Question: differential gene expression analysis
1
gravatar for  DataFanatic
14 months ago by
DataFanatic130
DataFanatic130 wrote:

I'm trying to do differential gene expression analysis (RNA-Seq data) using Limma and EdgeR How can I adjust my count matrix for technical confounders(RIN, batch)?

a. Is it possible to adjust for confounders within Limma?

b. Can I adjust the count matrix for covariates using linear regression and import the residuals for downstream differential expression analysis using Limma or EdgeR? Thanks!

ADD COMMENTlink modified 14 months ago • written 14 months ago by DataFanatic130

"Yes, typically one simply adds batch and RIN as covariates if they seem relevant. " Do you mean that you can add covariates into the model matrix e.g. design<-model.matrix(~group+batch+RIN) Thanks!

ADD REPLYlink written 14 months ago by DataFanatic130
1

Yes, correct

ADD REPLYlink written 14 months ago by Devon Ryan91k

Is there a way to visualize the data (residuals) using PCA or MDS plot after performing the linear modeling? I want to do this to confirm the samples no longer cluster by batch.

ADD REPLYlink written 14 months ago by DataFanatic130
1
gravatar for Devon Ryan
14 months ago by
Devon Ryan91k
Freiburg, Germany
Devon Ryan91k wrote:
  1. Yes, typically one simply adds batch and RIN as covariates if they seem relevant. You could also use combat from the SVA package if really necessary. In limma there's also a removeBatchEffects() function, though that wouldn't handle RIN.
  2. See the SVA package in R.
ADD COMMENTlink written 14 months ago by Devon Ryan91k
0
gravatar for  DataFanatic
14 months ago by
DataFanatic130
DataFanatic130 wrote:

Devon, How can I plot the data going into the analysis? design<-model.matrix(~group+batch+RIN) will include RIN in the model and this should change the way samples cluster by PCA or MDS plot. Is there a way to extract the corrected counts matrix? Thank you!

ADD COMMENTlink written 14 months ago by DataFanatic130
1

Including things in a model will have absolutely no effect on PCA or MDS. Check some residuals or some scatter plots if you want to QC how well the correction is being done.

ADD REPLYlink written 14 months ago by Devon Ryan91k
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