to derive that value? 

ah never mind you just answered it. Thank you so much for your help. 

 

ADD REPLYlink modified 3.4 years ago • written 3.4 years ago by pt23950
2

Yeah, model.matrix will choose the first level as the "reference". You should be able to flip it around by setting levels=c("2","1") in the construction of the Congestion variable, in which case you should end up with Congestion1 as the relevant coefficient to drop for your DE testing.

ADD REPLYlink modified 3.4 years ago • written 3.4 years ago by Aaron Lun23k

Thank you so much! This was really helpful. 

ADD REPLYlink written 3.4 years ago by pt23950
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 16.09
Traffic: 125 users visited in the last hour