compare the structures of isoforms within a gene

[Oliverfeudj]

Hello @EricKutschera,

is there a way to do this classify_isoform_differences.py for all isoforms? not doing individually for each isoform so that I have at the end all isoform in a single tsv file, not individual tsv for each isoform

Thank you in advance for your reply

[EricKutschera]

By default rmats_long.py outputs a separate .tsv for each of the most significant isoforms showing the differences of that isoform to the most significant isoform in that gene with a delta proportion in the opposite direction

If you run rmats_long.py with --compare-all-within-gene then when it calls classify_isoform_differences.py it won't use --second-transcript-id and each .tsv will have the differences of the most significant isoform to all other isoforms in the gene

I added a branch which changes classify_isoform_differences.py to do a pairwise comparison of isoforms in a gene when run without --second-transcript-id: https://github.com/Xinglab/rMATS-long/commit/bc2bdfb59972ad7090047024b006d9c7128b7b44

Doing a pairwise comparison can take a long time for genes with many isoforms

[kwonej0617]

Hi @EricKutschera

I have used --compare-all-within-gene when running rmats_long.py and I have a question regarding the output. Here's the command line I used:

python ./rMATS-long/scripts/rmats_long.py --abundance espresso_data/HEK293T-all/HEK293T-all_N2_R0_abundance.esp --updated-gtf espresso_data/HEK293T-all/HEK293T-all_N2_R0_updated.gtf --gencode-gtf ${gtf} --group-1 rMATS-long_data/input/HEK293T-all_group1.txt --group-2 rMATS-long_data/input/HEK293T-all_group2.txt --group-1-name WT --group-2-name KO --out-dir ${output_path} --plot-file-type .png --compare-all-within-gene

In the differential_transcript.tsv, the lines containing ENSG00000004455.17 (just randomly picked) were 10 lines as follows:

grep ENSG00000004455.17 rMATS-long_data/differential_transcripts.tsv 
ENSG00000004455.17  ENST00000354858.11  0.281545888814435   1   0.595689772079638   1   0.0064  0.0129  0.0081  0.0066  0.0073  0.0047  0.0091  0.0062  0.0029
ENSG00000004455.17  ENST00000373449.7   56.7319289594579    1   4.99459681293473e-14    2.34608698795577e-10    0.3385  0.354   0.4015  0.529   0.6199  0.6675  0.3647  0.6055  -0.2408
ENSG00000004455.17  ENST00000467905.5   1.41173459743368    1   0.234768653534498   1   0.0128  0.0102  0.0079  0.0 0.0036  0.0093  0.0103  0.0043  0.006
ENSG00000004455.17  ENST00000480134.5   0.462403563942644   1   0.496502801619222   1   0.1079  0.0688  0.0312  0.0548  0.0578  0.0794  0.0693  0.064   0.0053
ENSG00000004455.17  ENST00000487289.1   12.9953227365513    1   0.000312270022764174    0.0371344396057979  0.0222  0.0203  0.0391  0.0032  0.0 0.0 0.0272  0.0011  0.0261
ENSG00000004455.17  ENST00000548033.5   4.71189004554435    1   0.0299547012700919  0.555825341913299   0.0 0.0025  0.0 0.0064  0.0108  0.0187  0.0008  0.01-0.0111
ENSG00000004455.17  ENST00000550338.5   1.87958598265686    1   0.170381210967297   1   0.019   0.0228  0.0469  0.0128  0.0144  0.014   0.0296  0.0137  0.0159
ENSG00000004455.17  ENST00000629371.2   3.9830445464795 1   0.0459604191091984  0.679867100930521   0.0 0.0 0.0 0.0032  0.0109  0.0094  0.0 0.0079  -0.0079
ENSG00000004455.17  ENST00000672715.1   49.5163324181758    1   1.96727117889871e-12    7.39261163606559e-09    0.4766  0.4792  0.4491  0.3296  0.2418  0.1822  0.4683  0.2512  0.2171
ENSG00000004455.17  ESPRESSO:chr1:376:3 1.94993508482912    1   0.162593846125627   1   0.0165  0.0241  0.0163  0.0544  0.0335  0.0147  0.019   0.0342  -0.0152

I found the structure figure ENSG00000004455.17_structure.png only includes 5 transcripts. I wonder why it only has 5 transcripts, not all transcripts shown in differential_transcript.tsv?

In addition, I have another question. Could you please explain How the number next to the event type (for example, exon skipping 75) are calculated?

Thank you so much for your help.

[EricKutschera]

visualize_isoforms.py has a parameter --max-transcripts which defaults to 5: https://github.com/Xinglab/rMATS-long/blob/v1.0.0/scripts/visualize_isoforms.py#L82

If you want to plot more than 5 then you'll need to add more colors here: https://github.com/Xinglab/rMATS-long/blob/v1.0.0/scripts/visualize_isoforms.py#L17

Here's the code to get the number for each event type: https://github.com/Xinglab/rMATS-long/blob/592cb3268d16aa6bea3a6b79aedceac128563e3b/scripts/rmats_long.py#L517

It looks at the isoform_differences files and it checks each pair of transcripts to see what splicing events were found between those two transcripts. If there is only 1 event for a pair of transcripts then the count for that event type will increase. If there are multiple events for a pair then it will count as combinatorial