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no alignment #10

Closed carolinehey closed 1 year ago

carolinehey commented 1 year ago

What happened?

Hi,

I provided a ubam with Ml and MM tags to the workflow but get an error with sniffles2 which i think is because it did not perform alignment. Should it not start with aligning before performing any of the other steps or am i missing something? nextflow run epi2me-labs/wf-human-variation \ -w workspace \ -profile standard \ --sv --methyl \ --bam sample1_ubam.bam \ --ref T2T-CHM13v2_reference.fasta \ --sample_name sample1 \ --out_dir output

Operating System

ubuntu 18.04

Workflow Execution

Command line

Workflow Execution - EPI2ME Labs Versions

No response

Workflow Execution - Execution Profile

No response

Workflow Version

nextflow version 22.04.5 workflow version 0.3.0

Relevant log output

Workflow execution completed unsuccessfully!
The exit status of the task that caused the workflow execution to fail was: 1.

The full error message was:

Error executing process > 'sv:variantCall:sniffles2 (1)'

Caused by:
  Process `sv:variantCall:sniffles2 (1)` terminated with an error exit status (1)

Command executed:

  export REF_PATH=ref_cache/%2s/%2s/%s:null
  sniffles         --threads 4         --sample-id sample1         --output-rnames         --cluster-merge-pos 150         --input sample1.filtered.cram                           --vcf sample1.sniffles.vcf
  sed -i '/.:0:0:0:NULL/d' sample1.sniffles.vcf

Command exit status:
  1

Command output:
  Running Sniffles2, build 2.0.3
    Run Mode: call_sample
    Start on: 2022/10/13 09:25:45
    Working dir: /longread/Caroline/sample1/workspace/8a/1697b3b6ad787ce1bcff330f1df651
    Used command: /home/epi2melabs/conda/bin/sniffles --threads 4 --sample-id sample1 --output-rnames --cluster-merge-pos 150 --input sample1.filtered.cram --vcf sample1.sniffles.vcf
  ==============================
  Opening for reading: sample1.filtered.cram

Command error:
  Traceback (most recent call last):
    File "/home/epi2melabs/conda/bin/sniffles", line 607, in 
      Sniffles2_Main(config.from_cmdline(),processes)
    File "/home/epi2melabs/conda/bin/sniffles", line 117, in Sniffles2_Main
      bam_in=pysam.AlignmentFile(config.input, config.input_mode)
    File "pysam/libcalignmentfile.pyx", line 751, in pysam.libcalignmentfile.AlignmentFile.__cinit__
    File "pysam/libcalignmentfile.pyx", line 1000, in pysam.libcalignmentfile.AlignmentFile._open
  ValueError: file has no sequences defined (mode='rc') - is it SAM/BAM format? Consider opening with check_sq=False

Work dir:
  /longread/Caroline/sample1/workspace/8a/1697b3b6ad787ce1bcff330f1df651

Tip: view the complete command output by changing to the process work dir and entering the command `cat .command.out`
SamStudio8 commented 1 year ago

Hi @carolinehey! The pipeline tries to determine if the input BAM needs alignment by checking the BAM header's SQ lines against the reference. Can you confirm with your .nextflow.log that the minimap2_ubam step was not called (I can't see that from this log).

Can you sanity check the format of the file /longread/Caroline/sample1/workspace/8a/1697b3b6ad787ce1bcff330f1df651.sample1.filtered.cram? Does it look like a CRAM file? Does it have zero sequences as the error message reports?

