Sorry for the long post but i am lost and need suggestions I want to use publicly available human-RNA seq data to know which splicing isoform of certain gene s more abundant in that data and which one is less abundant. I don't know how to pull that information from RNA seq data. What I tried so far is below:
I downloaded some RNA seq data ( t-RNA >200bp) and use hisat2. Then open these bam file in IGV and make sashimi plot. but the issues with this approach are the following.
- Gene is big and have long introns which make visual in IGV difficult.
- Introns also show alot of aligned reads. ( So could it be because i am using t-RNA? As The alignment quality in the introns area is mostly 60)
- Is this is the right approach to use sashimi plot to estimate the abundance of the splicing isoform ?
- I look for other approaches and read about the MISO (looks abandon, no recent updates)or ggsashimi (just make plot but does not provide quantification). please guide me towards most recent and well developed source which i can use.