下流码头问题--码头工人的许可问题？无法运行pomoxis容器

Question

我对nextflow和码头集装箱很陌生。我正在尝试重新组装一些读取数据，并将其映射到一个参考基因组中，但始终得到以下错误：

shaun@shaun-HP-Z6-G4-Workstation:~/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble$ nextflow pomoxis_map_assemble_nextflow.nf
N E X T F L O W  ~  version 22.04.5
Launching `pomoxis_map_assemble_nextflow.nf` [goofy_hoover] DSL1 - revision: 6e4be1e0bd
executor >  local (1)
executor >  local (1)
[cf/bc2b69] process > pomoxis (1) [100%] 1 of 1, failed: 1 ✘
WARN: There's no process matching config selector: fastqc
WARN: There's no process matching config selector: porechop
WARN: There's no process matching config selector: bioawk
WARN: There's no process matching config selector: fastqconvert
WARN: There's no process matching config selector: blast_raw
Error executing process > 'pomoxis (1)'

Caused by:
  Process `pomoxis (1)` terminated with an error exit status (127)

Command executed:

  mini_align -i output.fastq -r /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble/ref/*.fasta -o results -p > output_test_final.fa

Command exit status:
  127

Command output:
  (empty)

Command error:
  .command.sh: line 2: mini_align: command not found

Work dir:
  /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble/work/cf/bc2b696aea0863d76c2c9221315c39

