Error executing process > 'mod:modkit_phase (1)'

[cihaterdogan]

Operating System

Other Linux (please specify below)

Other Linux

Red Hat Enterprise Linux 8.9

Workflow Version

v2.2.0

Workflow Execution

Command line (Cluster)

Other workflow execution

No response

EPI2ME Version

No response

CLI command run

module load singularity/3.7.2 module load java/15.0.2

export NXF_SINGULARITY_CACHEDIR=Path/.singularity export SINGULARITY_TMPDIR=Path/.singularity export NXF_HOME=Path

OUTPUT=s4059246577_results

./nextflow run epi2me-labs/wf-human-variation \ -w ${OUTPUT}/workspace \ -profile singularity \ --bam 'data/s4059246577_merged.bam' \ --basecaller_cfg 'dna_r10.4.1_e8.2_400bps_sup@v4.3.0' \ --mod \ --ref 'hg38/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna' \ --sample_name 's4059246577' \ --sv \ --snp \ --str \ --cnv \ --phased \ --threads 16 \ --bam_min_coverage 5 \ --out_dir ${OUTPUT}

Workflow Execution - CLI Execution Profile

None

What happened?

I was running the wf-human-variation pipeline as a job (with Slurm) on my institution's clusters using the above command. But got the following error

ERROR ~ Error executing process > 'mod:modkit_phase (1)'

Caused by: Process mod:modkit_phase (1) terminated with an error exit status (2)

Command executed:

modkit pileup \ chrY_hp.bam \ s4059249500 \ --ref GCA_000001405.15_GRCh38_no_alt_analysis_set.fna \ --partition-tag HP \ --interval-size 1000000 \ --prefix s4059249500.wf_mods.chrY \ --log-filepath modkit.log \ --region chrY \ --filter-threshold 0.78125 0.8613281 \ --threads 4 --combine-strands --cpg

Compress all

for i in ls s4059249500/; do root_name=$( basename $i '.bed' )

modkit saves the file as params.sample_name.wf_mods_haplotype.bed

  # create a new name with the patter params.sample_name.wf_mods.haplotype.bedmethyl
  new_name=$( echo ${root_name} | sed 's/wf_mods_/wf_mods\./' )
  mv s4059249500/${root_name}.bed s4059249500/${new_name}.bedmethyl
  bgzip s4059249500/${new_name}.bedmethyl

done

Command exit status: 2

Command output: (empty)

Command error: error: unexpected argument '0.8613281' found

Usage: modkit pileup [OPTIONS]

For more information, try '--help'.

