Splice aware or not for RNA sequence data

Question

[Nemo]

Hello,

I have paired fastq rna sequences. I am using hisat2 in order to align the reads to my reference based on the following command:

hisat2 --no-spliced-alignment --no-unal -p 2 -x ./reference.fasta -1 ./Data/D10_R1.fastq.gz -2 ./Data/D10_R2.fastq.gz -S D10.sam

Though I am not sure should I keep --no-spliced-alignment tag or not. For RNA data should I consider splices?

Answer 1

[Rob]

What is in reference.fasta? Is it the reference genome, or the collection of extracted and spliced transcripts. Using HISAT2, it is most common to align against the genome directly. In this case, you should absolutely remove --no-spliced-alignment, as you are aligning RNA-seq reads, and you expect any read crossing a junction to align in a spliced fashion to the underlying genome.

[Nemo]

Thanks Rob for your quick reply. The reference.fasta is Wuhan-Hu-1.fasta related to wuhan genome.