Hi,

I'm doing RNA seq and at the alignment step. I'm using HISAT2 aligner with Ensembl reference genome. I don't know if I should use top level or primary assembly file (both are unmasked). I know that primary assembly file contains all toplevel sequence regions excluding haplotypes and patches while top level file includes haplotype/patch regions.

According to this website https://bioinformatics.stackexchange.com/questions/540/what-ensembl-genome-version-should-i-use-for-alignments-e-g-toplevel-fa-vs-p, one user said that primary assembly file should be used for HISAT2. Another user said they usually use top level file for RNA seq.

I'm using HISAT2 and RNA seq, so I'm not sure what to do. What are your thoughts on this?

Also, for FastQ Screen, should I use top level or primary assembly reference genome, or it doesn't matter?

Thank you.