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`

警告是因为我对所有的.nf脚本都使用了一个主配置文件。

下面是上面错误的日志文件：

Oct.-12 10:33:04.759 [main] DEBUG nextflow.cli.Launcher - $> nextflow pomoxis_map_assemble_nextflow.nf
Oct.-12 10:33:04.822 [main] INFO  nextflow.cli.CmdRun - N E X T F L O W  ~  version 22.04.5
Oct.-12 10:33:04.837 [main] DEBUG nextflow.config.ConfigBuilder - Found config local: /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble/nextflow.config
Oct.-12 10:33:04.838 [main] DEBUG nextflow.config.ConfigBuilder - Parsing config file: /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble/nextflow.config
Oct.-12 10:33:04.856 [main] DEBUG nextflow.config.ConfigBuilder - Applying config profile: `standard`
Oct.-12 10:33:05.414 [main] DEBUG nextflow.cli.CmdRun - Applied DSL=1 by probing script field
Oct.-12 10:33:05.428 [main] INFO  nextflow.cli.CmdRun - Launching `pomoxis_map_assemble_nextflow.nf` [goofy_hoover] DSL1 - revision: 6e4be1e0bd
Oct.-12 10:33:05.439 [main] DEBUG nextflow.plugin.PluginsFacade - Setting up plugin manager > mode=prod; plugins-dir=/home/shaun/.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.-12 10:33:05.440 [main] DEBUG nextflow.plugin.PluginsFacade - Plugins default=[]
Oct.-12 10:33:05.449 [main] INFO  org.pf4j.DefaultPluginStatusProvider - Enabled plugins: []
Oct.-12 10:33:05.449 [main] INFO  org.pf4j.DefaultPluginStatusProvider - Disabled plugins: []
Oct.-12 10:33:05.452 [main] INFO  org.pf4j.DefaultPluginManager - PF4J version 3.4.1 in 'deployment' mode
Oct.-12 10:33:05.478 [main] INFO  org.pf4j.AbstractPluginManager - No plugins
Oct.-12 10:33:05.527 [main] DEBUG nextflow.Session - Session uuid: ddab5124-cc54-4c39-840d-6cc1bf664773
Oct.-12 10:33:05.527 [main] DEBUG nextflow.Session - Run name: goofy_hoover
Oct.-12 10:33:05.527 [main] DEBUG nextflow.Session - Executor pool size: 16
Oct.-12 10:33:05.547 [main] DEBUG nextflow.cli.CmdRun - 
  Version: 22.04.5 build 5708
  Created: 15-07-2022 16:09 UTC (16-07-2022 01:39 ACDT)
  System: Linux 5.13.0-52-generic
  Runtime: Groovy 3.0.10 on OpenJDK 64-Bit Server VM 11.0.15+10-Ubuntu-0ubuntu0.21.10.1
  Encoding: UTF-8 (UTF-8)
  Process: 665521@shaun-HP-Z6-G4-Workstation [127.0.1.1]
  CPUs: 16 - Mem: 62.5 GB (32.1 GB) - Swap: 2 GB (2 GB)
Oct.-12 10:33:05.564 [main] DEBUG nextflow.Session - Work-dir: /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble/work [ext2/ext3]
Oct.-12 10:33:05.564 [main] DEBUG nextflow.Session - Script base path does not exist or is not a directory: /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble/bin
Oct.-12 10:33:05.573 [main] DEBUG nextflow.executor.ExecutorFactory - Extension executors providers=[]
Oct.-12 10:33:05.583 [main] DEBUG nextflow.Session - Observer factory: DefaultObserverFactory
Oct.-12 10:33:05.605 [main] DEBUG nextflow.cache.CacheFactory - Using Nextflow cache factory: nextflow.cache.DefaultCacheFactory
Oct.-12 10:33:05.613 [main] DEBUG nextflow.util.CustomThreadPool - Creating default thread pool > poolSize: 17; maxThreads: 1000
Oct.-12 10:33:05.700 [main] DEBUG nextflow.Session - Session start invoked
Oct.-12 10:33:05.706 [main] DEBUG nextflow.trace.TraceFileObserver - Flow starting -- trace file: /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble/pipeline_trace.txt
Oct.-12 10:33:05.921 [main] DEBUG nextflow.script.ScriptRunner - > Launching execution
Oct.-12 10:33:05.969 [PathVisitor-1] DEBUG nextflow.file.PathVisitor - files for syntax: glob; folder: /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble/; pattern: *.fastq; options: [:]
Oct.-12 10:33:05.998 [PathVisitor-1] DEBUG nextflow.file.PathVisitor - files for syntax: glob; folder: /home/shaun/nextflow_pipelines/pipelines/pomoxis_map_assemble/ref/; pattern: *.fasta; options: [:]
Oct.-12 10:33:06.048 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: null
Oct.-12 10:33:06.048 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'local'
Oct.-12 10:33:06.052 [main] DEBUG nextflow.executor.Executor - [warm up] executor > local
Oct.-12 10:33:06.056 [main] DEBUG n.processor.LocalPollingMonitor - Creating local task monitor for executor 'local' > cpus=16; memory=62.5 GB; capacity=16; pollInterval=100ms; dumpInterval=5m
Oct.-12 10:33:06.121 [main] DEBUG nextflow.Session - Workflow process names [dsl1]: pomoxis
Oct.-12 10:33:06.133 [main] WARN  nextflow.Session - There's no process matching config selector: fastqc
Oct.-12 10:33:06.134 [main] WARN  nextflow.Session - There's no process matching config selector: porechop
Oct.-12 10:33:06.135 [main] WARN  nextflow.Session - There's no process matching config selector: bioawk
Oct.-12 10:33:06.135 [main] WARN  nextflow.Session - There's no process matching config selector: fastqconvert
Oct.-12 10:33:06.135 [main] WARN  nextflow.Session - There's no process matching config selector: blast_raw
Oct.-12 10:33:06.135 [main] DEBUG nextflow.script.ScriptRunner - > Await termination 
Oct.-12 10:33:06.135 [main] DEBUG nextflow.Session - Session await
Oct.-12 10:33:06.344 [Task submitter] DEBUG nextflow.executor.LocalTaskHandler - Launch cmd line: /bin/bash -ue .command.run
Oct.-12 10:33:06.349 [Task submitter] INFO  nextflow.Session - [cf/bc2b69] Submitted process > pomoxis (1)
Oct.-12 10:33:06.457 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 1; name: pomoxis (1); status: COMPLETED; exit: 127; error: -; workDir: /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble/work/cf/bc2b696aea0863d76c2c9221315c39]
Oct.-12 10:33:06.491 [Task monitor] ERROR nextflow.processor.TaskProcessor - Error executing process > 'pomoxis (1)'

Caused by:
  Process `pomoxis (1)` terminated with an error exit status (127)

Command executed:

  mini_align -i output.fastq -r /home/shaun/nextflow_pipelines/pipelines/pomoxis_map_assemble/ref/*.fasta -o results -p > output_test_final.fa

Command exit status:
  127

Command output:
  (empty)

Command error:
  .command.sh: line 2: mini_align: command not found

Work dir:
  /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble/work/cf/bc2b696aea0863d76c2c9221315c39