Relevant log output

executor >  local (1297)
[3d/dfdce2] process > index_ref_fai (1)              [100%] 1 of 1 ✔
[44/668649] process > ingress:checkBamHeaders (1)    [100%] 1 of 1 ✔
[-        ] process > ingress:sortBam                -
[-        ] process > ingress:mergeBams              -
[-        ] process > ingress:catSortBams            -
[-        ] process > ingress:validateIndex          -
[08/1493a4] process > ingress:samtools_index (1)     [100%] 1 of 1 ✔
[50/f5b016] process > ingress:check_for_alignment... [100%] 1 of 1 ✔
[6a/f368e9] process > ingress:minimap2_alignment (1) [100%] 1 of 1 ✔
[0e/2a4326] process > getGenome (1)                  [100%] 1 of 1 ✔
[05/8f26cb] process > cram_cache (1)                 [100%] 1 of 1 ✔
[5f/8bfbd4] process > getAllChromosomesBed (1)       [100%] 1 of 1 ✔
[a3/1f727a] process > mosdepth_input (1)             [100%] 1 of 1 ✔
[27/18c41e] process > getVersions                    [100%] 1 of 1 ✔
[1a/f64212] process > getParams                      [100%] 1 of 1 ✔
[e0/83cba9] process > readStats (1)                  [100%] 1 of 1 ✔
[3c/66bfbc] process > infer_sex (1)                  [100%] 1 of 1 ✔
[fb/1bb250] process > makeAlignmentReport            [100%] 1 of 1 ✔
[-        ] process > failedQCReport                 -
[34/c1685c] process > lookup_clair3_model (1)        [100%] 1 of 1 ✔
[dd/026ccb] process > snp:make_chunks (1)            [100%] 1 of 1 ✔
[6f/037a89] process > snp:pileup_variants (622)      [100%] 632 of 632 ✔
[40/d9f4cd] process > snp:aggregate_pileup_varian... [100%] 1 of 1 ✔
[73/5db1df] process > snp:select_het_snps (10)       [100%] 25 of 25 ✔
[93/510285] process > snp:phase_contig (25)          [100%] 25 of 25 ✔
[1b/27cfdd] process > snp:get_qual_filter (1)        [100%] 1 of 1 ✔
[c1/f06fc8] process > snp:create_candidates (13)     [100%] 25 of 25 ✔
[f2/af49e1] process > snp:evaluate_candidates (465)  [100%] 472 of 472 ✔
[e4/4883d7] process > snp:aggregate_full_align_va... [100%] 1 of 1 ✔
[ed/16e203] process > snp:merge_pileup_and_full_v... [100%] 25 of 25 ✔
[59/d38a0d] process > snp:post_clair_phase_contig... [ 96%] 24 of 25
[07/e52bfe] process > snp:post_clair_contig_haplo... [ 54%] 13 of 24
[-        ] process > snp:cat_haplotagged_contigs    -
[-        ] process > snp:aggregate_all_variants     -
[-        ] process > snp:haploblocks_snp            -
[-        ] process > sv:variantCall:sniffles2       -
[-        ] process > sv:variantCall:filterCalls     -
[-        ] process > sv:variantCall:sortVCF         -
[-        ] process > sv:annotate_sv_vcf             -
[4e/7ef5bc] process > sv:runReport:getVersions       [100%] 1 of 1 ✔
[7e/5b72f4] process > sv:runReport:getParams         [100%] 1 of 1 ✔
[-        ] process > sv:runReport:report            -
[-        ] process > output_sv                      -
[-        ] process > refine_with_sv                 -
[-        ] process > concat_refined_snp             -
[-        ] process > annotate_snp_vcf               -
[-        ] process > concat_snp_vcfs                -
[-        ] process > sift_clinvar_snp_vcf           -
[-        ] process > vcfStats                       -
[f1/0dc753] process > report_snp:getVersions         [100%] 1 of 1 ✔
[16/6362ce] process > report_snp:getParams           [100%] 1 of 1 ✔
[-        ] process > report_snp:makeReport          -
[-        ] process > output_snp                     -
[17/f6cb0c] process > validate_modbam (1)            [100%] 1 of 1 ✔
[b8/5d85ab] process > sample_probs (1)               [100%] 1 of 1 ✔
[5f/71013a] process > mod:modkit_phase (1)           [  0%] 0 of 13
[-        ] process > mod:concat_bedmethyl           -
[be/456d47] process > cnv_spectre:mosdepth (1)       [100%] 1 of 1 ✔
[-        ] process > cnv_spectre:callCNV            -
[-        ] process > cnv_spectre:bgzip_and_index... -
[-        ] process > cnv_spectre:annotate_vcf       -
[63/c5cb97] process > cnv_spectre:getVersions        [100%] 1 of 1 ✔
[3e/c52b63] process > cnv_spectre:add_snp_tools_t... [100%] 1 of 1 ✔
[3f/7686fd] process > cnv_spectre:getParams          [100%] 1 of 1 ✔
[-        ] process > cnv_spectre:makeReport         -
[-        ] process > output_cnv                     -
[8a/a3236e] process > str:call_str (2)               [ 15%] 2 of 13
[-        ] process > str:annotate_repeat_expansions -
[d2/eb19fc] process > str:getVersions                [100%] 1 of 1 ✔
[ff/3a927d] process > str:getParams                  [100%] 1 of 1 ✔
[90/73337b] process > str:bam_region_filter (5)      [ 23%] 3 of 13
[-        ] process > str:bam_read_filter            -
[-        ] process > str:generate_str_content       -
[-        ] process > str:concat_str_vcfs            -
[-        ] process > str:merge_tsv                  -
[-        ] process > str:make_report                -
[-        ] process > output_str                     -
[72/15cdff] process > configure_jbrowse (1)          [100%] 1 of 1 ✔
[-        ] process > combine_metrics_json           -
[c9/18f5d8] process > publish_artifact (8)           [100%] 8 of 8
ERROR ~ Error executing process > 'mod:modkit_phase (1)'

