Question: Anova-like in Limma
0
5 months ago by
ivan.vechetti0 wrote:

Hello guys,

I currently have the following design: (Knockout=TAm and WT-Veh) in a time course (0,3,5 and 7 days)

> design
fTam.0d fTam.1d fTam.3d fTam.5d fTam.7d fVeh.0d fVeh.1d fVeh.3d fVeh.5d fVeh.7d
1        1       0       0       0       0       0       0       0       0       0
2        1       0       0       0       0       0       0       0       0       0
3        0       1       0       0       0       0       0       0       0       0
4        0       1       0       0       0       0       0       0       0       0
5        0       0       1       0       0       0       0       0       0       0
6        0       0       1       0       0       0       0       0       0       0
7        0       0       0       1       0       0       0       0       0       0
8        0       0       0       1       0       0       0       0       0       0
9        0       0       0       0       1       0       0       0       0       0
10       0       0       0       0       1       0       0       0       0       0
11       0       0       0       0       0       1       0       0       0       0
12       0       0       0       0       0       1       0       0       0       0
13       0       0       0       0       0       0       1       0       0       0
14       0       0       0       0       0       0       1       0       0       0
15       0       0       0       0       0       0       0       1       0       0
16       0       0       0       0       0       0       0       1       0       0
17       0       0       0       0       0       0       0       0       1       0
18       0       0       0       0       0       0       0       0       1       0
19       0       0       0       0       0       0       0       0       0       1
20       0       0       0       0       0       0       0       0       0       1


I want to compare within the genotype as well as time-course. I've been following the Limma section 9.6.1 (Time course with replicates) and my strategy looks like:

 > colnames(design)
[1] "Tam.0d" "Tam.1d" "Tam.3d" "Tam.5d" "Tam.7d" "Veh.0d" "Veh.1d" "Veh.3d" "Veh.5d" "Veh.7d"

> fit<- lmFit(dat, design)

> cont.Tam<- makeContrasts("Tam.1d-Tam.0d", "Tam.3d-Tam.0d", "Tam.5d-Tam.0d", "Tam.7d-Tam.0d", levels = design)

> fit2<- contrasts.fit(fit, cont.Tam)

> fit2<- eBayes(fit2)


I would do the same for the WT as in the manual. My question is, am I doing this right, to have an Anova-like test? If so, how can I get the differences? Based on I saw it would be:

>topTable(fit2, coef = 1, number=Inf, adjust.method = "BH")


I just want to make sure if I'm doing the proper design and comparisons and how to get what is actually changing.

limma • 119 views
modified 5 months ago by Gordon Smyth38k • written 5 months ago by ivan.vechetti0
Answer: Anova-like in Limma
1
5 months ago by
Gordon Smyth38k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth38k wrote:

Your analysis seems fine from the code you've shown. So far you have obtained a list of differentially expressed genes for KO at 1 day vs KO at 0 days. You can perform any other comparison you want in the same way.