carolinehey commented 1 year ago

The nextflow.log. I think minimap2_ubam step was called, but i'm not sure. Oct-13 11:16:25.053 [main] DEBUG nextflow.cli.Launcher - $> nextflow run epi2me-labs/wf-human-variation -w workspace -profile standard --sv --methyl --bam sample1_ubam.bam --ref T2T-CHM13v2_reference.fasta --sample_name sample1 --out_dir output Oct-13 11:16:25.124 [main] INFO nextflow.cli.CmdRun - N E X T F L O W ~ version 22.04.5 Oct-13 11:16:25.933 [main] DEBUG nextflow.scm.AssetManager - Git config: /root/.nextflow/assets/epi2me-labs/wf-human-variation/.git/config; branch: master; remote: origin; url: https://github.com/epi2me-labs/wf-human-variation.git Oct-13 11:16:25.945 [main] DEBUG nextflow.scm.AssetManager - Git config: /root/.nextflow/assets/epi2me-labs/wf-human-variation/.git/config; branch: master; remote: origin; url: https://github.com/epi2me-labs/wf-human-variation.git Oct-13 11:16:27.388 [main] INFO nextflow.scm.AssetManager - NOTE: Your local project version looks outdated - a different revision is available in the remote repository [627b26b3e7] Oct-13 11:16:27.401 [main] DEBUG nextflow.config.ConfigBuilder - Found config base: /root/.nextflow/assets/epi2me-labs/wf-human-variation/nextflow.config Oct-13 11:16:27.404 [main] DEBUG nextflow.config.ConfigBuilder - Parsing config file: /root/.nextflow/assets/epi2me-labs/wf-human-variation/nextflow.config Oct-13 11:16:27.415 [main] DEBUG nextflow.config.ConfigBuilder - Applying config profile:standard Oct-13 11:16:27.634 [main] DEBUG nextflow.cli.CmdRun - Applied DSL=2 from script declararion Oct-13 11:16:27.634 [main] INFO nextflow.cli.CmdRun - Launchinghttps://github.com/epi2me-labs/wf-human-variation` [nauseous_solvay] DSL2 - revision: 898280e1ea [master] Oct-13 11:16:27.650 [main] DEBUG nextflow.plugin.PluginsFacade - Setting up plugin manager > mode=prod; plugins-dir=/root/.nextflow/plugins; core-plugins: nf-amazon@1.7.2,nf-azure@0.13.2,nf-console@1.0.3,nf-ga4gh@1.0.3,nf-google@1.1.4,nf-sqldb@0.4.0,nf-tower@1.4.0 Oct-13 11:16:27.651 [main] DEBUG nextflow.plugin.PluginsFacade - Plugins default=[] Oct-13 11:16:27.682 [main] INFO org.pf4j.DefaultPluginStatusProvider - Enabled plugins: [] Oct-13 11:16:27.683 [main] INFO org.pf4j.DefaultPluginStatusProvider - Disabled plugins: [] Oct-13 11:16:27.688 [main] INFO org.pf4j.DefaultPluginManager - PF4J version 3.4.1 in 'deployment' mode Oct-13 11:16:27.699 [main] INFO org.pf4j.AbstractPluginManager - No plugins Oct-13 11:16:27.752 [main] DEBUG nextflow.Session - Session uuid: 2f6bd1b4-d7d9-4bcf-95bc-6960b54ffee4 Oct-13 11:16:27.753 [main] DEBUG nextflow.Session - Run name: nauseous_solvay Oct-13 11:16:27.753 [main] DEBUG nextflow.Session - Executor pool size: 64 Oct-13 11:16:27.794 [main] DEBUG nextflow.cli.CmdRun - Version: 22.04.5 build 5708 Created: 15-07-2022 16:09 UTC (18:09 CEST) System: Linux 4.15.0-187-generic Runtime: Groovy 3.0.10 on OpenJDK 64-Bit Server VM 11.0.1-internal+0-adhoc..src Encoding: UTF-8 (UTF-8) Process: 50937@srvodeappgsq02 [10.161.6.233] CPUs: 64 - Mem: 376.5 GB (65.5 GB) - Swap: 8 GB (7.5 GB) Oct-13 11:16:27.860 [main] DEBUG nextflow.Session - Work-dir: /longread/Caroline/sample1/workspace [UNKNOWN (0xfe534d42)] Oct-13 11:16:27.874 [main] DEBUG nextflow.executor.ExecutorFactory - Extension executors providers=[] Oct-13 11:16:27.886 [main] DEBUG nextflow.Session - Observer factory: DefaultObserverFactory Oct-13 11:16:27.985 [main] DEBUG nextflow.cache.CacheFactory - Using Nextflow cache factory: nextflow.cache.DefaultCacheFactory Oct-13 11:16:27.998 [main] DEBUG nextflow.util.CustomThreadPool - Creating default thread pool > poolSize: 65; maxThreads: 1000 Oct-13 11:16:28.230 [main] DEBUG nextflow.Session - Session start invoked Oct-13 11:16:28.240 [main] DEBUG nextflow.trace.TraceFileObserver - Flow starting -- trace file: /longread/Caroline/sample1/output/execution/trace.txt Oct-13 11:16:28.288 [main] DEBUG nextflow.Session - Using default localLib path: /root/.nextflow/assets/epi2me-labs/wf-human-variation/lib Oct-13 11:16:28.293 [main] DEBUG nextflow.Session - Adding to the classpath library: /root/.nextflow/assets/epi2me-labs/wf-human-variation/lib Oct-13 11:16:28.293 [main] DEBUG nextflow.Session - Adding to the classpath library: /root/.nextflow/assets/epi2me-labs/wf-human-variation/lib/nfcore_external_java_deps.jar Oct-13 11:16:29.542 [main] DEBUG nextflow.script.ScriptRunner - > Launching execution Oct-13 11:16:31.455 [main] INFO nextflow.Nextflow - Core Nextflow options revision : master runName : nauseous_solvay containerEngine : docker container : ontresearch/wf-human-variation:sha90e1c9ae1b7cd51548c4c9b66df424fe3c3a81a8 launchDir : /longread/Caroline/sample1 workDir : /longread/Caroline/sample1/workspace projectDir : /root/.nextflow/assets/epi2me-labs/wf-human-variation userName : root profile : standard configFiles : /root/.nextflow/assets/epi2me-labs/wf-human-variation/nextflow.config

