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Question: upperquartile with voom
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gravatar for bilcodygm
6 months ago by
bilcodygm0
bilcodygm0 wrote:

Hi all,

I was just wondering whether it would make sense to combine the upperquartile normalization with limma-voom analysis of RNAseq data.

this is just out of interest, not driven by the data or so. At first, I did

uq <- DGEList(counts=initDGE, group=responseStatus)
uq <- calcNormFactors(uq, method="upperquartile")
uq <- estimateDisp(uq, design)

 

uq.v <- voom(uq, design, normalize.method="none")

cat("Limma::voom & eBayes after UpperQuartile\n")
fit <- lmFit(uq.v, design)
v.UQ <- contrasts.fit(fit, contrasts=contrastMat.R)
v.UQ <- eBayes(v.UQ)

then extracting a toptable from this object.

Ofcourse I get a toptable, but is this a correct approach. first, I am generating upperquartile normalized counts, then I feed this to 'voom' to log-transform the data without further normalization by voom, then I do the modelfitting etc.

Thank you for your advice.

ADD COMMENTlink modified 6 months ago • written 6 months ago by bilcodygm0

Thank you for taking the time to reply!

It was just my uncertainty of using the commands. 

I am trying to compare different normalization methods with different statistical testings (basically TMM, upperquartile, quantile and DESeq2's default method across with RLT, QLF (both edgeR) and with voom-eBayes).

ADD REPLYlink written 6 months ago by bilcodygm0

Comparing methods using experimental data is usually not very instructive. If you don't already know which genes are differentially expressed, and you get different results for each normalization method, what criteria are you planning to use to compare the different normalization methods?

ADD REPLYlink written 6 months ago by James W. MacDonald47k
0
gravatar for Gordon Smyth
6 months ago by
Gordon Smyth34k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth34k wrote:

voom works fine with any scale normalization method that you might do using calcNormFactors. This is just standard use of the function. Why did you think there would be any problem?

Why do you want to do upperquartile normalization? What do you want to over-ride calcNormFactor's default method?

ADD COMMENTlink modified 6 months ago • written 6 months ago by Gordon Smyth34k
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