Does Gene length corrected TMM [GeTMM] violate any assumptions of TMM normalization?
1
2
Entering edit mode
5.1 years ago
O.rka ▴ 720

I've read and been told that edgeR must take in counts only. I noticed that the RPK values are being fed into the TMM normalization procedure. Is this a correct usage assuming all of the assumptions?

Should this be used for downstream DGE analysis?

Note: I am no expert with these methods but I just wanted to ask the community

https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-018-2246-7

# calculate RPK
rpk <- (x[,2:ncol(x)]/x[,1])
# remove length col in x
x <- x[,-1]
# for normalization purposes, no grouping of samples
group <- c(rep("A",ncol(x)))
#EdgeR
x.norm.edger <- DGEList(counts=x,group=group)
x.norm.edger <- calcNormFactors(x.norm.edger)
norm.counts.edger <- cpm(x.norm.edger)

#GeTMM
rpk.norm <- DGEList(counts=rpk,group=group)
rpk.norm <- calcNormFactors(rpk.norm)
norm.counts.rpk_edger <- cpm(rpk.norm)

# Source:
# https://static-content.springer.com/esm/art%3A10.1186%2Fs12859-018-2246-7/MediaObjects/12859_2018_2246_MOESM4_ESM.docx
RNA-Seq • 13k views
ADD COMMENT
1
Entering edit mode

For DGE, use raw counts, like the software demands. Other normalizations can be used for things like visualizations.

ADD REPLY
3
Entering edit mode
5.1 years ago

Technically, RPK values do not violate assumptions of TMM.

TMM is just a technique that tries to find the non-DE portion of the expression distribution by very liberally trimming off outliers. It doesn't matter what kind of expression units you are using.

However, RPK values do violate assumptions for DE analysis. So you cannot use it for downstream DGE.

ADD COMMENT
2
Entering edit mode

I understand your point, but why then in the article they use GeTMM for DE as well? See figure 7 here

ADD REPLY

Login before adding your answer.

Traffic: 1823 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6