Error in rule run_kmers_gwas

[DavideGue]

Hi @akcorut ,
I am running kGWASflow on a VM hosted on our server with 7TB Hard Disk and 180 G RAM.
I got this message:
[Thu Oct 12 01:53:07 2023]
Job 2972: Running k-mers based GWAS on /mnt/Data-out/kgwasflow_wd/Flowering_Time.pheno...
Reason: Missing output files: results/kmers_gwas/Flowering_Time/kmers/pass_threshold_5per; Input files updated by another job: results/kmers_table/kmers_table.table, results/kmers_table/kmers_table.kinship, scripts/external/kmers_gwas/kmers_gwas.py

Activating conda environment: .snakemake/conda/66a0fa76bc7c3da7cd9f3a46e7e64c95_
[Thu Oct 12 01:53:22 2023]
Error in rule run_kmers_gwas:
jobid: 2972
input: scripts/external/kmers_gwas/kmers_gwas.py, /mnt/Data-out/kgwasflow_wd/Flowering_Time.pheno, results/kmers_table/kmers_table.table, results/kmers_table/kmers_table.kinship
output: results/kmers_gwas/Flowering_Time/kmers/pass_threshold_5per
log: logs/kmers_gwas/Flowering_Time/Flowering_Time_kmers_gwas_run.log (check log file(s) for error details)
conda-env: /mnt/Data-out/kgwasflow_wd/.snakemake/conda/66a0fa76bc7c3da7cd9f3a46e7e64c95_
shell:

    export LD_LIBRARY_PATH=$CONDA_PREFIX/lib

    rm -r results/kmers_gwas/Flowering_Time

    python2 scripts/external/kmers_gwas/kmers_gwas.py          --min_data_points 30         --pheno /mnt/Data-out/kgwasflow_wd/Flowering_Time.pheno         --kmers_table results/kmers_table/kmers_table         --kmers_number 10001         --permutations 100         --maf 0.05 --mac 5         -l 31 -p 12         --outdir results/kmers_gwas/Flowering_Time >>logs/kmers_gwas/Flowering_Time/Flowering_Time_kmers_gwas_run.log 2>&1

    (one of the commands exited with non-zero exit code; note that snakemake uses bash strict mode!)

Shutting down, this might take some time.
Exiting because a job execution failed. Look above for error message
Error! The Snakemake workflow aborted.
Complete log: .snakemake/log/2023-10-03T162604.308859.snakemake.log
ERROR:root:Error running Snakemake: Command '['snakemake', '--use-conda', '--conda-frontend', 'conda', '--cores', '12', '--snakefile', '/home/davide/miniconda3/envs/kgwasflow/lib/python3.11/site-packages/workflow/Snakefile', '--rerun-triggers', 'mtime', 'params', 'input', 'software-env', 'code']' returned non-zero exit status 1.

Attached you can find the log file logs/kmers_gwas/Flowering_Time/Flowering_Time_kmers_gwas_run.log
Flowering_Time_kmers_gwas_run.LOG

[akcorut]

Hi @DavideGue,

This error seems to be related to the phenotype file. Do you mind sharing your .pheno file with me so I can have a look?

Best,
Kivanc

[DavideGue]

Here the .pheno file
Flowering_Time.pheno.txt
Thank you for your help,
Davide

[akcorut]

Okay, it looks like there is one missing phenotype information for your accession WB-359. If you remove the row with the missing pheno (WB-359) and run the pipeline again, it should work without a problem.

WB-355	86.411
WB-356	78.509
WB-357	82.411
WB-359	
WB-360	79.404
WB-361	74.411

WB-153 seems to have no phenotype information as well.

In the future, make sure to check your .pheno files and simply remove any samples with missing phenotype information from the .pheno file.

Hope this will solve the issue.

