Question: normalization of RNA-seq data after over-expressing the transcription factors
0
gravatar for Bogdan
28 days ago by
Bogdan570
Palo Alto, CA, USA
Bogdan570 wrote:

Dear all,

talking about the standard normalization methods of RNA-seq data as implemented in limma, edgeR or DeSeq2 :

is there any modification we shall do in the normalization method when analyzing the RNA-seq data of these 3 sets of samples :

a) control b) over-expressing X protein (Wild Type) WT b) over-expressing X protein (Mutant) MUT

taking into consideration the fact that XproteinWT may have very different levels of expression that XproteinMUTANT

(i.e. 1.5-2x fold difference) ?

thanks a lot,

-- bogdan

limma edger deseq2 voom • 90 views
ADD COMMENTlink modified 28 days ago by Aaron Lun24k • written 28 days ago by Bogdan570

I must say that I don't understand your question at all. What exactly is the problem here?

ADD REPLYlink written 28 days ago by Aaron Lun24k

Hi Aaron, great to hear from you. If I may re-phrase the question please :

considering the RNA-seq for a set of 3 treatments :

  • (a) control

  • (b) over-expressing X protein (Wild Type) i.e. X_WT

  • (c) over-expressing X protein (Mutant) i.e. X_MUT

I have used limma and edgeR for normalization and differential expression. So far, so good ...

However some colleagues mentioned that, :

as the expression of X_WT is 2*higher than X_MUT (after over-expressing these proteins), the "standard" normalization pipelines shall be adjusted / changed to include this fact.

The question would be : is this a legitimate point ?

if the answer may be yes, the next question is : how shall we include the differences in the expression of XWT and XMUT in the pipelines ?

(we do not have SPIKE-IN controls in the bulk RNA-seq data) ;

thank you !

bogdan

ADD REPLYlink modified 28 days ago • written 28 days ago by Bogdan570
Answer: normalization of RNA-seq data after over-expressing the transcription factors
3
gravatar for Aaron Lun
28 days ago by
Aaron Lun24k
Cambridge, United Kingdom
Aaron Lun24k wrote:

is this a legitimate point ?

No. I can only assume that something was lost in translation because that doesn't make sense to me. The relative levels of X - in and of themselves - have no bearing on the normalization for other genes. (You would only encounter problems if the change in expression of X caused a majority of other genes to be DE, but that would be rare.)

ADD COMMENTlink written 28 days ago by Aaron Lun24k

thanks a lot, Aaron. it is great and very helpful to have your advise ;)

ADD REPLYlink written 28 days ago by Bogdan570
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