Analize RNA-seq data using Hisat2

Question

[Maurice]

Hi all, I'm trying to analyze RNA-seq data. I performed paired-end sequencing and to prepare the libraries I have used Illumina Stranded mRNA Prep, so my reads are stranded. After quality check I used Hisat2 to perform the alignment and I selected the option unstranded. I also tried to perform the aligment using the strand information (forward/reverse) but nothing change. So I was wondering am I missing something? Did I perform the aligment correctly?

Thank you for everyone that will help me

[biofalconch]

I think providing the actual command you used could help to assess if this was done correctly :)

[Maurice]

I performed it on the Galaxy platform. Selected parameters are those in the attached picture

Thank you for your help

[Sara]

It would significantly affect your output if your reads were "reverse-stranded" and you chose "forward-stranded" for alignment instead. In this scenario, it would be much more difference in the output because your reads were reverse and you chose forward stranded as alignment so, it couldn't align the right reads.

But now I think it is expected that it was not a lot of change, because it aligned the reads regardless of the orientation. but of course, for downstream analysis, it is better to choose the correct parameters as you know.

ps: if your library is reverse stranded choose RF; and if it is forward-stranded choose FR in Galaxy.

[benformatics]

HISAT2 rna-strandness option

Probably: RF (reverse-forward) and fr (firststrand)