Question: Interpreting Fold Change Calculation in Limma to Non-Statisticians: Sanity Check
0
5 months ago by
JMallory0
JMallory0 wrote:

I have seen this question posed elsewhere, but wanted to double check for some of my colleagues who want to make sure. It is the following: when defining contrasts in Limma, how is the syntax to be interpreted? I've included some code. If clarification is required, I can provide that as needed.

#### edata is expression matrix of aptamer probes; design is the design matrix; Used a repeated measures design so estimated the patient-wise correlation in expression over visits where StudyID defines this pairing ; pheno_table details sample metadata

fit<-lmFit(edata,design,
block=pheno_table$StudyID, correlation=corfit$consensus)

cm <- makeContrasts(
Diabetpost.PUFA.Met-Diabetpre.PUFA.Met = Diabetpost.PUFA.Met-Diabetpre.PUFA.Met,
Diabetpost.MUFA.Met-Diabetpre.MUFA.Met = Diabetpost.MUFA.Met-Diabetpre.MUFA.Met,
Diabetpost.PUFA.NoMet-Diabetpre.PUFA.NoMet = Diabetpost.PUFA.NoMet-Diabetpre.PUFA.NoMet,
Diabetpost.MUFA.NoMet-Diabetpre.MUFA.NoMet = Diabetpost.MUFA.NoMet-Diabetpre.MUFA.NoMet,
levels=design)

fit2 <- contrasts.fit(fit, cm)
fit2 <- eBayes(fit2,robust=TRUE)
T<-topTable(fit2,number=100000)



In plain English, are the above comparisons made "between post with respect to pre" or "between pre with respect to post"? That is, would a positive logFC indicate post > pre or pre > post? Again, I believe I know the answer, but I would like confirmation for my colleagues. Thanks.

proteomics limma • 196 views
modified 4 months ago by LKal0 • written 5 months ago by JMallory0
Answer: Interpreting Fold Change Calculation in Limma to Non-Statisticians: Sanity Check
1
5 months ago by
Aaron Lun25k
Cambridge, United Kingdom
Aaron Lun25k wrote:

It's easy to understand in terms of how you called makeContrasts. Take your first comparison:

    Diabetpost.PUFA.Met-Diabetpre.PUFA.Met = Diabetpost.PUFA.Met-Diabetpre.PUFA.Met,


The log-fold change will be computed as Diabetpost.PUFA.Met-Diabetpre.PUFA.Met, i.e., the difference between the two coefficients. So if the log-fold change is positive, you've basically got:

Diabetpost.PUFA.Met - Diabetpre.PUFA.Met > 0 # i.e.,
Diabetpost.PUFA.Met > Diabetpre.PUFA.Met


So a positive log-fold change would represent an increase in "Post" relative to "Pre". And so forth.

Thanks Aaron. This is exactly what I was looking for.

Answer: Interpreting Fold Change Calculation in Limma to Non-Statisticians: Sanity Check
0
4 months ago by
LKal0
LKal0 wrote:

I have also wondered this question. How I came to my solution was to check a single gene that had a log-fold change and then go look at the CPM values for the same gene. It is very easy to tell by this method and there is basically no chance to misinterpret the answer.