Question: Help interpreting IGV sashimi plots for paired-end RNAseq data
1
gravatar for spykexxx
16 months ago by
spykexxx10
spykexxx10 wrote:

Hi everyone,

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).

Image of a two exons in IGV, together with the read coverage and splicing event quantification

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.

igv rna-seq sashimi • 812 views
ADD COMMENTlink written 16 months ago by spykexxx10

Sorry, the image did not add correctly.

This is another link https://i.ibb.co/X4xC0Cs/IGV-splice-junction.png

ADD REPLYlink written 16 months ago by spykexxx10

I think I agree with MISO's default setting of only counting actual split-reads. With PE reads that both fully align to single exons you could, in principle, also be sequencing fragments that still contain the intron. That being said, I can see why you would want to be able to change that behavior -- maybe take it up with the MISO developers on their google mailing list?

ADD REPLYlink written 16 months ago by Friederike5.8k

Thank you for the clarification. I've emailed them directly to ask about the issue also. Thanks again.

ADD REPLYlink written 16 months ago by spykexxx10
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