Splicing in virus using long read sequencing

Question

[snishtala03]

Hello,

I am working on a project where long read sequencing was done using PacBio for a virus genome. I aligned the reads using minimap2, a splice aware aligner and can see splices when I view the bam files on IGV.

Now, I am trying to explore these results further by trying to generate figures showing the splicing events, sashimi plots, psi scores, etc.

I tried MISO but for some reason, am not able to get it working.

Are there any tools/ methods you can suggest?

Thanks!

[GenoMax]

Have you tried IGV?

[snishtala03]

I did but what I am looking for is those plots showing exons and other alternative splicing events and also number of reads supporting it.

[GenoMax]

I tried MISO but for some reason, am not able to get it working.

What seems to be the issue?

[snishtala03]

So my Reference genome is about 3000bp long. It is a predicted reference generated by our team, so we know of a few potential transcripts and exons and their start and end sites but no genes or their locations. MISO needs a gff file. I created a gff with these transcripts and putative splice sites and tried to use MISO. However, the index does not create as it needs genes as well (I guess). So I added the whole reference as a gene and tried to run it, then it gives me errors like -

seq_lens[len(bam_read.seq)] = True TypeError: object of type 'NoneType' has no len()

I just also tried StringTie, I do get some exons and their quantification which matches somewhat with our putative splice sites but I am still hoping to generate some visualization.

[snishtala03]

Since these are PacBio reads the read lengths are variable and MISO cannot handle variable read lengths currently. Can you suggest any other tool or method to do this?