Why is the line between two exons not shown when the inclusive isoform count is 0?

[bioPG]

Hello, I have two questions about the sashimiplot:

1. Why is the line between two exons not shown when the inclusive isoform count is 0?

2. Why is there a lack of corresponding data display in the 23Y381-1 case section?

[EricKutschera]

The junction count is adjusted for the number of bam files in the group here: https://github.com/Xinglab/rmats2sashimiplot/blob/v3.0.0/src/MISO/misopy/sashimi_plot/plot_utils/plot_gene.py#L91

That adjustment could result in a junction count of 0. For example if there are 3 bam files in the group and only 1 junction count in just 1 of the bam files. That situation would result in the '0' being shown but without a line. It looks like the line width would be calculated as 0: https://github.com/Xinglab/rmats2sashimiplot/blob/v3.0.0/src/MISO/misopy/sashimi_plot/plot_utils/plot_gene.py#L174

A different situation is if there are actually no counts for that junction. In that case nothing would be displayed

[bioPG]

Thank you so much for your response! I believe I have a clearer understanding now. Your explanation helped me grasp the adjustment made for junction counts in the code and why '0' might appear without a line. I appreciate your help!

[Saida2019]

Hello,
I have two naive questions

1.Why the anatomy of plot in my example is different than shown on https://github.com/Xinglab/rmats2sashimiplot. > Example>Using a group file?
2.The isoforms drawn at the bottom are the mirror view of plot isn`t it?

My code is like
rmats2sashimiplot --b1 /Users/RMATS/Drought.txt --b2 /Users/RMATS/Control.txt --event-type RI -e /Users/RMATS/RI.MATS.JC.txt --l1 Drought --l2 Control --exon_s 1 --intron_s 5 -o /Users/RMATS/test_grouped_output --group-info grouping.gf

Thank you.

7_Solyc02g032120.2.1_Scaffold_12632_Chr11_46700344_46700397_-@Scaffold_12632_Chr11_46699185_46700397_-@Scaffold_12632_Chr11_46699185_46699644_-.pdf

[EricKutschera]

1. It's a retained intron (RI) event, but the example in the README is a skipped exon (SE) event
2. The x-axis shows the coordinates. At the left is 46,700,397 and the right is 46,699,187. The x-axis label indicates it's a "-" strand event. Minus strand events are shown with the coordinates flipped

[Saida2019]

Hi Eric,
Thank you very much for your response. I am sorry, I am new with this type analysis, that is why I have such "easy questions". I have 8 control and 8 drought stress experiment repeats. I have filtered rmats results for FDR<0.05 and InclLevel Diff >0.1. This identified around 220 AS events but still almost in all of the Sashimi plots read counts are less than 10 and I am thinking that this might not be statistically enough to be sure on that event. Does those 4 and 8 counts means just 4 and 8 read counts or they are somehow normalized counts? I think that my RNA seq data is not big enough, that is why I have such less amount of counts.

[EricKutschera]

The code posted before used --group-info. In that case the junction count shown in the sashimiplot is the average over all bams in the group: https://github.com/Xinglab/rmats2sashimiplot/blob/v3.0.0/src/MISO/misopy/sashimi_plot/plot_utils/plot_gene.py#L91

You can plot without --group-info to see the counts in each bam. Also the supporting read counts from rMATS are in RI.MATS.JC.txt. The read counting is different between rMATS and rmats2sashimiplot: #33