Strange Mean-Variance plot after Voom Normalization (RNAseq)

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3.8 years ago

grayapply2009 ▴ 280

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

RNA-Seq Normalizaton Voom Limma • 1.9k views

ADD COMMENT • link updated 3.8 years ago by Biostar 20 • written 3.8 years ago by grayapply2009 ▴ 280

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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 REPLY • link 3.8 years ago by Kristoffer Vitting-Seerup ★ 4.0k

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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 REPLY • link 3.8 years ago by grayapply2009 ▴ 280

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

ADD REPLY • link 3.8 years ago by Fabio Marroni ★ 3.0k

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