Error with sqanti_qc2.py command

[tfiskur]

Hello!

I have tried to run the sqanti_qc2.py command with the "all_samples.chained.gtf", annotation and genome fasta files. The program stopped with the next error in the end:

FileNotFoundError: [Errno 2] No such file or directory: '/home/smrtanalysis/Downloads/SQANTI2-master/killifish/chain_truncated_isoforms_as_different_filtered/splits/0/all_samples.chained_corrected.faa

That is strange because the corrected.faa file should be the output of the SQANTI2. Why does the program require its presence?

Also, in the terminal I see this error prior to the final one:

Traceback (most recent call last): File "/home/smrtanalysis/anaconda3/envs/anaCogen/lib/python3.7/multiprocessing/process.py", line 297, in _bootstrap self.run() File "/home/smrtanalysis/anaconda3/envs/anaCogen/lib/python3.7/multiprocessing/process.py", line 99, in run self._target(*self._args, **self._kwargs) File "sqanti_qc2.py", line 1654, in run orfDict = correctionPlusORFpred(args, genome_dict) File "sqanti_qc2.py", line 591, in correctionPlusORFpred if subprocess.check_call(cmd, shell=True, cwd=gmst_dir)!=0: File "/home/smrtanalysis/anaconda3/envs/anaCogen/lib/python3.7/subprocess.py", line 363, in check_call raise CalledProcessError(retcode, cmd) subprocess.CalledProcessError: Command 'perl /home/smrtanalysis/Downloads/SQANTI2-master/killifish/chain_truncated_isoforms_as_different_filtered/utilities/gmst/gmst.pl -faa --strand direct --fnn --output /home/smrtanalysis/Downloads/SQANTI2-master/killifish/chain_truncated_isoforms_as_different_filtered/splits/2/GMST/GMST_tmp /home/smrtanalysis/Downloads/SQANTI2-master/killifish/chain_truncated_isoforms_as_different_filtered/splits/2/all_samples.chained_corrected.fasta' returned non-zero exit status 2. Skipping aligning of sequences because GTF file was provided.

Would be grateful for any help.

[Magdoll]

Hi @tfiskur ,

The error message and the lack of an output .faa means GMST (ORF prediction) failed.

You can try to troubleshoot GMST by running the failed command:

perl /home/smrtanalysis/Downloads/SQANTI2-master/killifish/chain_truncated_isoforms_as_different_filtered/utilities/gmst/gmst.pl -faa --strand direct --fnn --output /home/smrtanalysis/Downloads/SQANTI2-master/killifish/chain_truncated_isoforms_as_different_filtered/splits/2/GMST/GMST_tmp /home/smrtanalysis/Downloads/SQANTI2-master/killifish/chain_truncated_isoforms_as_different_filtered/splits/2/all_samples.chained_corrected.fasta

If ORF prediction is not necessary you can turn it off with --skipORF

--Liz

[tfiskur]

Dear Liz, thanks a lot. I will try.

Upd. Thanks! It worked.

On Fri, 17 Apr 2020 at 19:42, Elizabeth Tseng notifications@github.com wrote:

Hi @tfiskur https://github.com/tfiskur ,

The error message and the lack of an output .faa means GMST (ORF prediction) failed.

You can try to troubleshoot GMST by running the failed command:

perl /home/smrtanalysis/Downloads/SQANTI2-master/killifish/chain_truncated_isoforms_as_different_filtered/utilities/gmst/gmst.pl -faa --strand direct --fnn --output /home/smrtanalysis/Downloads/SQANTI2-master/killifish/chain_truncated_isoforms_as_different_filtered/splits/2/GMST/GMST_tmp /home/smrtanalysis/Downloads/SQANTI2-master/killifish/chain_truncated_isoforms_as_different_filtered/splits/2/all_samples.chained_corrected.fasta

If ORF prediction is not necessary you can turn it off with --skipORF

--Liz

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/Magdoll/SQANTI2/issues/66#issuecomment-615377103, or unsubscribe https://github.com/notifications/unsubscribe-auth/APHIGKU7CWD3UHRV2ZRNZKDRNCIH3ANCNFSM4MK3BXRQ .

