ERROR:psiCalculator:PSI not calculated for event

[maronem]

When running psiPerEvent, I get NA values for all events. I used the GENCODE GRCh38.p14 gtf for my event annotation as well as for my transcript abundance quantification using StringTie. I have seen that most issues arise with the expression file and I have tried where the transcript ids match what is in the .ioe file and the annotation .gtf file and both give me the same PSI not calculated error. If I search for a transcript from my expression file I do get matches in the .ioe file so I am not sure why it is not calculating a PSI value for those that are within both files. Any suggestions?

.ioe file:

(SUPPA2.3) Michaels-MacBook-Pro-3:SUPPA-2.3 michael$ head GENCODE_genEvent_SE_strict.ioe
seqname	gene_id	event_id	alternative_transcripts	total_transcripts
chr1	ENSG00000286448.2	ENSG00000286448.2;SE:chr1:267056-270855:270984-273153:+	ENST00000784886.1	ENST00000784886.1,ENST00000784885.1
chr1	ENSG00000286448.2	ENSG00000286448.2;SE:chr1:267056-267946:268020-268122:+	ENST00000784890.1	ENST00000784890.1,ENST00000669836.1
chr1	ENSG00000228794.13	ENSG00000228794.13;SE:chr1:826923-829003:829104-847654:+	ENST00000744865.1	ENST00000744870.1,ENST00000744868.1,ENST00000744865.1
chr1	ENSG00000228794.13	ENSG00000228794.13;SE:chr1:829104-847654:847806-851927:+	ENST00000744865.1,ENST00000659124.3,ENST00000449005.8,ENST00000658846.2,ENST00000744874.1,ENST00000691316.1,ENST00000692194.1,ENST00000702098.2,ENST00000691234.3,ENST00000702847.1,ENST00000744891.1,ENST00000744878.1,ENST00000657175.1,ENST00000744888.1	ENST00000744890.1,ENST00000744881.1,ENST00000658846.2,ENST00000702847.1,ENST00000744888.1,ENST00000449005.8,ENST00000623070.6,ENST00000744878.1,ENST00000744879.1,ENST00000657837.1,ENST00000744891.1,ENST00000692194.1,ENST00000623808.4,ENST00000622921.1,ENST00000744882.1,ENST00000702557.2,ENST00000659124.3,ENST00000416570.7,ENST00000744873.1,ENST00000702273.1,ENST00000702098.2,ENST00000691316.1,ENST00000445118.7,ENST00000744874.1,ENST00000691234.3,ENST00000657175.1,ENST00000688309.1,ENST00000744880.1,ENST00000685764.3,ENST00000744865.1
chr1	ENSG00000228794.13	ENSG00000228794.13;SE:chr1:847806-851927:852110-852671:+	ENST00000744865.1,ENST00000744868.1,ENST00000744869.1,ENST00000659124.3,ENST00000701598.2,ENST00000670780.1,ENST00000661237.3,ENST00000691316.1,ENST00000691234.3,ENST00000702847.1,ENST00000744878.1,ENST00000744888.1,ENST00000744894.1,ENST00000744896.1,ENST00000688420.2	ENST00000691316.1,ENST00000744868.1,ENST00000702847.1,ENST00000744888.1,ENST00000744896.1,ENST00000691234.3,ENST00000670780.1,ENST00000701598.2,ENST00000661237.3,ENST00000744878.1,ENST00000744894.1,ENST00000659124.3,ENST00000744876.1,ENST00000744869.1,ENST00000688420.2,ENST00000744865.1
chr1	ENSG00000228794.13	ENSG00000228794.13;SE:chr1:852110-852671:852766-853391:+	ENST00000744865.1,ENST00000685334.2,ENST00000744868.1,ENST00000744879.1,ENST00000744869.1,ENST00000744870.1,ENST00000659124.3,ENST00000666741.3,ENST00000685764.3,ENST00000701598.2,ENST00000445118.7,ENST00000670780.1,ENST00000688008.3,ENST00000688309.1,ENST00000685566.2,ENST00000608189.6,ENST00000448975.7,ENST00000661237.3,ENST00000686238.2,ENST00000609139.7,ENST00000609009.6,ENST00000691316.1,ENST00000691234.3,ENST00000702557.2,ENST00000702847.1,ENST00000744886.1,ENST00000744880.1,ENST00000744887.1,ENST00000744878.1,ENST00000744881.1,ENST00000744883.1,ENST00000744884.1,ENST00000744888.1,ENST00000744892.1,ENST00000744894.1,ENST00000744895.1,ENST00000744896.1,ENST00000688420.2,ENST00000744899.1,ENST00000744898.1,ENST00000744904.1	ENST00000744881.1,ENST00000744883.1,ENST00000744904.1,ENST00000744899.1,ENST00000701598.2,ENST00000744894.1,ENST00000702847.1,ENST00000744887.1,ENST00000744888.1,ENST00000623070.6,ENST00000449005.8,ENST00000744870.1,ENST00000688008.3,ENST00000744892.1,ENST00000744895.1,ENST00000448975.7,ENST00000744886.1,ENST00000744879.1,ENST00000744878.1,ENST00000685334.2,ENST00000744891.1,ENST00000666741.3,ENST00000744868.1,ENST00000744896.1,ENST00000609139.7,ENST00000686238.2,ENST00000685566.2,ENST00000702557.2,ENST00000701768.1,ENST00000661237.3,ENST00000744869.1,ENST00000608189.6,ENST00000744898.1,ENST00000691316.1,ENST00000445118.7,ENST00000691234.3,ENST00000670780.1,ENST00000744877.1,ENST00000609009.6,ENST00000659124.3,ENST00000688309.1,ENST00000744880.1,ENST00000685764.3,ENST00000688420.2,ENST00000744865.1,ENST00000744884.1
chr1	ENSG00000228794.13	ENSG00000228794.13;SE:chr1:826923-851927:852110-852671:+	ENST00000685334.2	ENST00000685334.2,ENST00000744866.1
chr1	ENSG00000228794.13	ENSG00000228794.13;SE:chr1:826923-847654:847806-851927:+	ENST00000744868.1	ENST00000685334.2,ENST00000744868.1
chr1	ENSG00000228794.13	ENSG00000228794.13;SE:chr1:826923-852671:852766-853391:+	ENST00000744866.1	ENST00000744867.1,ENST00000744866.1

