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Question: limma- time course multiple treatments
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gravatar for harelarik
27 days ago by
harelarik0
harelarik0 wrote:

Hi,

We would like to test: control, treatment1, and treatment2 in a time course experiment using only few time points.

However,  chapter 9.6.1 of limma guide that discusses time-course for few time points, illustrates how to test only two treatments (WT vs. a mutant).

Questions:

1. I wonder if the described approach in this chapter is true also for multiple treatments?

2. If the answer is yes please see what I wrote in section 1-2 below. Is that the correct way to compare multiple treatments over a time course experiment?
Is the 
contrast.matrix I have used below correct?
 

 

-----------------------------------------------------------------

DETAILS:

0. Data from RNA-seq experiment.

 

1. Design matrix (two time points :1,6 hr; treatments: control, coldStress, heatStress; triplicate for each treatment):

  cont_1hr cont_6hr cold_1hr cold_6hr heat_1hr heat_6hr

1        1       0      0      0      0      0

2        1       0      0      0      0      0

3        1       0      0      0      0      0

4        0       1      0      0      0      0

5        0       1      0      0      0      0

6        0       1      0      0      0      0

7        0       0      1      0      0      0

8        0       0      1      0      0      0

9        0       0      1      0      0      0

10       0       0      0      1      0      0

11       0       0      0      1      0      0

12       0       0      0      1      0      0

13       0       0      0      0      1      0

14       0       0      0      0      1      0

15       0       0      0      0      1      0

16       0       0      0      0      0      1

17       0       0      0      0      0      1

18       0       0      0      0      0      1

 

 

2. Please see below, Is that a correct way to compare multiple treatments in a time course? 

The series of commands below correspond to chapter 9.6.1 of limma guide.

Please note the contrast.matrix.

 

lev<-c("cont_1hr","cont_6hr","heat_1hr","heat_6hr","cold_1hr","cold_6hr")

f <- factor(all_groups_repeats, levels=lev)

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

colnames(design) <- lev

 

v <- voom(rawCounts, design, plot=FALSE)

fit <- lmFit(v , design)

 

#Is this a correct contrast matrix?

contrast.matrix <- makeContrasts(heat_1hr-cont_1hr,heat_6hr- cont_6hr, cold_1hr- cont_1hr, cold_6hr- cont_1hr,  levels=design)

 

fit2 <- contrasts.fit(fit, contrast.matrix)

fit2 <- eBayes(fit2)

 

fit2<-treat(fit2,lfc=1)#Check parameters!!!

wt<-decideTests(fit2,p.value = 0.1, adjust.method = "BH",  method = "nested")

summary(wt)#Produce summary table

 

Thank you very  much for your support!!!

 

 

ADD COMMENTlink written 27 days ago by harelarik0

Help anyone?

ADD REPLYlink written 24 days ago by harelarik0
  • Help anyone??
ADD REPLYlink modified 15 days ago • written 21 days ago by harelarik0

Help anyone???

ADD REPLYlink written 23 hours ago by harelarik0
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