[BioC] multiple level factors contrast in nbinomLRT, DESeq2.

[Michael Love]

Hi Chunxuan,

On Aug 20, 2014 12:38 PM, "sh. chunxuan" <hibergo at outlook.com> wrote:
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> Dear list,
> I am not sure the correct way to interpret the nbinomLRT results in
multiple level factors condition.
> Here is a toy example. I would to find DE genes after controlling batch
effect in the experiments, in which there are multiple levels.
> ## make data library(DESeq2)dds <- makeExampleDESeqDataSet(m = 18)colData
<- data.frame(row.names = rownames(colData(dds)), sample =
colData(dds)$sample, condition = rep(LETTERS[1:6], each = 3), batch =
factor(rep(c(1, 1, 2), 6)))dds <- DESeqDataSetFromMatrix(counts(dds),
colData = colData, design = ~ batch + condition)dds <-
estimateSizeFactors(dds)dds <- estimateDispersions(dds)## LRTestdds <-
nbinomLRT(dds, reduced = formula(~ batch ))resultsNames(dds)[1]
"Intercept"        "batch_2_vs_1"     "condition_B_vs_A" "condition_C_vs_A"
"condition_D_vs_A" "condition_E_vs_A" "condition_F_vs_A"res.1 <-
results(dds, name = "condition_B_vs_A")res.2 <- results(dds, contrast =
list("condition_B_vs_A", "batch_2_vs_1")
> Questions:1.) I would like to get the DE genes between B and A, while
controlling for the batch effect.

You should use the default Wald test for such a comparison, not the LRT.
See the vignette for an explanation of the difference between the two tests.

So simply use DESeq() and then use:

results(dds, contrast = c("condition", "B", "A"))

> Should I take "res.1" or "res.2", or both are wrong? and what is the
correct way to do it?
> 2.) Why "res.3 <- results(dds, contrast = c("condition", "A", "b"))" gave
error: "Error in normalizeSingleBracketSubscript(j, x) :   subscript
contains invalid names"

This code gives an error because lowercase "b" is not a level. (It should
be giving a more understandable error message, not sure why it is not doing
so here.)

> 3.) In order to get DE genes between conditions not directly listed in
the "resultsNames", is the following codes correct? should "batch_2_vs_1"
be included int the contrast?##for example, DE between C and F, controlling
for batch effect;res.4 <- results(dds, contrast = list("condition_C_vs_A",
"condition_F_vs_A")

Check the help for ?results and the examples there. You should simply use:

contrast = c("condition", "C", "F")

Mike

> Best, Chunxuan
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