Basic Input/Output Options bam : sample1_ubam.bam ref : T2T-CHM13v2_reference.fasta ubam_sort_threads : 3 ubam_bam2fq_threads: 1

wf-human-sv options sv : true

wf-human-snp options sample_name : sample1

Methylation calling options methyl : true

Other parameters process_label : wfdefault

!! Only displaying parameters that differ from the pipeline defaults !! ------------------------------------------------------ If you use epi2me-labs/wf-human-variation for your analysis please cite:

Oct-13 11:16:31.543 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.544 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.548 [main] DEBUG nextflow.executor.Executor - [warm up] executor > local Oct-13 11:16:31.554 [main] DEBUG n.processor.LocalPollingMonitor - Creating local task monitor for executor 'local' > cpus=16; memory=8 GB; capacity=64; pollInterval=100ms; dumpInterval=5m Oct-13 11:16:31.922 [main] DEBUG nextflow.script.ProcessConfig - Config settings withLabel:wf_human_methyl matches labels wf_human_methyl for process with name methyl:modbam2bed Oct-13 11:16:31.923 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.923 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.930 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.930 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.933 [main] DEBUG nextflow.script.ProcessConfig - Config settings withLabel:wf_human_sv matches labels wf_human_sv for process with name sv:getAllChromosomesBed Oct-13 11:16:31.933 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.933 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.937 [main] DEBUG nextflow.script.ProcessConfig - Config settings withLabel:wf_human_sv matches labels wf_human_sv for process with name sv:variantCall:filterBam Oct-13 11:16:31.937 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.937 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.942 [main] DEBUG nextflow.script.ProcessConfig - Config settings withLabel:wf_human_sv matches labels wf_human_sv for process with name sv:variantCall:sniffles2 Oct-13 11:16:31.942 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.943 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.948 [main] DEBUG nextflow.script.ProcessConfig - Config settings withLabel:wf_human_sv matches labels wf_human_sv for process with name sv:variantCall:mosdepth Oct-13 11:16:31.949 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.949 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.953 [main] DEBUG nextflow.script.ProcessConfig - Config settings withLabel:wf_human_sv matches labels wf_human_sv for process with name sv:variantCall:filterCalls Oct-13 11:16:31.954 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.954 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.957 [main] DEBUG nextflow.script.ProcessConfig - Config settings withLabel:wf_human_sv matches labels wf_human_sv for process with name sv:variantCall:sortVCF Oct-13 11:16:31.957 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.957 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.959 [main] DEBUG nextflow.script.ProcessConfig - Config settings withLabel:wf_human_sv matches labels wf_human_sv for process with name sv:variantCall:indexVCF Oct-13 11:16:31.960 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.960 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.968 [main] DEBUG nextflow.script.ProcessConfig - Config settings withLabel:wf_human_sv matches labels wf_human_sv for process with name sv:runReport:getVersions Oct-13 11:16:31.969 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.969 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.970 [main] DEBUG nextflow.script.ProcessConfig - Config settings withLabel:wf_human_sv matches labels wf_human_sv for process with name sv:runReport:getParams Oct-13 11:16:31.971 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.971 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.975 [main] DEBUG nextflow.script.ProcessConfig - Config settings withLabel:wf_human_sv