[anilkanthi]

Hi,

I am having the following gmst.pl error:

python sqanti_qc2.py -t 30 --gtf bc1001.gtf gencode.v33.annotation.gtf /gpfs/data/skoklab/home/shared-ali-anil/ref_data/ref/hg38/hg38.fa --fl_count tofu.collapsed.abundance.txt R scripting front-end version 3.6.1 (2019-07-05) Write arguments to /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/bc1001.params.txt... Running SQANTI2... Parsing provided files.... Reading genome fasta /gpfs/data/skoklab/home/shared-ali-anil/ref_data/ref/hg38/hg38.fa.... Skipping aligning of sequences because GTF file was provided.

Indels will be not calculated since you ran SQANTI2 without alignment step (SQANTI2 with gtf format as transcriptome input). ** Predicting ORF sequences... GeneMarkS: error on last system call, error code 7 Abort program!!! Traceback (most recent call last): File "sqanti_qc2.py", line 2198, in main() File "sqanti_qc2.py", line 2191, in main run(args) File "sqanti_qc2.py", line 1654, in run orfDict = correctionPlusORFpred(args, genome_dict) File "sqanti_qc2.py", line 591, in correctionPlusORFpred if subprocess.check_call(cmd, shell=True, cwd=gmst_dir)!=0: File "/gpfs/home/kantha01/.conda/envs/anaCogent3/lib/python3.7/subprocess.py", line 363, in check_call raise CalledProcessError(retcode, cmd) subprocess.CalledProcessError: Command 'perl /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/utilities/gmst/gmst.pl** -faa --strand direct --fnn --output /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/GMST/GMST_tmp /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/bc1001_corrected.fasta' returned non-zero exit status 1.

After which I ran the failed command and it worked!: perl /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/utilities/gmst/gmst.pl -faa --strand direct --fnn --output /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/GMST/GMST_tmp /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/bc1001_corrected.fasta

But the main command gives the same error. I do require the ORF analysis. How do I go ahead with this?

[anilkanthi]

When I run it with --skipORF, I get a Rscript error:

python sqanti_qc2.py -t 30 --gtf bc1001.gtf gencode.v33.annotation.gtf /gpfs/data/skoklab/home/shared-ali-anil/ref_data/ref/hg38/hg38.fa --fl_count tofu.collapsed.abundance.txt --skipORF R scripting front-end version 3.6.1 (2019-07-05) Write arguments to /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/bc1001.params.txt... Running SQANTI2... Parsing provided files.... Reading genome fasta /gpfs/data/skoklab/home/shared-ali-anil/ref_data/ref/hg38/hg38.fa.... Error corrected FASTA /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/bc1001_corrected.fasta already exists. Using it... Predicting ORF sequences... WARNING: Skipping ORF prediction because user requested it. All isoforms will be non-coding! WARNING: All input isoforms were predicted as non-coding Parsing Reference Transcriptome.... Parsing Isoforms.... Splice Junction Coverage files not provided. Performing Classification of Isoforms.... Number of classified isoforms: 24533 RT-switching computation.... Reading Full-length read abundance files... Single-sample PacBio FL count format detected. Isoforms expression files not provided. Writing output files.... Generating SQANTI2 report.... Traceback (most recent call last): File "sqanti_qc2.py", line 2198, in main() File "sqanti_qc2.py", line 2191, in main run(args) File "sqanti_qc2.py", line 1844, in run if subprocess.check_call(cmd, shell=True)!=0: File "/gpfs/home/kantha01/.conda/envs/anaCogent3/lib/python3.7/subprocess.py", line 363, in check_call raise CalledProcessError(retcode, cmd) subprocess.CalledProcessError: Command '/gpfs/share/apps/R/3.6.1/bin/Rscript /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/utilities//SQANTI_report2.R /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/bc1001_classification.txt /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/bc1001_junctions.txt /gpfs/data/skoklab/home/kantha01/iso_seq/softwares/SQANTI2/bc1001.params.txt' died with <Signals.SIGKILL: 9>.