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`
Oct.-12 10:33:06.499 [Task monitor] DEBUG nextflow.Session - Session aborted -- Cause: Process `pomoxis (1)` terminated with an error exit status (127)
Oct.-12 10:33:06.503 [main] DEBUG nextflow.Session - Session await > all process finished
Oct.-12 10:33:06.520 [main] DEBUG nextflow.Session - Session await > all barriers passed
Oct.-12 10:33:06.540 [main] DEBUG nextflow.trace.WorkflowStatsObserver - Workflow completed > WorkflowStats[succeededCount=0; failedCount=1; ignoredCount=0; cachedCount=0; pendingCount=0; submittedCount=0; runningCount=0; retriesCount=0; abortedCount=0; succeedDuration=0ms; failedDuration=1.2s; cachedDuration=0ms;loadCpus=0; loadMemory=0; peakRunning=1; peakCpus=16; peakMemory=60 GB; ]
Oct.-12 10:33:06.540 [main] DEBUG nextflow.trace.TraceFileObserver - Flow completing -- flushing trace file
Oct.-12 10:33:06.715 [main] DEBUG nextflow.cache.CacheDB - Closing CacheDB done
Oct.-12 10:33:06.728 [main] DEBUG nextflow.script.ScriptRunner - > Execution complete -- Goodbye

当我试图使用柚子来组装读的时候，我也犯了同样的错误：

shaun@shaun-HP-Z6-G4-Workstation:~/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble$ nextflow pomoxis_denovo_nextflow.nf
N E X T F L O W  ~  version 22.04.5
Launching `pomoxis_denovo_nextflow.nf` [intergalactic_sammet] DSL1 - revision: 1927bae9ab
executor >  local (1)
[fb/3868bc] process > pomoxis (1) [100%] 1 of 1, failed: 1 ✘
WARN: There's no process matching config selector: fastqc
WARN: There's no process matching config selector: porechop
WARN: There's no process matching config selector: bioawk
WARN: There's no process matching config selector: fastqconvert
WARN: There's no process matching config selector: blast_raw
Error executing process > 'pomoxis (1)'

Caused by:
  Process `pomoxis (1)` terminated with an error exit status (127)

Command executed:

  mini_assemble -i output.fastq -o results -p > output_test_final.fa

Command exit status:
  127

Command output:
  (empty)

Command error:
  .command.sh: line 2: mini_assemble: command not found

Work dir:
  /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_map_assemble/work/fb/3868bc97cabfa0d2934f4344c13c89

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`

这是.command.sh的输出

#!/bin/bash -ue
mini_assemble -i output.fastq -o results -p > output_test_final.fa

我用旗子做了这里的柚子--> https://nanoporetech.github.io/pomoxis/programs.html

下面是两个.nf文件：

#!/usr/bin/env nextflow

//data_location

params.outdir = './results'
params.in = "$PWD/*.fastq"
datasetA = Channel.fromPath(params.in)
params.ref = "$PWD/ref/*.fasta"
datasetB = Channel.fromPath(params.ref)


//map and assemble

process pomoxis {

    publishDir "${params.outdir}", mode:'copy'

    input:
    path (c) from datasetA

    output:
    path "${c.baseName}_test_final.fa" into mapped_ch
  
    script:
    """
    mini_align -i $c -r $params.ref -o results -p > ${c.baseName}_test_final.fa 
    """
}

#!/usr/bin/env nextflow

//data_location

params.outdir = './results'
params.in = "$PWD/*.fastq"
dataset = Channel.fromPath(params.in)


//devno only

process pomoxis {
    
    publishDir "${params.outdir}", mode:'copy'
        
    input:
    path (c) from dataset

    output:
    path "${c.baseName}_test_final.fa" into mapped_ch
  
    script:
    """
    mini_assemble -i $c -o results -p > ${c.baseName}_test_final.fa 
    """
}

下面是配置文件，我尝试过来自码头集线器的多个对接容器，但是我得到了相同的错误消息。

//resume = true

process {
            cpus = 16
            accelerator = 'Quadro-RTX-5000'
              memory = 60. GB
}

trace {
        enabled = true
        file = 'pipeline_trace.txt'
        fields = 'task_id,hash,process,name,status,exit,module,container,cpus,time,disk,memory,attempt,submit,start,complete,duration,realtime,queue,%cpu,%mem'
}

docker {
            enabled = true
            temp = 'auto'
            runOption = '--user root' 
}

params {
            nt_db_20221011 = '/home/shaun/blast/nt_db_20221011/nt'
}

process {
            withName:fastqc      {container = 'staphb/fastqc:latest'                                               }
            withName:porechop    {container = 'quay.io/biocontainers/porechop:0.2.3_seqan2.1.1--py36h2d50403_3'    }
            withName:bioawk      {container = 'wslhbio/bioawk:1.0-wslh-signed'                                     }
            withName:fastqconvert{container = 'staphb/seqtk:1.3'                                                   }
            withName:blast_raw   {container = 'staphb/blast:2.13.0'                                                }
            withname:pomoxis    {container = 'dpirdmk/pomoxis:0.1.11'                          }
}