matches labels wf_human_sv for process with name sv:runReport:report Oct-13 11:16:31.976 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.976 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.986 [main] DEBUG nextflow.script.ProcessConfig - Config settings withLabel:wf_human_sv matches labels wf_human_sv for process with name output_sv Oct-13 11:16:31.986 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null Oct-13 11:16:31.987 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local' Oct-13 11:16:31.988 [main] DEBUG nextflow.Session - Workflow process names [dsl2]: filterBam, get_qual_filter, create_candidates, sortVCF, sort_calls, sv:variantCall:sniffles2, index_ref_gzi, indexVCF, makeReport, hap, select_het_snps, getVersions, sv:runReport:getVersions, sv:runReport:report, output_snp, pileup_variants, methyl:modbam2bed, guppy, phase_contig, aggregate_full_align_variants, minimap2_ubam, getAllChromosomesBed, post_clair_phase_contig, getParams, sv:variantCall:mosdepth, merge_pileup_and_full_vars, sv:variantCall:indexVCF, sv:runReport:getParams, sniffles2, output_sv, readStats, evaluate_candidates, aggregate_pileup_variants, index_ref_fai, output_methyl, filterCalls, sv:variantCall:sortVCF, make_chunks, cram_cache, sv:variantCall:filterCalls, vcfStats, merge_calls, mosdepth, sv:getAllChromosomesBed, decompress_ref, aggregate_all_variants, report, modbam2bed, sv:variantCall:filterBam Oct-13 11:16:31.989 [main] DEBUG nextflow.Session - Ignite dataflow network (18) Oct-13 11:16:31.994 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > cram_cache Oct-13 11:16:31.995 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > methyl:modbam2bed Oct-13 11:16:31.996 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > output_methyl Oct-13 11:16:31.998 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > sv:getAllChromosomesBed Oct-13 11:16:31.999 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > sv:variantCall:filterBam Oct-13 11:16:31.999 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > sv:variantCall:sniffles2 Oct-13 11:16:32.000 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > sv:variantCall:mosdepth Oct-13 11:16:32.002 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > sv:variantCall:filterCalls Oct-13 11:16:32.005 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > sv:variantCall:sortVCF Oct-13 11:16:32.005 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > sv:variantCall:indexVCF Oct-13 11:16:32.007 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > sv:runReport:getVersions Oct-13 11:16:32.012 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > sv:runReport:getParams Oct-13 11:16:32.014 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > sv:runReport:report Oct-13 11:16:32.016 [main] DEBUG nextflow.processor.TaskProcessor - Starting process > output_sv Oct-13 11:16:32.018 [main] DEBUG nextflow.script.ScriptRunner - > Await termination Oct-13 11:16:32.019 [main] DEBUG nextflow.Session - Session await Oct-13 11:16:32.248 [Task submitter] DEBUG nextflow.executor.LocalTaskHandler - Launch cmd line: /bin/bash -ue .command.run Oct-13 11:16:32.255 [Task submitter] INFO nextflow.Session - [f0/ef99b0] Submitted process > cram_cache (1) Oct-13 11:16:32.292 [Task submitter] DEBUG nextflow.executor.LocalTaskHandler - Launch cmd line: /bin/bash -ue .command.run Oct-13 11:16:32.292 [Task submitter] INFO nextflow.Session - [a1/6b41ba] Submitted process > sv:runReport:getVersions Oct-13 11:16:32.332 [Task submitter] DEBUG nextflow.executor.LocalTaskHandler - Launch cmd line: /bin/bash -ue .command.run Oct-13 11:16:32.333 [Task submitter] INFO nextflow.Session - [d3/5599da] Submitted process > sv:runReport:getParams Oct-13 11:16:34.373 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 2; name: sv:runReport:getParams; status: COMPLETED; exit: 0; error: -; workDir: /longread/Caroline/sample1/workspace/d3/5599da8e958a11abeee36faaf85c3c] Oct-13 11:16:39.383 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 3; name: