understanding the dup isoforms in the classification file

[alexyfyf]

Hi team,

I have recently run sqanti3 with fasta as input, and the result table contains some duplicate isoforms.
I had a quick check and found it stems from the corrected.gtf file first, which seems been generated after aligning with minimap2, the isoform is splitted because of supplement alignment.

I did not find much detail about that, could you explain a bit? Does it make sense to consider them separately, because sometimes the dup is far away say in a different chromosome, but sometimes quite close.

I can give an example here:
In same chromosome (430kb apart)

Cross chromosome:

Cheers,
Alex

[aarzalluz]

Hi @alexyfyf,

I am not sure why this is happening (SQANTI3 runs minimap2 with --secondary=no, but I guess there still could be some supplementary alignemtns), have you tried any of the other implemented mappers?

If you want more control over the process, I would suggest mapping isoforms outside of SQANTI3, using more stringent parameters or filtering supplementary alignments, and then run the QC script using the recommended GTF input.

Best,

Ángeles

[alexyfyf]

Thank you for you prompt reply and suggestion. I think setting secondary=no does not prevent the supplementary alignment. I will try to process them outside SQANTI. Also from my point of view, these should be considered fusion genes.

However, I do see fusion genes in SQANTI3 classification output, but it is always two genes nearby that are chained together. I am also wondering if that should be the appropriate interpretation. To me, they are more likely to be either read-through transcripts or due to overlap of gene annotations. BTW, I use gencode as suggested in your wiki.

I also ran gencode gtf into SQANTI3, and it does classify some transcripts as fusion as a mistake.
The first one looks like a readthrough, the second one is because GLYATL1 (ENST00000534063) and GLYATL1P4 (ENST00000529326) have a shared exon.

I'd like some suggestions about how to understand this.

Cheers,
Alex