Sashimi plot not showing up M1 mac

[Harshangda Karan Puri]

Hi I am using IGV 2.17.0 on M1 mac Ventura 13.0.1. I can use the app to get the alignment track, coverage track, and splice junction tracks. However, when I click on sashimi plot I get an empty window. PFA the screen shots.

I have also tried doing the visualization using GRCm39.primary_assembly.genome.fa as reference geneome which I supplemented with gencode.vM33.primary_assembly.annotation.gtf.gz for annotations and I get the same problem. 

I tried different levels of zoom but still doesn't work.

Please let me know how to fix this.

[igv-help]

Hi,

I'm not able to reproduce the issue.  Could you try the following as a test.  Select genome human hg19, then select "File > Load from Server... >  Available Datasets > RNA-Seq (Body Map) > Heart.   After loading, jump to gene "SLC25A3" by typing its name in the go box.   Right-click and view the Sashimi Plot, you should see something like the screenshot below.

This will tell us if there is a general problem, or a problem specific to you data.

[Harshangda Karan Puri]

Hi, 

Yes, using the heart dataset and human genome, I am able to reproduce the sashimi plot. 

When I use my data with mouse mm39, I don't get sashimi plots. I am using Bam files generated from Minimap 2 using Oxoford long read data. 

What could be wrong?

Harsha

[igv-help]

No I don't, but we have never tested with ONT long read data, and in fact don't have any data to test with.   Could you share your dataset or a portion of if?  If you email igv-...@broadinstitute.org I can send you a secure dropbox link.   You don't need to share the whole file, I can also send instructions for extracting a sample.

[Harshangda Karan Puri]

Hi, I have sent the email. Please send me the dropbox link there and I could send some Bam files and genome that I am using. I have sent email from my University of Manchester email address. 

Harsha

[igv-help]

Hi, sorry for the delay.    I think the issue is just bad default settings for "junction coverage min".     After right clicking and setting the the value to zero I see junctions in the sashimi plot (see screenshot).

First I had to change the experiment type to "RNA".    This is a new type in IGV, long read RNA data, so we need to make some changes to accomodate it, but for now you can manually change the experiment type which I assume you have figured out.

[Harshangda Karan Puri]

Hi,

I am not very sure how to change the experiment type to "RNA". I am using IGV app version 2.17.0 on M1 mac. I am using  GRCm39.primary_assembly.genome.fa as reference genome which I supplemented with gencode.vM33.primary_assembly.annotation.gtf.gz for annotations. 

When I go to the sashimi plot window, it doesn't allow me to right click. I can only right click on the splice junction track and from there I can change the minimum junction coverage to zero. But even if I change that, I still don't get the sashimi. Please see below. 

Are you using IGV browser or app. Are you using a different genome and annotations? Not sure where I am going wrong.  

[James Robinson]

You change experiment type from the alignments track, the menu looks like its maybe from the junctions track.   

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[James Robinson]

I am using IGV 2.17.2 (desktop application).   I suggest you update, there were some important bug fixes.  See https://igv.org/doc/desktop/#ReleaseNotes/2.17.x/

[Harshangda Karan Puri]

Thanks,

I changed the experiment type to "RNA" from alignment track and it worked. Thanks a lot!

Harsha