Question: Reordering factor levels in DESeq2 causes results error in cleanContrast
gravatar for Chris Stubben
8 months ago by
Salt Lake City, Utah
Chris Stubben40 wrote:

I recently updated to R version 3.4.2 and noticed that DESeq2 results now returns an error if I relevel the factors.  

cnts <- matrix(rnbinom(n=1000, mu=100, size=1/0.5), ncol=10)
samp <- data.frame( cond=factor(rep(c("a", "b"), each=5)))
dds <- DESeqDataSetFromMatrix(cnts, samp, ~ cond)
dds <- DESeq(dds)

# [1] a a a a a b b b b b
# Levels: a b

## works
res <- results(dds, c("cond", "a", "b"))

dds$cond <- factor(dds$cond, levels = c("b","a"))
# [1] a a a a a b b b b b
#Levels: b a

# Error
results(dds, c("cond", "a", "b"))

# Error in cleanContrast(object, contrast, expanded = isExpanded, listValues = listValues,  : 
  as b is the reference level, was expecting cond_a_vs_b to be present in 'resultsNames(object)'


ps.  Trying to submit this question, why do I get Language "ca" is not one of the supported languages ['en']!

ADD COMMENTlink modified 8 months ago by Michael Love18k • written 8 months ago by Chris Stubben40
gravatar for Michael Love
8 months ago by
Michael Love18k
United States
Michael Love18k wrote:

I'm not sure I understand the problem. You can pull out whatever comparison you like using 'contrast' and then specifying exactly what you want in the numerator and denominator of the LFC.

Regarding the error: you should not relevel the factor after running DESeq(), as the software uses the reference level of the variables to look up which coefficients were estimated. You've estimated coefficients under one setting, and then changed the level orders, so the software is confused. You should either perform re-leveling before running DESeq(), or just use 'contrast' afterward to specify explicitly what comparison you want.

ADD COMMENTlink written 8 months ago by Michael Love18k

It's not very clear in the Note on Factor Levels in the DESeq2 vignette that you should not reorder factors after running DESeq2.   I will only reorder factors in sample tables from now on.

ADD REPLYlink written 8 months ago by Chris Stubben40

It occurs in the vignette before one has run DESeq(), so I guess it was implied, but I can certainly make it explicit by adding a sentence to the vignette. Thanks for your suggestion.

ADD REPLYlink written 8 months ago by Michael Love18k

I've added a sentence to this section of the vignette to make it clear that one shouldn't change factor levels during or after running DESeq().

ADD REPLYlink written 8 months ago by Michael Love18k
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