更新：

在纠正语法错误后，我按照建议更改了容器，并将-h函数添加到脚本部分。以下是由此产生的输出：

shaun@shaun-HP-Z6-G4-Workstation:~/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_align$ nextflow pomoxis_align_nextflow.nf
N E X T F L O W  ~  version 22.04.5
Launching `pomoxis_align_nextflow.nf` [voluminous_solvay] DSL1 - revision: 4dca1d1d46
executor >  local (1)
[55/4dad0b] process > pomoxis (1) [  0%] 0 of 1
WARN: There's no process matching config selector: fastqc
WARN: There's no process matching config selector: porechop
WARN: There's no process matching config selector: bioawk
WARN: There's no process matching config selector: fastqconvert
WARN: There's no process matching config selector: blast_raw
Error executing process > 'pomoxis (1)'

Caused by:
  Missing output file(s) `output` expected by process `pomoxis (1)`

Command executed:

  mini_align -h

Command exit status:
  0

Command output:
  (empty)
executor >  local (1)
[55/4dad0b] process > pomoxis (1) [100%] 1 of 1, failed: 1 ✘
WARN: There's no process matching config selector: fastqc
WARN: There's no process matching config selector: porechop
WARN: There's no process matching config selector: bioawk
WARN: There's no process matching config selector: fastqconvert
WARN: There's no process matching config selector: blast_raw
Error executing process > 'pomoxis (1)'

Caused by:
  Missing output file(s) `output` expected by process `pomoxis (1)`

Command executed:

  mini_align -h

Command exit status:
  0

Command output:
  (empty)

Command error:
  mini_align [-h] -r <reference> -i <fastq>

  Align fastq/a formatted reads to a genome using minimap2.

      -h  show this help text.
      -r  reference, should be a fasta file. If correspondng minimap indices
          do not exist they will be created. (required).
      -i  fastq/a input reads (required).
      -I  split index every ~NUM input bases (default: 16G, this is larger
          than the usual minimap2 default).
      -d  set the minimap2 preset, e.g. map-ont, asm5, asm10, asm20 [default: map-ont]
      -f  force recreation of index file.
      -a  aggressively extend gaps (sets -A1 -B2 -O2 -E1 for minimap2).
      -P  filter to only primary alignments (i.e. run samtools view -F 2308).
          Deprecated: this filter is now default and can be disabled with -A.
      -y  filter to primary and supplementary alignments (i.e. run samtools view -F 260)
      -A  do not filter alignments, output all.
      -n  sort bam by read name.
      -c  chunk size. Input reads/contigs will be broken into chunks
          prior to alignment.
      -t  alignment threads (default: 1).
      -p  output file prefix (default: reads).
      -m  fill MD tag.
      -s  fill cs(=long) tag.
      -X  only create reference index files.
      -x  log all commands before running.
      -M  match score
      -S  mismatch score
      -O  open gap penalty
      -E  extend gap penalty.

Work dir:
  /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_align/work/55/4dad0b1df7105e31362f58b0c67f1f

Tip: view the complete command output by changing to the process work dir and entering the command `cat .command.out`

虽然有一个错误，我假设这是可行的，因为我可以看到帮助菜单？

然后，我按照Pomoxis网站向.nf的脚本部分添加了标志。

#!/usr/bin/env nextflow

//data_location

params.outdir = './results'
params.in = "$PWD/*.fastq"
datasetA = Channel.fromPath(params.in)
params.ref = "$PWD/SLCMV.fasta"
datasetB = Channel.fromPath(params.ref)


//map and assemble; input fastq only
//output; pomoxis is very particular about names, minimap can out put 
//.sam or .paf 

process pomoxis {

    publishDir "${params.outdir}", mode:'copy'
        
    input:
    path (c) from datasetA
    path (d) from datasetB

    output:
    path "${c.simpleName}" into mapped_ch
  
    script:
    """
    mini_align -r $d -i $c -p ${c.simpleName}
    """
}

结果如下：

shaun@shaun-HP-Z6-G4-Workstation:~/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_align$ nextflow pomoxis_align_nextflow.nf
N E X T F L O W  ~  version 22.04.5
Launching `pomoxis_align_nextflow.nf` [determined_bassi] DSL1 - revision: 1ad3e75c1b
executor >  local (1)
[50/860f9f] process > pomoxis (1) [  0%] 0 of 1
WARN: There's no process matching config selector: fastqc
WARN: There's no process matching config selector: porechop
WARN: There's no process matching config selector: bioawk
WARN: There's no process matching config selector: fastqconvert
WARN: There's no process matching config selector: blast_raw
Error executing process > 'pomoxis (1)'

