HISAT2 _spliced_sites with a gff3 file

[htetre]

Hello,

I am trying to extract_splice_sites using the python script provided by HISAT2 to use in the downstream alignment in HISAT2. However the reference genome that I am using, the most recent version, has a gff3 file with the gene annotation. I am unable to extract spliced sites with this gff file. Does anyone know a way to extract splice sites from a gff3 that can be used with the Hisat2 program? I have a gtf file but it was built on an older version of the genome assembly so I would prefer to use the newest (gff3).

Thank you for any help you can provide

[Macspider]

Hi, I know how.
First, quoting the hisat2-align manual you will read that:

--ss <path>
Note this option should be used with the following --exon option. Provide a list of splice sites (in the HISAT2's own format) as follows (four columns).

chromosome name <tab> zero-offset based genomic position of the flanking base on the left side of an intron <tab> zero-offset based genomic position of the flanking base on the right <tab> strand

Use hisat2_extract_splice_sites.py (in the HISAT2 package) to extract splice sites from a GTF file.

Since GFF / GTF files are usually prone to mis-formatting errors given that for a long time there has been no unification of the format and hardly there is one actually today (sigh) it's better if you homebrew your file. That said, you can do that with awk.

Commands

Exons:

awk '{if ($3=="exon") {print $1"\t"$4-1"\t"$5-1}}' genes.gtf > exonsFile.table

Splice Sites:

awk '{if ($3=="intron") {print $1"\t"$4-2"\t"$5}}' genes.gtf > ssFile.table

[Haledm]

Hi, I know how.
First, quoting the hisat2-align manual you will read that:



Since GFF / GTF files are usually prone to mis-formatting errors given that for a long time there has been no unification of the format and hardly there is one actually today (sigh) it's better if you homebrew your file. That said, you can do that with awk.

Commands

Exons:

awk '{if ($3=="exon") {print $1"\t"$4-1"\t"$5-1}}' genes.gtf > exonsFile.table

Splice Sites:

awk '{if ($3=="intron") {print $1"\t"$4-2"\t"$5}}' genes.gtf > ssFile.table

Hi! This has been immensely helpful, as I'm addressing the same issue as OP. But do you have a similar recommendation on how to generate the SS table if the reference annotations do not contain explicit intron coordinates? I'm working from a .gff3 file that only contains exon coordinates. I know the information I need is technically in that file, but I don't have the command line know-how to easily get that information using awk.

Thanks!