Caused by:
  Process `mod:modkit_phase (1)` terminated with an error exit status (2)

Command executed:

  modkit pileup \
      chrY_hp.bam \
      s4059249500 \
      --ref GCA_000001405.15_GRCh38_no_alt_analysis_set.fna \
      --partition-tag HP \
      --interval-size 1000000 \
      --prefix s4059249500.wf_mods.chrY \
      --log-filepath modkit.log \
      --region chrY \
      --filter-threshold 0.78125 0.8613281 \
      --threads 4 --combine-strands --cpg

  # Compress all
  for i in `ls s4059249500/`; do
      root_name=$( basename $i '.bed' )
      # modkit saves the file as params.sample_name.wf_mods_haplotype.bed
      # create a new name with the patter params.sample_name.wf_mods.haplotype.bedmethyl
      new_name=$( echo ${root_name} | sed 's/wf_mods_/wf_mods\./' )
      mv s4059249500/${root_name}.bed s4059249500/${new_name}.bedmethyl
      bgzip s4059249500/${new_name}.bedmethyl
  done

Command exit status:
  2

Command output:
  (empty)

Command error:
  error: unexpected argument '0.8613281' found

  Usage: modkit pileup [OPTIONS] <IN_BAM> <OUT_BED>

  For more information, try '--help'.

Application activity log entry

No response

Were you able to successfully run the latest version of the workflow with the demo data?

yes

Other demo data information

No response

[cihaterdogan]

Please also find the log file here log.txt

[RenzoTale88]

@cihaterdogan could you please share the content of folder:

${OUTPUT}/workspace/b8/5d85ab*/

In particular, can you share the content of .command.env and .command.sh please?

[cihaterdogan]

Hi @RenzoTale88, Thank you for your reply. I was able to run the wf-human-variation pipeline with v2.0.0 and removed the old output due to space limitations. Unfortunately, I am unable to share the files you requested.

[RenzoTale88]

Ok no worries, I'll close this ticket then. We just released v2.2.2, so if you come across the issue again please re-open this issue with the new comment. Thanks again Andrea

[agatafant]

Hi @RenzoTale88 I am using v2.2.2 but I have exactly the same error. Thanks

[RenzoTale88]

@agatafant we will need more investigation into this then. Can you please share the nextflow log?

[agatafant]

nextflow.log

[RenzoTale88]

Can you please check the content of the following files:

/archive/s2/genomics/afant/NANOPORE_analysis/PDM0001NBLDN1R/pipe_wf_human_variation_threshold_latest/work/97/8923b6fe6fde356317df755ccedf3f/.command.env
/archive/s2/genomics/afant/NANOPORE_analysis/PDM0001NBLDN1R/pipe_wf_human_variation_threshold_latest/work/97/8923b6fe6fde356317df755ccedf3f/.command.err

[agatafant]

command.env probs=0.8183594 0.7421875

command.err is empty

thanks

[RenzoTale88]

Thanks for confirming this. Before progressing with some more tests, did you -resume the run after updating the workflow, or did you start from the beginning?