Caused by:
  Missing output file(s) `output` expected by process `pomoxis (1)`
executor >  local (1)
[50/860f9f] process > pomoxis (1) [100%] 1 of 1, failed: 1 ✘
WARN: There's no process matching config selector: fastqc
WARN: There's no process matching config selector: porechop
WARN: There's no process matching config selector: bioawk
WARN: There's no process matching config selector: fastqconvert
WARN: There's no process matching config selector: blast_raw
Error executing process > 'pomoxis (1)'

Caused by:
  Missing output file(s) `output` expected by process `pomoxis (1)`

Command executed:

  mini_align -r SLCMV.fasta -i output.fastq -p output

Command exit status:
  0

Command output:
  (empty)

Command error:
  Creating fai index file SLCMV.fasta.fai
  Creating mmi index file SLCMV.fasta.map-ont.mmi
  [M::mm_idx_gen::0.003*2.10] collected minimizers
  [M::mm_idx_gen::0.005*2.42] sorted minimizers
  [M::main::0.009*1.77] loaded/built the index for 1 target sequence(s)
  [M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 1
  [M::mm_idx_stat::0.009*1.74] distinct minimizers: 522 (100.00% are singletons); average occurrences: 1.000; average spacing: 5.282; total length: 2757
  [M::main] Version: 2.24-r1122
  [M::main] CMD: minimap2 -I 16G -x map-ont -d SLCMV.fasta.map-ont.mmi SLCMV.fasta
  [M::main] Real time: 0.010 sec; CPU: 0.017 sec; Peak RSS: 0.003 GB
  [M::main::0.005*1.39] loaded/built the index for 1 target sequence(s)
  [M::mm_mapopt_update::0.005*1.37] mid_occ = 10
  [M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 1
  [M::mm_idx_stat::0.005*1.35] distinct minimizers: 522 (100.00% are singletons); average occurrences: 1.000; average spacing: 5.282; total length: 2757
  [M::worker_pipeline::9.159*1.00] mapped 87369 sequences
  [M::main] Version: 2.24-r1122
  [M::main] CMD: minimap2 -x map-ont --secondary=no -L -t 1 -a SLCMV.fasta.map-ont.mmi output.fastq
  [M::main] Real time: 9.182 sec; CPU: 9.145 sec; Peak RSS: 0.299 GB

Work dir:
  /home/shaun/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_align/work/50/860f9ffe37f7a17a4d5e216f67b512

Tip: when you have fixed the problem you can continue the execution adding the option `-resume` to the run command line

我不知道如何解决这个问题，我尝试过更改输出名，但这并没有帮助。

有趣的是，当我运行mini_assemble .nf时：

#!/usr/bin/env nextflow

//data_location

params.outdir = './results'
params.in = "$PWD/*.fastq"
dataset = Channel.fromPath(params.in)

//devno only; input is fastq
//output; pomoxis is very particular about names, the name that it out puts is
//prefix_test_final.fa where prefic can be anything and it will add _test_final.fa
//to the end of the file


process pomoxis {

    tag "$c"
    publishDir "${params.outdir}", mode:'copy'
        
    input:
    path (c) from dataset

    output:
    path ("${c.simpleName}_test_final.fa") into mapped_ch
  
    script:
    """
    mini_assemble -i $c -p ${c.simpleName}
    """ 
}

我得到以下错误：

shaun@shaun-HP-Z6-G4-Workstation:~/nextflow_pipelines/pipelines/WA_MomicaKehoe/Asad/nf_pipeline/pomoxis_assemble$ nextflow pomoxis_assemble_nextflow.nf
N E X T F L O W  ~  version 22.04.5
Launching `pomoxis_assemble_nextflow.nf` [intergalactic_fermi] DSL1 - revision: ce7a466307
executor >  local (1)
[04/28f5e6] process > pomoxis (output.fastq) [  0%] 0 of 1


Error executing process > 'pomoxis (output.fastq)'

Caused by:
  Missing output file(s) `output_test_final.fa` expected by process `pomoxis (output.fastq)`

Command executed:

  mini_assemble -i output.fastq -p output

Command exit status:
  0

Command output:
  Copying FASTX input to workspace: output.fastq > assm/output.fa.gz
  Skipped adapter trimming.
  Skipped pre-assembly correction.
  Overlapping reads...
  Assembling graph...
  Running racon read shuffle 1...
  Running round 1 consensus...
  Running round 2 consensus...
  Running round 3 consensus...
  Running round 4 consensus...
  Waiting for cleanup.
  Final assembly written to assm/output_final.fa. Have a nice day.