Kivanc

[DavideGue]

Hi,
I removed the samples with missing genotypes and it apparently solved that issue.
However, I got a different error message now:
[Thu Oct 12 14:20:04 2023]
Job 2972: Running k-mers based GWAS on /mnt/Data-out/kgwasflow_wd/Flowering_Time.pheno...
Reason: Missing output files: results/kmers_gwas/Flowering_Time/kmers/pass_threshold_5per

Activating conda environment: .snakemake/conda/66a0fa76bc7c3da7cd9f3a46e7e64c95_
[Thu Oct 12 15:41:52 2023]
Error in rule run_kmers_gwas:
jobid: 2972
input: scripts/external/kmers_gwas/kmers_gwas.py, /mnt/Data-out/kgwasflow_wd/Flowering_Time.pheno, results/kmers_table/kmers_table.table, results/kmers_table/kmers_table.kinship
output: results/kmers_gwas/Flowering_Time/kmers/pass_threshold_5per
log: logs/kmers_gwas/Flowering_Time/Flowering_Time_kmers_gwas_run.log (check log file(s) for error details)
conda-env: /mnt/Data-out/kgwasflow_wd/.snakemake/conda/66a0fa76bc7c3da7cd9f3a46e7e64c95_
shell:

    export LD_LIBRARY_PATH=$CONDA_PREFIX/lib

    rm -r results/kmers_gwas/Flowering_Time

    python2 scripts/external/kmers_gwas/kmers_gwas.py          --min_data_points 30         --pheno /mnt/Data-out/kgwasflow_wd/Flowering_Time.pheno         --kmers_table results/kmers_table/kmers_table         --kmers_number 10001         --permutations 100         --maf 0.05 --mac 5         -l 31 -p 12         --outdir results/kmers_gwas/Flowering_Time >>logs/kmers_gwas/Flowering_Time/Flowering_Time_kmers_gwas_run.log 2>&1

    (one of the commands exited with non-zero exit code; note that snakemake uses bash strict mode!)

Shutting down, this might take some time.
Exiting because a job execution failed. Look above for error message
Error! The Snakemake workflow aborted.
Complete log: .snakemake/log/2023-10-12T141754.479754.snakemake.log
ERROR:root:Error running Snakemake: Command '['snakemake', '--use-conda', '--conda-frontend', 'conda', '--cores', '12', '--snakefile', '/home/davide/miniconda3/envs/kgwasflow/lib/python3.11/site-packages/workflow/Snakefile', '--rerun-triggers', 'mtime', 'params', 'input', 'software-env', 'code']' returned non-zero exit status 1.

Here log files:

log_file.txt
Flowering_Time_kmers_gwas_run.log.txt

I hope that what I add below can help:
I also tried to run kmer_gwas.py from https://github.com/voichek/kmersGWAS/tree/master using the docker image at the link he provides on its giuthub page. In this case I used the k-mers table generated by kgwasflow. Doing this I got the following error message:
root@3613c691355a:/app# python2.7 kmers_gwas.py --pheno ./manual_kgwas/pheno.phenotypes --kmers_table ./manual_kgwas/kmers_table -l 31 -p 4 --outdir ./results
affective MAF = 0.05
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
/app/external_programs/gemma_0_96: error while loading shared libraries: libgfortran.so.3: cannot open shared object file: No such file or directory
Traceback (most recent call last):
File "kmers_gwas.py", line 278, in
main()
File "kmers_gwas.py", line 247, in main
th_5per = get_threshold_from_perm(res, "P", args.n_permutations, 0.05)
File "/app/src/py/functions.py", line 110, in get_threshold_from_perm
pvals.append(best_pvals[prefix + str(i)])
KeyError: 'P1'

[akcorut]

Hi @DavideGue,

Can you share the content of results/kmers_gwas/Flowering_Time/kmers/. You can just name the files that exist in that directory. This is most likely an issue with kmersGWAS and GEMMA, but I will try to find a solution for you.

Unfortunately, I cannot really comment on the issues with that docker image because it is not part of the kGWASflow pipeline.

