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Question: Global expression shift using RUVSeq
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gravatar for Leon
10 weeks ago by
Leon0
Leon0 wrote:

Hi all,

Does the Section 2.3 Differential expression analysis in RUVSeq vignette work in case of a global shift in exprssion? And can normalized counts obtained by normCounts(set1) be comparable directly between samples? Or should be transformed to CPM, TPM...?

Thanks!

ADD COMMENTlink modified 10 weeks ago by davide risso740 • written 10 weeks ago by Leon0
1
gravatar for davide risso
10 weeks ago by
davide risso740
Weill Cornell Medicine
davide risso740 wrote:

Hi,

in the case of a global shift of expression the preliminary scaling step that we show in Section 2.3 should be avoided, as it assumes that the majority of the genes are not DE.

In practice, it means to avoid the betweenLaneNormalization and the calcNormFactor steps. You may want to set the all the size factors to 1 in edgeR so that no offset is added (the factors inferred by RUV should be enough).

As for your second question: yes, the normCounts() function returns normalized expression levels that are comparable across samples in the same dataset (but not across genes).

ADD COMMENTlink written 10 weeks ago by davide risso740

Thanks, Davide! The last thing I wanna confirm is "across samples" means comparing the same gene in different samples, and "across genes" means comparing different genes in one sample, right?

ADD REPLYlink modified 10 weeks ago • written 10 weeks ago by Leon0

That's correct!

ADD REPLYlink written 10 weeks ago by davide risso740
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