sv:runReport:getVersions; status: COMPLETED; exit: 0; error: -; workDir: /longread/Caroline/sample1/workspace/a1/6b41ba8e9baf28e5c4ef1e65d7cba3] Oct-13 11:17:58.250 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 1; name: cram_cache (1); status: COMPLETED; exit: 0; error: -; workDir: /longread/Caroline/sample1/workspace/f0/ef99b0497c67b2800cb442274b367f] Oct-13 11:17:58.557 [Task submitter] DEBUG nextflow.executor.LocalTaskHandler - Launch cmd line: /bin/bash -ue .command.run Oct-13 11:17:58.558 [Task submitter] INFO nextflow.Session - [82/44af20] Submitted process > sv:getAllChromosomesBed (1) Oct-13 11:17:58.599 [Task submitter] DEBUG nextflow.executor.LocalTaskHandler - Launch cmd line: /bin/bash -ue .command.run Oct-13 11:17:58.600 [Task submitter] INFO nextflow.Session - [ec/92fec2] Submitted process > methyl:modbam2bed (1) Oct-13 11:17:58.639 [Task submitter] DEBUG nextflow.executor.LocalTaskHandler - Launch cmd line: /bin/bash -ue .command.run Oct-13 11:17:58.639 [Task submitter] INFO nextflow.Session - [b6/377bff] Submitted process > sv:variantCall:filterBam (1) Oct-13 11:18:28.905 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 6; name: sv:getAllChromosomesBed (1); status: COMPLETED; exit: 0; error: -; workDir: /longread/Caroline/sample1/workspace/82/44af20c24ab4dd6b385ec6105d79b5] Oct-13 11:21:08.265 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 5; name: methyl:modbam2bed (1); status: COMPLETED; exit: 0; error: -; workDir: /longread/Caroline/sample1/workspace/ec/92fec2a616889feb6c208845c7cdc4] Oct-13 11:21:08.386 [Task submitter] DEBUG nextflow.executor.LocalTaskHandler - Launch cmd line: /bin/bash -ue .command.run Oct-13 11:21:08.387 [Task submitter] INFO nextflow.Session - [c0/5dadbb] Submitted process > output_methyl (1) Oct-13 11:21:10.222 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 7; name: output_methyl (1); status: COMPLETED; exit: 0; error: -; workDir: /longread/Caroline/sample1/workspace/c0/5dadbb6daf134873b5212f8e08e372] Oct-13 11:21:10.397 [Task monitor] DEBUG nextflow.util.ThreadPoolBuilder - Creating thread pool 'FileTransfer' minSize=4; maxSize=4; workQueue=LinkedBlockingQueue[10000]; allowCoreThreadTimeout=false Oct-13 11:21:31.757 [Task monitor] DEBUG n.processor.TaskPollingMonitor - !! executor local > tasks to be completed: 1 -- submitted tasks are shown below ~> TaskHandler[id: 4; name: sv:variantCall:filterBam (1); status: RUNNING; exit: -; error: -; workDir: /longread/Caroline/sample1/workspace/b6/377bff5ca8712d3bdc95ff34feca69] Oct-13 11:25:39.903 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 4; name: sv:variantCall:filterBam (1); status: COMPLETED; exit: 0; error: -; workDir: /longread/Caroline/sample1/workspace/b6/377bff5ca8712d3bdc95ff34feca69] Oct-13 11:25:44.219 [Actor Thread 65] DEBUG nextflow.util.CacheHelper - Hash asset file sha-256: /root/.nextflow/assets/epi2me-labs/wf-human-variation/data/OPTIONAL_FILE Oct-13 11:25:44.258 [Task submitter] DEBUG nextflow.executor.LocalTaskHandler - Launch cmd line: /bin/bash -ue .command.run Oct-13 11:25:44.259 [Task submitter] INFO nextflow.Session - [2d/c5a4f5] Submitted process > sv:variantCall:mosdepth (1) Oct-13 11:25:44.305 [Task submitter] DEBUG nextflow.executor.LocalTaskHandler - Launch cmd line: /bin/bash -ue .command.run Oct-13 11:25:44.306 [Task submitter] INFO nextflow.Session - [8a/1697b3] Submitted process > sv:variantCall:sniffles2 (1) Oct-13 11:25:46.209 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 8; name: sv:variantCall:mosdepth (1); status: COMPLETED; exit: 0; error: -; workDir: /longread/Caroline/sample1/workspace/2d/c5a4f5ed9e249785aa67b56f7dcf64] Oct-13 11:25:46.389 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 9; name: sv:variantCall:sniffles2 (1); status: COMPLETED; exit: 1; error: -; workDir: /longread/Caroline/sample1/workspace/8a/1697b3b6ad787ce1bcff330f1df651] Oct-13 11:25:46.430 [Task monitor] ERROR nextflow.processor.TaskProcessor - Error executing process > 'sv:variantCall:sniffles2 (1)'