Best,
Kivanc

[DavideGue]

Here the content of results/kmers_gwas/Flowering_Time/kmers/
best_pvals
pheno.31.P31.bed
pheno.54.P54.fam
pheno.78.P78.bed
output
pheno.31.P31.bim
pheno.54.P54.fam.orig
pheno.78.P78.bim
pheno.0.phenotype_value.bed
pheno.31.P31.fam
pheno.55.P55.bed
pheno.78.P78.fam
pheno.0.phenotype_value.bim
pheno.31.P31.fam.orig
pheno.55.P55.bim
pheno.78.P78.fam.orig
pheno.0.phenotype_value.fam
pheno.32.P32.bed
pheno.55.P55.fam
pheno.79.P79.bed
pheno.0.phenotype_value.fam.orig
pheno.32.P32.bim
pheno.55.P55.fam.orig
pheno.79.P79.bim
pheno.100.P100.bed
pheno.32.P32.fam
pheno.56.P56.bed
pheno.79.P79.fam
pheno.100.P100.bim
pheno.32.P32.fam.orig
pheno.56.P56.bim
pheno.79.P79.fam.orig
pheno.100.P100.fam
pheno.33.P33.bed
pheno.56.P56.fam
pheno.7.P7.bed
pheno.100.P100.fam.orig
pheno.33.P33.bim
pheno.56.P56.fam.orig
pheno.7.P7.bim
pheno.10.P10.bed
pheno.33.P33.fam
pheno.57.P57.bed
pheno.7.P7.fam
pheno.10.P10.bim
pheno.33.P33.fam.orig
pheno.57.P57.bim
pheno.7.P7.fam.orig
pheno.10.P10.fam
pheno.34.P34.bed
pheno.57.P57.fam
pheno.80.P80.bed
pheno.10.P10.fam.orig
pheno.34.P34.bim
pheno.57.P57.fam.orig
pheno.80.P80.bim
pheno.11.P11.bed
pheno.34.P34.fam
pheno.58.P58.bed
pheno.80.P80.fam
pheno.11.P11.bim
pheno.34.P34.fam.orig
pheno.58.P58.bim
pheno.80.P80.fam.orig
pheno.11.P11.fam
pheno.35.P35.bed
pheno.58.P58.fam
pheno.81.P81.bed
pheno.11.P11.fam.orig
pheno.35.P35.bim
pheno.58.P58.fam.orig
pheno.81.P81.bim
pheno.12.P12.bed
pheno.35.P35.fam
pheno.59.P59.bed
pheno.81.P81.fam
pheno.12.P12.bim
pheno.35.P35.fam.orig
pheno.59.P59.bim
pheno.81.P81.fam.orig
pheno.12.P12.fam
pheno.36.P36.bed
pheno.59.P59.fam
pheno.82.P82.bed
pheno.12.P12.fam.orig
pheno.36.P36.bim
pheno.59.P59.fam.orig
pheno.82.P82.bim
pheno.13.P13.bed
pheno.36.P36.fam
pheno.5.P5.bed
pheno.82.P82.fam
pheno.13.P13.bim
pheno.36.P36.fam.orig
pheno.5.P5.bim
pheno.82.P82.fam.orig
pheno.13.P13.fam
pheno.37.P37.bed
pheno.5.P5.fam
pheno.83.P83.bed
pheno.13.P13.fam.orig
pheno.37.P37.bim
pheno.5.P5.fam.orig
pheno.83.P83.bim
pheno.14.P14.bed
pheno.37.P37.fam
pheno.60.P60.bed
pheno.83.P83.fam
pheno.14.P14.bim
pheno.37.P37.fam.orig
pheno.60.P60.bim
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pheno.14.P14.fam
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Best,
Davide

[akcorut]

Hi @DavideGue,

Is the output/ folder empty?

[DavideGue]

Hi @akcorut
yes the output folder is empty!
Which version of gemma does the pipeline use?
I noticed problems running gemma v0.96, while I can run succesfully gemma v0.98 on my machine.
Could that be the problem?