I'm quite new to RNAseq paired end analysis so apologies if this comes across as a basic question.
I have been visualising paired end RNAseq data in Integrated Genome Viewer (IGV). I am confused as to how IGV quantifies splicing junctions. The RNAseq had been aligned using HiSAT2, then the bam file loaded into IGV.
In the image below, I am visualising two exons, the read coverage and the splicing events between them. However when IGV quantifies this splicing event, it only counts 24 reads that span this junction. Further inspection revealed that it is only counting split reads, i.e. one read that is spanning an exon junction (blue lines). It is not counting reads where the first read aligns to the initial exon, then it's respective pair aligns to the next exon (grey lines).
I do not understand why this is the case. Surely if each read pair is mapping within separate exons, then this should also count as a legit splicing event? Why is it only counting split reads as splicing events?
Thank you very much, I appreciate the help.