[agatafant]

I had to run -resume because:

1. I had to add sniffles options in nextflow.config
2. when running the first time it gives error when pulling from the repository so I manually did singularity pull --name ontresearch-wf-human-variation-sv-shac591518dd32ecc3936666c95ff08f6d7474e9728.img.pulling.1717507122239 docker://ontresearch/wf-human-variation-sv:shac591518dd32ecc3936666c95ff08f6d7474e9728

[RenzoTale88]

The issue with this is that it will not retry the sample-probs process, and therefore will fail again with the same error. Can you try removing the following file and try again?

/archive/s2/genomics/afant/NANOPORE_analysis/PDM0001NBLDN1R/pipe_wf_human_variation_threshold_latest/work/97/8923b6fe6fde356317df755ccedf3f/.command.env

[RenzoTale88]

@agatafant can you provide more details on how you generated the input BAM?

[agatafant]

As you suggested I removed /archive/s2/genomics/afant/NANOPORE_analysis/PDM0001NBLDN1R/pipe_wf_human_variation_threshold_latest/work/97/8923b6fe6fde356317df755ccedf3f/.command.env, I have launched again the pipeline with -resume but I have obtained same error. The bam is obtained with dorado basecaller v.0.6.0

[RenzoTale88]

@agatafant I was mostly interested in knowing which remora model(s) did you use. Did you use a single model, multiple models, etc

[agatafant]

dorado basecaller -x cuda:all --reference /archive/s1/sconsRequirements/databases/reference/resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta /archive/s2/genomics/afant/PCG_Nanopore/out_PCG_PDM_PDM0001NBLDN1R/dorado0.6.0/dna_r10.4.1_e8.2_400bps_sup@v4.3.0 /archive/genomic_data/Collaboration/PCG_ParkinsonDiseaseMilano/FAST5/PCG_PDM_PDM0001NBLDN1R/PDM0001NBLDN1R/20240409_1716_1G_PAS38058_469a1fd2/pod5/ --modified-bases-models /archive/s2/genomics/afant/PCG_Nanopore/out_PCG_PDM_PDM0001NBLDN1R/dorado0.6.0/dna_r10.4.1_e8.2_400bps_sup@v4.3.0_6mA@v2,/archive/s2/genomics/afant/PCG_Nanopore/out_PCG_PDM_PDM0001NBLDN1R/dorado0.6.0/dna_r10.4.1_e8.2_400bps_sup@v4.3.0_5mC_5hmC@v1

[RenzoTale88]

Ok this confirms the issue, i.e. the workflow currently can't handle multiple modified bases, and causes to compute multiple filtering thresholds. We will work on a patch, and notify when ready.

[agatafant]

Ok great, thank you. In the meanwhile I re-obtain the BAM with only one modified bases

[RenzoTale88]

@agatafant we just released wf-human-variation v2.2.4, that should support multiple modification types (e.g. modA and modC). Can you try with this release and let us know if it works for you?

[agatafant]

Thank you @RenzoTale88. I still have this problem when launching the pipeline: I have to remove folder ~/.nextflow/assets/epi2me-labs otherwise it fails with: Pulling epi2me-labs/wf-human-variation... epi2me-labs/wf-human-variation contains uncommitted changes -- cannot pull from repository

But since I have to modify some options in nextflow.config I have to stop the pipeline (is there a better way to do this?), modify the config and then resume, but when resuming it fails again with: Pulling epi2me-labs/wf-human-variation... epi2me-labs/wf-human-variation contains uncommitted changes -- cannot pull from repository

[RenzoTale88]

Hi @agatafant try do first drop the workflow:

nextflow drop epi2me-labs/wf-human-variation

And then try the analysis again, specifying version 2.2.4:

nextflow run epi2me-labs/wf-human-variation -r v2.2.4 [OPTIONS HERE]

[agatafant]

Hi, I have launched the pipeline, but it continues failing because of: sort: write failed: /var/tmp/pbs.1385700.5kgpsmhpcfe/sortCqvMDv: No space left on device why is it writing in /var/tmp?

attached the log nextflow.log

[RenzoTale88]

From what I can see, you are using a PBS distributed system right? If so, you need to discuss with your IT support to define an appropriate configuration for your system to avoid this issue.