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Strange Mean-Variance plot after Voom Normalization (RNAseq)

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
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updated by Biostar Biostar • written by ngs101.com ngs101.com

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?

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written by Kristoffer Vitting-Seerup Kristoffer Vitting-Seerup

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

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written by ngs101.com ngs101.com

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

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written by Fabio Marroni Fabio Marroni

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