Question: How to normalize RNA-seq data if majority of transcripts expected to be differentially expressed vs control condition?
0
gravatar for gdeniz
7 weeks ago by
gdeniz0
gdeniz0 wrote:

Hi, I am using edgeR to analyze a knockout cell line vs WT control and expect a major fraction of transcripts differentially expressed. Using default settings with edgeR I could confirm significant downregulation of my knocked out transcript. However, given the following statement in the edgeR manual I am worried I am using incorrect assumptions for the analysis:

"TMM is recommended for most RNA-Seq data where the majority (more than half) of the genes are believed not differentially expressed between any pair of the samples. The following commands perform the TMM normalization and display the normalization factors."

What would be an acceptable strategy for data where most genes are believed to be differentially expressed?

rnaseq edger • 96 views
ADD COMMENTlink modified 7 weeks ago by Gordon Smyth39k • written 7 weeks ago by gdeniz0
Answer: How to normalize RNA-seq data if majority of transcripts expected to be differen
2
gravatar for Gordon Smyth
7 weeks ago by
Gordon Smyth39k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth39k wrote:

The problem only occurs when all the DE genes are changed in the same direction. There is no automatic RNA-seq analysis procedure that can accurately handle cases where all or most genes are DE in the same direction. This is an intrinsic limitation of the RNA-seq technology.

In most cases, the best procedure is just to do the usual analysis and see what you get. It would be very unusual for (say) 75% of all genes in the genome to be truly DE in the same direction but, even in this case, the edgeR results will not be disastrous. In the worst case that 75% of genes are truly up-regulated (say) and none are truly down-regulated, then edgeR will slightly underestimate the amount of up-regulation and will introduce a few extra down-regulated genes that might be spurious.

ADD COMMENTlink modified 7 weeks ago • written 7 weeks ago by Gordon Smyth39k

Thanks for your reply.

ADD REPLYlink modified 6 weeks ago • written 6 weeks ago by gdeniz0

I am working with a factor that is part of the general transcription machinery thus I would expect most genes to be downregulated. I see a reduction in the meta gene analysis for all genes from TSS to TTS, but strangely it is not really evident in the MA plots after using edgeR.

ADD REPLYlink modified 6 weeks ago • written 6 weeks ago by gdeniz0
1

It is impossible for RNA-seq to show a reduction in expression for all genes. Any global change in expression is indistinguishable from variations in sequencing depth. RNA-seq only measures relative expression within each sample.

ADD REPLYlink modified 6 weeks ago • written 6 weeks ago by Gordon Smyth39k
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