kal's test on rpkm vs raw counts

Question

[from the mountains]

For comparisons with 0 replicates, I am using Kal's Z-test on proportions. I am wondering, does it make more sense to run Kal's on RPKM or on raw counts, and why? I would think that RPKM is better because it is normalized by gene length. We are more interested in knowing the (transcripts_of_geneA/total_transcripts) rather than (reads_of_geneA/total_reads), aren't we? I can see however that they can yield slightly different results in some cases.

I see conflicting information on each alternative. On the one hand, the Kal's paper writes the equation as (n-specific mRNA reads/cell)/(N-total mRNA reads/cell), but on the other hand, when I try to run the function on RPKM in CLC it warns me that proportions tests are aimed at count data.

Which is better?

[Kristoffer Vitting-Seerup]

Could you elaborate on what it is you actually want to do? Then we migth be better able to help you.

Answer 1

[theobroma22]

CLC Bio is a licensed platform, so it may be best for you to contact CLC to get an answer to your question. However, Karl's test is for the count data and not RPKM.

[from the mountains]

good to know. but why? and doesn't that seem in conflict with the formula stated in the kal's test paper? : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC25383/

[theobroma22]

Not at all. You have to consider the experimental design of SAGE vs. RNA-seq, which is what you used.