expression file showing no extra tabs in header:

head TPMs.txt | cat -t
VEH_A
ENST00000831248.1^I1.793814
ENST00000831153.1^I2.304462
ENST00000831668.1^I1.410982
ENST00000416931.1^I89.168289
ENST00000457540.1^I58.015953
ENST00000414273.1^I19.674627
ENST00000427426.1^I51.452415
ENST00000467115.1^I66.111641
ENST00000416718.2^I4.859468

Here is my command when running psiPerEvent:

$ python3.11 suppa.py psiPerEvent -e TPMs.txt -i GENCODE_genEvent_SE_strict.ioe -o VEH_SE

[EduEyras]

Hi,
it's hard to see from the data what could be wrong.
Perhaps you don't have values for all transcripts so that's what's failing?
E.

[EduEyras]

If a transcript does not have a value, it is not assumed to be a zero, the event will produce an NA.

[maronem]

To clarify, for each event in the respective .ioe file, such as:

chr1	ENSG00000228794.13	ENSG00000228794.13;SE:chr1:829104-847654:847806-851927:+	ENST00000744865.1,ENST00000659124.3,ENST00000449005.8,ENST00000658846.2,ENST00000744874.1,ENST00000691316.1,ENST00000692194.1,ENST00000702098.2,ENST00000691234.3,ENST00000702847.1,ENST00000744891.1,ENST00000744878.1,ENST00000657175.1,ENST00000744888.1	

Every transcript in the final column shown above needs to also be present in the TPM file, otherwise, it will produce an NA?

[bbhinder]

Hi ! I have the same issue - were you able to resolve this? Thanks!

[EduEyras]

Hi,

Yes, every transcript in the final column needs to have a value in the TPM file for the event to be not NA. A lack of value is not interpreted as zero, as zero is a meaningful value (no expression).

But if these are meant to have zero expression, then yes, you can put those values to zero.

Your head output of the expression has some hidden symbol "^I", is that supposed to be there? Just checking that there are not hidden symbols that might be misinterpreted. It happens often when expression file generation and python analysis occur on different OSs, e.g. windows OS vs mac OS

I hope this helps

Eduardo