hisat-3n for RNA align

[xieyy46]

Hi developer! Thank you for your state-of-the-art tools hisat-3n! I want to know when I use hisat-3n for RNA-Seq in which C convert to T, should I make alignment twice one for C to T , and another for G to A for those RNA transcripted from reverse strand?

[imzhangyun]

Hello @xieyy46 ,

Thank you for using HISAT-3N. No, you don't need to align the read twice. HISAT-3N will automatically convert and map the reads to both C-to-T and G-to-A internally during the mapping process.

Best, Leo

[xieyy46]

Hi Leo! If I use hisat-3n for A to G conversion RNA-seq alignment in which the canonical splice sites will also change such as GT/AG to GT/GG, I want to know if hisat-3n will sets penalty for this? Is there a setting only consider the splice sites derived from GTF?

[imzhangyun]

Hello @xieyy46 ,

If you use HISAT-3N for A-to-G conversion, the canonical splice sites with conversion will not have any penalty. For I know HISAT2/HISAT-3N cannot only consider splice site from GTF. However, if you provide GTF file, HIAST2/HISAT-3N will use its splice site from GTF with high priority.

Best, Leo

[xieyy46]

Hello Leo! I want to know how hisat-3n determine primary alignment for multi-map reads? according to the alignment score in AS tag? however, in my result, I saw the low score one was treated as primary alignment!

[imzhangyun]

Hello @xieyy46 ,

This is a very good question. If HISAT-3N output multiple positions, they are considered equally important. For paired-end reads, HISAT-3N calculates the score by alignment score(AS) and paired distance. In this case, the first pair are closer to each other compared to the second pair. Internally, these two pairs have the same score, and HISAT-3N will randomly choose one for primary alignment.

Best, Leo