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[eladzis]

Hello, I was able to run PSI-Sigma with no errors (via singularity), but the run return an empty table and the log data of the run said that zero events where found. Also I noted that when the program tried to generate IR files the files where also empty. I added the log file. run_psi_sigma.sh.3682796.log I tried running the testsample data and a similar thing happened- the log file is added also run_tests_psi_sigma.sh.3685825.log I'd appreciate help in this matter, Best,

Elad

[wososa]

Hi @eladzis ,

happy testing. Is your own bam file using the same “chr” name as the .gtf? For testsamples, I will update soon to fix this issue for long-read RNA-seq data.

Best, Woody

[wososa]

Hi @eladzis ,

I just tested PSI-Sigma-v2.0 with files in the testsample folder. It worked as expected. Please note that using --type 2 is required for .bam files in the testsample folder. You should get one record from the PUM2 gene if --type 2 is specified:

perl /path/to/PSI-Sigma-2.0/dummy.pl --gtf Homo_sapiens.GRCh38.100.sorted.gtf --name testsample --threads 10 --type 2 --nread 10

Best, Woody

[eladzis]

Hi @wososa I tried to run PSI-Sigma_v2.0 but still got the same results. Again the out.tab files are empty and also the final result table is empty. The log file is attached: run_tests_psi_sigma.sh.3687249.log I checked and I'm using know the correct reference genome. Can you pinpoint now what is the problem? Thanks,

Elad

[wososa]

Hi @eladzis ,

The log says that "awk" is not working properly (below). Could you try update or install awk in your environment?

Generating... testsample_WT.SJ.out.tab
awk: /sci/labs/milotem/eladzis/Michal_Loteme/SplicingAnalysis/PSI-Sigma-2.0/sjFromSAMcollapseUandM_inclOverlaps.awk: line 56: function and never defined

This is the 5th time user reported this issue in their environment. Are you using AWS environment? I probably should write a script to replace the awk script..

Best, Woody

[eladzis]

Hi @wososa I'm running the program on a cluster. The awk was installed by the IT department, and I can see it's working so I'm not sure what is causing the issue when PSI-Sigma is running. I'd appreciate replacing the awk script if possible.

Thanks again for all your help!

Elad

[wososa]

Hi @eladzis ,

It looks like that the error of awk is due to an undefined function named and(). The line in the awk script seems to be non-functional. I masked the line for now. Please try PSI-Sigma version 2.1 or Docker version 3.9.

Example for long-read RNA-seq:

singularity exec --bind /mnt:/mnt docker://woodydon/psi_sigma_pipeline:3.9 perl /usr/local/bin/PSI-Sigma-2.1/dummyai.pl --gtf Homo_sapiens.GRCh38.100.sorted.gtf --nread 10 --name PSIsigma2d1 --type 2 --fmode 3 --threads 6

Best, Woody

[eladzis]

Hi @wososa , The new version worked. Just to be sure I was supposed to get only 2 events of the gene PUM2 with PSI=1 , right?

Best, Elad

[wososa]

Hi @eladzis ,

Yes, you should get one record with +61% delta-PSI and the other record with 0% delta-PSI.

2:20308585-20312235     PUM2    2:20311494-20311663     SES     0       1       -       ENST00000319801 61.00   1.00000e+00     1.00000e+00     na      61.5384615384615        2_20308585_20312235_W_ENST00000319801_1
2:20318646-20327309     PUM2    2:20326261-20326385     SES     0       1       -       ENST00000319801 0.00    1.00000e+00     1.00000e+00     na      0       2_20318646_20327309_W_ENST00000319801_1

Best, Woody

[eladzis]

Hi @wososa That is exactly what I got.

Thanks for all your help!

Elad

[wososa]

Hi @eladzis ,

yes, MES (multiple exon splicing) events should have multiple records for each alternative exon.

Best, Woody