Question: Strange Mean-Variance plot after Voom Normalization (RNAseq)
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gravatar for grayapply2009
5 weeks ago by
grayapply2009210
United States
grayapply2009210 wrote:

I normalized my raw counts using Voom (limma package) and got a strange Mean-Variance plot https://ibb.co/C2RZXGk . It doesn't look right.

Any suggestions about why and how to deal with it?

enter image description here

voom rna-seq limma normalizaton • 140 views
ADD COMMENTlink modified 5 weeks ago by Biostar ♦♦ 20 • written 5 weeks ago by grayapply2009210

That does indeed look very stange! How did you quantify your samples? How many samples do you have? Did you do any filtering before using voom - if so how?

ADD REPLYlink written 5 weeks ago by kristoffer.vittingseerup3.4k

Thanks for your reply. Here is what I did: 1. Quantification: fastq -> count table (Star + featurecounts, mm10, N = 30) 2. Remove lowly expressed genes: genes showing less than or equal to 1 count in 30% samples were removed 3. Voom (TMM) normalization

The following plot is the voom normalization without removing any genes (https://ibb.co/6N8by9B). enter image description here

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by grayapply2009210

Probably this isn't the problem, but the variance is (standard deviation)^2 while you are plotting sqrt(standard deviation).

ADD REPLYlink written 5 weeks ago by Fabio Marroni2.6k
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