Command error:
  [M::mm_idx_stat::0.065*1.04] distinct minimizers: 69519 (92.83% are singletons); average occurrences: 1.101; average spacing: 5.337; total length: 408518
  [M::worker_pipeline::9.872*0.97] mapped 87369 sequences
  [M::main] Version: 2.24-r1122
  [M::main] CMD: minimap2 -L -K 500M -t 1 racon_1_3.fa.gz output.fa.gz
  [M::main] Real time: 9.880 sec; CPU: 9.620 sec; Peak RSS: 0.165 GB
  [racon::Polisher::initialize] loaded target sequences 0.011974 s
  [racon::Polisher::initialize] loaded sequences 2.047803 s
  [racon::Polisher::initialize] loaded overlaps 0.106485 s
  [racon::Polisher::initialize] aligning overlaps [=>                  ] 1.252726 s
  [racon::Polisher::initialize] aligning overlaps [==>                 ] 1.827141 s
  [racon::Polisher::initialize] aligning overlaps [===>                ] 2.413558 s
  [racon::Polisher::initialize] aligning overlaps [====>               ] 2.974846 s
  [racon::Polisher::initialize] aligning overlaps [=====>              ] 3.609222 s
  [racon::Polisher::initialize] aligning overlaps [======>             ] 4.263570 s
  [racon::Polisher::initialize] aligning overlaps [=======>            ] 4.934294 s
  [racon::Polisher::initialize] aligning overlaps [========>           ] 5.554714 s
  [racon::Polisher::initialize] aligning overlaps [=========>          ] 6.164404 s
  [racon::Polisher::initialize] aligning overlaps [==========>         ] 6.746845 s
executor >  local (1)
[04/28f5e6] process > pomoxis (output.fastq) [100%] 1 of 1, failed: 1 ✘
Error executing process > 'pomoxis (output.fastq)'

Caused by:
  Missing output file(s) `output_test_final.fa` expected by process `pomoxis (output.fastq)`

Command executed:

  mini_assemble -i output.fastq -p output

Command exit status:
  0

Command output:
  Copying FASTX input to workspace: output.fastq > assm/output.fa.gz
  Skipped adapter trimming.
  Skipped pre-assembly correction.
  Overlapping reads...
  Assembling graph...
  Running racon read shuffle 1...
  Running round 1 consensus...
  Running round 2 consensus...
  Running round 3 consensus...
  Running round 4 consensus...
  Waiting for cleanup.
  Final assembly written to assm/output_final.fa. Have a nice day.

