Dear Community members,
I found several informative posts on the difference between scaled TPM, lengthScaledTPM, and no scaling but I am unsure of how the options apply for Kallisto considering that the output from Kallisto is in TPM. So far, I have been using scaled TPM for my analysis in EdgeR. The following is the way I import my counts into EdgeR:
data_<- tximport(files, type = "kallisto", tx2gene=tx2gene, txOut=F, ignoreAfterBar= T, countsFromAbundance="scaledTPM")
Any recommendations on the correct method (for differential analysis) and explanation would be greatly appreciated. Thank you
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written
by
IM •
1 answer
Are you wanting to do a gene level or transcript level analysis in edgeR? To input transcript-level kallisto output into edgeR, simply use:
library(edgeR)
y <- catchKallisto(paths)
where paths is a character vector specifying the directories containing the kallisto output.
See
Baldoni et al (2024). Dividing out quantification uncertainty allows efficient assessment of differential transcript expression with edgeR. Nucleic Acids Research 52(3), e13. https://doi.org/10.1093/nar/gkad1167
or Sections 2.18 and 4.6 of the edgeR User's Guide: https://doi.org/doi:10.18129/B9.bioc.edgeR
I think complete instructions are in tximport vignette: https://bioconductor.org/packages/devel/bioc/vignettes/tximport/inst/doc/tximport.html
I only intended catchKallisto for transcript-level analyses, although it is easy enough to convert it to gene-level by aggregating the expected counts over genes, and we did that in fact to obtain gene-level overdispersions for the Baldoni et al (2024) paper.
For gene level analyses, I personally prefer not to rely on transcript annotation at all because my own (unpublished) research shows that transcript quantification tools are highly sensitive to incomplete or inaccurate annotation. I have plans to publish something on this but it's not at the top of my queue yet.
If you use tximport to read kallisto output into edgeR, then you must follow the instructions in the tximport vignette. The appropriate section of the vignette shows you explicitly how to combine edgeR TMM normalization factors with the tximport normalization.
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