Stranded or not stranded Fasq data

Question

[alruta13]

Hello everyone,

I am learning about RNAseq analysis data. I have began with kallisto-DESeq2 pipeline and I am stack with the kallisto psudoalligment because I don't know if my data is or not stranded.

What can I do to elucidate if my data is stranded ? How can I know if I have to use the fr or the rf mode of kallisto?

I have been testing and I notice that when I run and pseudoaligment with fr or rf mode, I get around 40.000 psudoaligment but when I run the program without fr or rf, I gen around 80.000. Is this telling me something?

Thank you very much!

[iraun]

Hi!what I usually do is to align the reads first with STAR or HISAT genome aligners, and then run infer_experiment.py to check the strandness of the RNASeq data. Once you know it, you can tune kallisto command accordingly.

[dsull]

You don't need a genome aligner for infer_experiment.py -- see here: https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-022-04572-7 (which uses kallisto)

[iraun]

Right, thank you for pointing out that tool! I did not know it. That tool uses kallisto with —genomebam argument anyway, which generates genome pseudoalignment. In any case you need some kind of alignment in order to infer the strandness of RNA-Seq data.