Command error:
  [M::mm_idx_stat::0.065*1.04] distinct minimizers: 69519 (92.83% are singletons); average occurrences: 1.101; average spacing: 5.337; total length: 408518
  [M::worker_pipeline::9.872*0.97] mapped 87369 sequences
  [M::main] Version: 2.24-r1122
  [M::main] CMD: minimap2 -L -K 500M -t 1 racon_1_3.fa.gz output.fa.gz
  [M::main] Real time: 9.880 sec; CPU: 9.620 sec; Peak RSS: 0.165 GB
  [racon::Polisher::initialize] loaded target sequences 0.011974 s
  [racon::Polisher::initialize] loaded sequences 2.047803 s
  [racon::Polisher::initialize] loaded overlaps 0.106485 s
  [racon::Polisher::initialize] aligning overlaps [=>                  ] 1.252726 s
  [racon::Polisher::initialize] aligning overlaps [==>                 ] 1.827141 s
  [racon::Polisher::initialize] aligning overlaps [===>                ] 2.413558 s
  [racon::Polisher::initialize] aligning overlaps [====>               ] 2.974846 s
  [racon::Polisher::initialize] aligning overlaps [=====>              ] 3.609222 s
  [racon::Polisher::initialize] aligning overlaps [======>             ] 4.263570 s
  [racon::Polisher::initialize] aligning overlaps [=======>            ] 4.934294 s
  [racon::Polisher::initialize] aligning overlaps [========>           ] 5.554714 s
  [racon::Polisher::initialize] aligning overlaps [=========>          ] 6.164404 s
  [racon::Polisher::initialize] aligning overlaps [==========>         ] 6.746845 s
  [racon::Polisher::initialize] aligning overlaps [===========>        ] 7.282895 s
  [racon::Polisher::initialize] aligning overlaps [============>       ] 7.878856 s
  [racon::Polisher::initialize] aligning overlaps [=============>      ] 8.459672 s
  [racon::Polisher::initialize] aligning overlaps [==============>     ] 8.984196 s
  [racon::Polisher::initialize] aligning overlaps [===============>    ] 9.558089 s
  [racon::Polisher::initialize] aligning overlaps [================>   ] 10.072586 s
  [racon::Polisher::initialize] aligning overlaps [=================>  ] 10.648603 s
  [racon::Polisher::initialize] aligning overlaps [==================> ] 11.220553 s
  [racon::Polisher::initialize] aligning overlaps [===================>] 11.753881 s
  [racon::Polisher::initialize] aligning overlaps [====================] 12.364460 s
  [racon::Polisher::initialize] transformed data into windows 0.018851 s
  [racon::Polisher::polish] generating consensus [=>                  ] 0.515508 s
  [racon::Polisher::polish] generating consensus [==>                 ] 1.077083 s
  [racon::Polisher::polish] generating consensus [===>                ] 1.561330 s
  [racon::Polisher::polish] generating consensus [====>               ] 1.967063 s
  [racon::Polisher::polish] generating consensus [=====>              ] 2.574379 s
  [racon::Polisher::polish] generating consensus [======>             ] 3.037187 s
  [racon::Polisher::polish] generating consensus [=======>            ] 3.740370 s
  [racon::Polisher::polish] generating consensus [========>           ] 20.112573 s
  [racon::Polisher::polish] generating consensus [=========>          ] 46.718507 s
  [racon::Polisher::polish] generating consensus [==========>         ] 48.913841 s
  [racon::Polisher::polish] generating consensus [===========>        ] 52.859126 s
  [racon::Polisher::polish] generating consensus [============>       ] 57.495382 s
  [racon::Polisher::polish] generating consensus [=============>      ] 74.111321 s
  [racon::Polisher::polish] generating consensus [==============>     ] 79.382051 s
  [racon::Polisher::polish] generating consensus [===============>    ] 113.129291 s
  [racon::Polisher::polish] generating consensus [================>   ] 115.971094 s
  [racon::Polisher::polish] generating consensus [=================>  ] 123.322513 s
  [racon::Polisher::polish] generating consensus [==================> ] 128.155934 s
  [racon::Polisher::polish] generating consensus [===================>] 130.509134 s
  [racon::Polisher::polish] generating consensus [====================] 131.095442 s
  [racon::Polisher::] total = 145.794273 s

Work dir:
  /home/shaun/nextflow_pipelines/pipelines/WA_MomicaKehoe/Asad/nf_pipeline/pomoxis_assemble/work/04/28f5e6eed13d3fc986739e287594d9

Tip: when you have fixed the problem you can continue the execution adding the option `-resume` to the run command line

但是，我需要的文件在：

shaun@shaun-HP-Z6-G4-Workstation:~/nextflow_pipelines/pipelines/nf_pipeline/pomoxis_assemble/work/04/28f5e6eed13d3fc986739e287594d9/assm$ cat output_final.fa

当我猫这个文件，我可以看到，在文件中有contig，如预期。

我可以看到，波波西斯期待output_test_final.fa，这确实是在工作中，但波波西斯不能看到它或使用它？

你对这些错误发生的原因有什么建议吗？我很感激你能抽出时间。

Answer 1

命令错误：.command.sh:第2行: mini_assemble:未找到命令

当您尝试在容器外部运行一个命令，并且您的$PATH中不存在可执行文件时，您将得到此错误。通常，当我们忘记向我们的docker.enabled = true添加docker.enabled = true(或者如果使用nextflow.config时添加singularity.enabled = true )，我们就会看到这一点。但是，在本例中，它只是由您的配置文件中的一个错误引起的。withName 配置选择器必须是骆驼箱。使用所选容器时，您还会得到一个“命令未找到”错误。如果我们使用柚木生物容器，就会得到预期的结果。例如：

nextflow.config含量

docker {
    enabled = true
}

process {

    withName: pomoxis {
        container = 'quay.io/biocontainers/pomoxis:0.3.10--pyhdfd78af_0'
    }
}

map_and_assembly.nf含量

process pomoxis {

    """
    mini_align -h
    """
}

结果：

$ nextflow run map_and_assembly.nf -dsl1
N E X T F L O W  ~  version 22.04.4
Launching `map_and_assembly.nf` [loving_thompson] DSL1 - revision: 6efbc53bf8
executor >  local (1)
[22/23c938] process > pomoxis [100%] 1 of 1 ✔

