Question

Ploting of normalized counts after batch removal

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altuda ▴ 10

@91f63ac0

Last seen 21 days ago

Czechia

Hello, I tried to search for a correct answer, but I don't think I've found one. I have an experiment with paired samples, I've performed DESeq2, and now I would like to plot DESeq2 normcounts. All the discussions and tutorials I found are regarding plotting heatmaps and PCA plots, where you remove the batch effect of the paired samples from vst. Now, how do you think I should proceed regarding the normcounts? Since I am introducing negative values, when I apply limma::removeBatchEffect it doesn't seem correct.

Thank you for your time.

DESeq2 • 253 views

ADD COMMENT • link updated 22 days ago by Gordon Smyth 51k • written 23 days ago by altuda ▴ 10

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You can use ComBat-seq from the sva package to regress covariates such as the pairing information. Advantage is that it returns "raw corrected" counts that are still integers, so you can put them into vst again and plot that.

ADD REPLY • link 23 days ago ATpoint ★ 4.2k

score 0 · Answer 1 · 2024-06-02

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Michael Love 42k

@mikelove

Last seen 19 hours ago

United States

You can't really plot counts with variance from nuisance covariates removed. I would go the route of using VST data instead (see FAQ).

ADD COMMENT • link 23 days ago Michael Love 42k

score 0 · Answer 2 · 2024-06-03

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Gordon Smyth 51k

@gordon-smyth

Last seen just now

WEHI, Melbourne, Australia

I am introducing negative values, when I apply limma::removeBatchEffect it doesn't seem correct.

The values input to removeBatchEffect() are always on the log-scale. Getting negative log-scale values is no problem. The unlogged expression values that they correspond to are always positive.

ADD COMMENT • link 22 days ago Gordon Smyth 51k