Caused by: Process sv:variantCall:sniffles2 (1) terminated with an error exit status (1)

Command executed:

export REF_PATH=ref_cache/%2s/%2s/%s:null sniffles --threads 4 --sample-id sample1 --output-rnames --cluster-merge-pos 150 --input sample1.filtered.cram --vcf sample1.sniffles.vcf sed -i '/.:0:0:0:NULL/d' sample1.sniffles.vcf

Command exit status: 1

Command output: Running Sniffles2, build 2.0.3 Run Mode: call_sample Start on: 2022/10/13 09:25:45 Working dir: /longread/Caroline/sample1/workspace/8a/1697b3b6ad787ce1bcff330f1df651 Used command: /home/epi2melabs/conda/bin/sniffles --threads 4 --sample-id sample1 --output-rnames --cluster-merge-pos 150 --input sample1.filtered.cram --vcf sample1.sniffles.vcf

Opening for reading: sample1.filtered.cram

Command error: Traceback (most recent call last): File "/home/epi2melabs/conda/bin/sniffles", line 607, in Sniffles2_Main(config.from_cmdline(),processes) File "/home/epi2melabs/conda/bin/sniffles", line 117, in Sniffles2_Main bam_in=pysam.AlignmentFile(config.input, config.input_mode) File "pysam/libcalignmentfile.pyx", line 751, in pysam.libcalignmentfile.AlignmentFile.cinit File "pysam/libcalignmentfile.pyx", line 1000, in pysam.libcalignmentfile.AlignmentFile._open ValueError: file has no sequences defined (mode='rc') - is it SAM/BAM format? Consider opening with check_sq=False

Work dir: /longread/Caroline/sample1/workspace/8a/1697b3b6ad787ce1bcff330f1df651

Tip: view the complete command output by changing to the process work dir and entering the command cat .command.out Oct-13 11:25:46.439 [Task monitor] DEBUG nextflow.Session - Session aborted -- Cause: Process sv:variantCall:sniffles2 (1) terminated with an error exit status (1) Oct-13 11:25:46.458 [Task monitor] DEBUG nextflow.Session - The following nodes are still active: [process] output_sv status=ACTIVE port 0: (queue) OPEN ; channel: fname port 1: (cntrl) - ; channel: $

Oct-13 11:25:46.462 [main] DEBUG nextflow.Session - Session await > all process finished Oct-13 11:25:46.657 [main] DEBUG nextflow.Session - Session await > all barriers passed Oct-13 11:25:46.720 [main] DEBUG nextflow.trace.WorkflowStatsObserver - Workflow completed > WorkflowStats[succeededCount=8; failedCount=1; ignoredCount=0; cachedCount=0; pendingCount=0; submittedCount=0; runningCount=0; retriesCount=0; abortedCount=0; succeedDuration=45m 8s; failedDuration=8s; cachedDuration=0ms;loadCpus=0; loadMemory=0; peakRunning=3; peakCpus=9; peakMemory=0; ] Oct-13 11:25:46.721 [main] DEBUG nextflow.trace.TraceFileObserver - Flow completing -- flushing trace file Oct-13 11:25:46.724 [main] DEBUG nextflow.trace.ReportObserver - Flow completing -- rendering html report Oct-13 11:25:46.780 [main] DEBUG nextflow.trace.ReportObserver - Execution report summary data: 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I made the ubam files using guppy and it surprised med that the header icluded SO:coordinate since it was run without a ref to obtain ubam. Maybe this is the cause?