Completed at: 13-Oct-2022 01:05:27
Duration    : 2m 46s
CPU hours   : (a few seconds)
Succeeded   : 1

$ cat work/22/23c938ca9312037ee10087ba5d48a2/.command.err 
Unable to find image 'quay.io/biocontainers/pomoxis:0.3.10--pyhdfd78af_0' locally
0.3.10--pyhdfd78af_0: Pulling from biocontainers/pomoxis
c1a16a04cedd: Already exists
4ca545ee6d5d: Already exists
af0d0c971daf: Pulling fs layer
af0d0c971daf: Verifying Checksum
af0d0c971daf: Download complete
af0d0c971daf: Pull complete
Digest: sha256:b42d95b742be3dc8333f57892c4aa2cc5cd739e796b33c7f310696856dcdea4d
Status: Downloaded newer image for quay.io/biocontainers/pomoxis:0.3.10--pyhdfd78af_0
mini_align [-h] -r <reference> -i <fastq>

Align fastq/a formatted reads to a genome using minimap2.

    -h  show this help text.
    -r  reference, should be a fasta file. If correspondng minimap indices
        do not exist they will be created. (required).
    -i  fastq/a input reads (required).
    -I  split index every ~NUM input bases (default: 16G, this is larger
        than the usual minimap2 default).
    -d  set the minimap2 preset, e.g. map-ont, asm5, asm10, asm20 [default: map-ont]
    -f  force recreation of index file.
    -a  aggressively extend gaps (sets -A1 -B2 -O2 -E1 for minimap2).
    -P  filter to only primary alignments (i.e. run samtools view -F 2308).
        Deprecated: this filter is now default and can be disabled with -A.
    -y  filter to primary and supplementary alignments (i.e. run samtools view -F 260)
    -A  do not filter alignments, output all.
    -n  sort bam by read name.
    -c  chunk size. Input reads/contigs will be broken into chunks
        prior to alignment.
    -t  alignment threads (default: 1).
    -p  output file prefix (default: reads).
    -m  fill MD tag.
    -s  fill cs(=long) tag.
    -X  only create reference index files.
    -x  log all commands before running.
    -M  match score 
    -S  mismatch score
    -O  open gap penalty
    -E  extend gap penalty.

使用dpirdmk/pomoxis:0.1.11容器的问题是，它指定了一个备用入口点，这使得在不修改要运行的命令或提供一些额外的Docker配置的情况下，很难开箱即用：

$ docker run --rm dpirdmk/pomoxis:0.1.11 bash -c 'cat /init.sh'
#!/bin/bash
. /apps/pomoxis/venv/bin/activate
exec "$@"

回答后续问题：

当命令成功完成(即退出状态为零)时，您将收到Missing output file(s) `output` expected by process错误，但是Nextflow (而不是pomoxis)无法找到工作目录中输出声明中指定的一个或多个输出文件。我想你想要的是以下。我冒昧地重命名了一些变量，以使事情更清楚一些。下面的代码当然是未经测试的：

params.outdir = './results'

params.ref_fasta = "SLCMV.fasta"
params.reads = "*.fastq"

reads = Channel.fromPath( params.reads )
ref_fasta = file( params.ref_fasta )


process mini_align {

    tag { fastq.name }

    publishDir "${params.outdir}/mini_align", mode:'copy'

    input:
    path fastq from reads
    path ref_fasta

    output:
    path "${fastq.simpleName}.bam{,.bai}" into mapped_ch

    script:
    """
    mini_align \\
        -r "${ref_fasta}" \\
        -i "${fastq}" \\
        -p "${fastq.simpleName}"
    """
}

params.outdir = './results'

params.reads = "*.fastq"

reads = Channel.fromPath( params.reads )


process mini_assemble {

    tag { fastq.name }

    publishDir "${params.outdir}/mini_assemble", mode:'copy'

    input:
    path fastq from reads

    output:
    path "assm/${fastq.simpleName}_final.fa" into mapped_ch

    script:
    """
    mini_assemble \\
        -i "${fastq}" \\
        -p "${fastq.simpleName}"
    """
}