edgeR: Correct pipeline for DE analysis with multiple conditions and batches

Question

[MT]

I'm using edgeR to test for DE genes in an expression matrix. For each sample if have a condition and a batch. I want to use the glm-functionality in edgeR to test for DE between conditions, while taking into account batches.

Some example data to show my problem. Say if have a count matrix, EM, of 10 sample with the following labels:

EM = EM # Imagine matrix of counts here...

conditions = c("con1", "con1", "con1", "con2", "con2", "con2", "con3", "con3", "con3", "con3")
batches = c("batch1", "batch2", "batch3", "batch1", "batch2", "batch3", "batch1", "con2", "con3", "con3")

The pipeline could look like this:

dge = DGEList(counts=EM) # Create object
dge = calcNormFactors(dge, method='TMM') # Normalize library sizes using TMM
design = model.matrix(~0+conditions+batches) # Create design matrix for glm
colnames(design) = c(levels(conditions), levels(batches)[2:length(levels(batches))]) # Set prettier column names
dge = estimateGLMCommonDisp(dge, design) # Estimate common dispersion
dge = estimateGLMTagwiseDisp(dge, design) # Estimate tagwise dispersion

fit = glmFit(dge,design) # Fit glm

pair_vector = sprintf("%s-%s", "con1", "con3") # Samples to be compared
pair_contrast = makeContrasts(contrasts=pair_vector, levels=design) # Make contrast

lrt = glmLRT(fit, contrast=pair_contrast) # Likelihood ratio test

My questions:

1. The design matrix: There is no baseline conditions, so I remove the intersect with the 0+. Is this necessary to do for batches as well, even though they are not directly used as contrasts?
2. Does the glmFit take into account norm-factors for library sizes?

[Devon Ryan]

Do you want to just compare "con3" and "con2" vs. "con1" or do all of the pairwise comparisons? Your setup is more targeted toward comparing things versus con1 and including an intercept rather than using contrasts.

[MT]

I want to test all pairwise comparisons between conditions (no condition can be considered baseline'). I omitted the for-loop for clarity.

Answer 1

[Devon Ryan]

There are two ways to go about this. Firstly, either allow an intercept and then just use a contrast for the cond3 to cond2 comparison, or don't allow an intercept and group things different (e.g., group=sprint("%s.%s",batch,condition)).

If we just allow an intercept then:

design <- model.matrix(~conditions+batches)
dge = estimateGLMCommonDisp(dge, design)
dge = estimateGLMTagwiseDisp(dge, design)
fit = glmFit(dge,design)
lrt2vs1 <- glmLRT(fit, coef=2)
lrt3vs1 <- glmLRT(fit, coef=3)
lrt3vs2 <- glmLRT(fit,contrast=c(0,-1,1,0,0))

BTW, have a read through the edgeR user's guide, particularly section 3.4.2, which has an almost identical example (it's a really well done user's guide).

[MT]

Thanks for the response! I would like to directly use the names of conditions when doing the testing, rather than using an Intercept column and coef - how would you go about that?

[Devon Ryan]

You can use names for the coef= argument, but I believe that contrast only takes numeric vectors and matrices. You should be able to use my example to determine how to do one without an intercept.

[super]

Hi Devon

I found a interesting but naive staff.

When I use

design1 <- model.matrix(~conditions) # with Intercept column
d <- estimateDisp(d, design, robust=TRUE)
fit <- glmFit(d, design)
lrt <- glmLRT(fit)

and

design2 <- model.matrix(~0+conditions) # without Intercept

e.g. design2 like

  conditionN conditionT
1          1          0
2          1          0
3          0          1
4          0          1

The design2 result is much more liberal than design1.

Why?

Which do you recommend? (I don't have muti-factor, the comparison is based on same timepoint).Thank you!

[Devon Ryan]

They end up being identical, though you MUST explicitly specify the contrast with the second method.

[super]

I got. design1 is

lrt <- glmLRT(fit)

coef = last column(default),

design2 is something like

lrt <- glmLRT(fit,contrast=(-1,1))

Am I right?

[Devon Ryan]

That looks correct at least

[dcrowe13]

I made a quick script for this.

pdacDGE <- function(group,ref.groups,comp.groups) {
  contrast <- c(0,0,0,0,0,0,0,0,0,0,0,0,0)
  for (i in 1:length(ref.groups)) {
    contrast[which(levels(group) == ref.groups[i], arr.ind = TRUE)] <- -(1/length(ref.groups))
  }
  for (j in 1:length(comp.groups)) {
    contrast[which(levels(group) == comp.groups[j], arr.ind = TRUE)] <- 1/length(comp.groups)
  }
  print(contrast)
  lrt<-glmLRT(glm.fit,contrast = contrast)
  tt<-topTags(lrt, n=500000, p.value = .05, sort.by = "logFC")
  print(contrast)
  print(length(rownames(tt)))
  return(tt)
}

[rse]

How can i run edgeR for multiple counts and sample info file using a for-loop? Thanks