SamStudio8 commented 1 year ago

Hi @carolinehey, thanks for the log output. It's not very clear but you're actually using v0.2.2 of the pipeline!

[main] INFO nextflow.scm.AssetManager - NOTE: Your local project version looks outdated - a different revision is available in the remote repository

Can you update to 0.3.0 with nextflow pull epi2me-labs/wf-human-variation and try again?

carolinehey commented 1 year ago

Thanks, unfortunately there is still something wrong with the file.

Error` executing process > 'check_for_alignment (1)'

Caused by: Process check_for_alignment (1) terminated with an error exit status (1)

Command executed:

realign=0 check_sq_ref.py --xam sample1_ubam.bam --ref T2T-CHM13v2_reference.fasta || realign=$?

Allow EX_OK and EX_DATAERR, otherwise explode if [ $realign -ne 0 ] && [ $realign -ne 65 ]; then exit 1 fi

Command exit status: 1

Command output: (empty)

Command error: Unable to find image 'ontresearch/wf-human-variation:shab3358c08b332f9d113caa258b6daea245184bc00' locally shab3358c08b332f9d113caa258b6daea245184bc00: Pulling from ontresearch/wf-human-variation 3b65ec22a9e9: Already exists 841971ce0eda: Already exists b3bf890f2514: Already exists b3bf890f2514: Already exists 27a9f84bae05: Already exists 0d9e0007c6ce: Already exists f34a55227e59: Pulling fs layer f8157626c138: Pulling fs layer 2802728cf048: Pulling fs layer f8157626c138: Waiting 2802728cf048: Waiting f34a55227e59: Download complete f34a55227e59: Pull complete f8157626c138: Download complete f8157626c138: Pull complete 2802728cf048: Verifying Checksum 2802728cf048: Download complete 2802728cf048: Pull complete Digest: sha256:ba793703036591653889ed598b514ca703e995b661863bbea1a5193ea8a2250c Status: Downloaded newer image for ontresearch/wf-human-variation:shab3358c08b332f9d113caa258b6daea245184bc00 Traceback (most recent call last): File "/root/.nextflow/assets/epi2me-labs/wf-human-variation/bin/check_sq_ref.py", line 26, in xam = pysam.AlignmentFile(args.xam) File "pysam/libcalignmentfile.pyx", line 751, in pysam.libcalignmentfile.AlignmentFile.cinit File "pysam/libcalignmentfile.pyx", line 1000, in pysam.libcalignmentfile.AlignmentFile._open ValueError: file has no sequences defined (mode='r') - is it SAM/BAM format? Consider opening with check_sq=False

Work dir: /longread/Caroline/sample1/workspace/b5/239e54598a4f2b8858b4417ff37aee

Tip: you can try to figure out what's wrong by changing to the process work dir and showing the script file named .command.sh

SamStudio8 commented 1 year ago

Thanks for trying that out @carolinehey, we're making progress! This is a bug and I can reproduce it internally. Hang tight and I can get a patch release out today.

SamStudio8 commented 1 year ago

I've now released wf-human-variation 0.3.1, please update your local copy with nextflow pull epi2me-labs/wf-human-variation and let me know if this fixes your issue.

carolinehey commented 1 year ago

Thank you very much for the help @SamStudio8, IT WORKS!

My only "issue" is that the output cram file is not in the output directory together with the other outputs but in the workspace directoy. At least i hope it is the final cram file that i find here.

SamStudio8 commented 1 year ago

Hi @carolinehey, I'm glad that's working for you now! Thanks for reporting your CRAM file issue, I've checked on this and that is also a bug (you're two for two). I've fixed this internally and this will be included in our next patch release.

SamStudio8 commented 1 year ago

Hi @carolinehey, thanks for your patience. The fix for the missing CRAM has now been released as part of wf-human-variation v0.4.0! Please use nextflow pull epi2me-labs/wf-human-variation to update.