Deseq: Multiple Conditions Testing
3
3
Entering edit mode
9.8 years ago
k.nirmalraman ★ 1.1k

I am finding it a bit hard to comprehend a case where multiple conditions are to be compared and find myself confused with the multifactor part of the DESeq Vignette..

Basically I have four tissues (conditions) and would like to find DE genes for all pairwise comparisons across all conditions. Is the following pairwise comparison approach in DESeq right?

>design
condition    libType
1            single-end
1            single-end
1            single-end
1            single-end
1            single-end
1            single-end
1            single-end
1            single-end
1            single-end
2            single-end
2            single-end
2            single-end
3            single-end
3            single-end
3            single-end
4            single-end
4            single-end
4            single-end
4            single-end
4            single-end
4            single-end

cds <- newCountDataSet( ceiling(RawCountData), conds )
cds <- estimateSizeFactors( cds )
sizeFactors( cds )
cds<-estimateDispersions(cds)

#contrasts for differential expression in DE
res12<-nbinomTest(cds,1,2)
res13<-nbinomTest(cds,1,3)
res23<-nbinomTest(cds,2,3)
res24<-nbinomTest(cds,2,4)
res34<-nbinomTest(cds,3,4)


Any better ways of doing it? Also as an extension, I am not sure yet, if the condition 1 and 4 have different time points (which I presume to be, however, I am not interested in time point specificty), will this by any chance change the approach?

Thanks!

4
Entering edit mode
9.8 years ago
venks ▴ 730

Hi,

It looks fine. But I guess if you read in your conditions as vector/matrix that would make life easier. Btw I am wondering if you are doing it that way?

0
Entering edit mode

Thanks!! That reassured... Yes, I am reading it as a phenoData Matrix and using it as vector

2
Entering edit mode

Perfect! I have never done DESeq with pairwise comparison but I have used DEXSeq which is almost same as DESeq. I guess you are doing it right. Good luck.

1
Entering edit mode
9.8 years ago
Toni ▴ 10

Is there any way to test for differential expression of all four conditions together instead of pairwise?

0
Entering edit mode

It would be quite good to know that! I've been trying to find that out as well

1
Entering edit mode
9.8 years ago

If you have multiple variables, you can use the GLM function in DESeq.

Linear regression can provide the analysis that you are looking for, so I would assume you could use the DESeq implementation to model a single factor with 4 levels. See the overview starting at page 10 for a description of the multi-factor analysis in DESeq:

http://bioconductor.org/packages/release/bioc/vignettes/DESeq/inst/doc/DESeq.pdf