Answers.Body: <p>When we run Bowtie2, we just need to make sure we supply the index basename using <code>-x</code>. This is the index filename prefix minus the <code>.X.bt2</code> extension. A safe assumption is that the first file in the collection of output files (sorted in lexicographical order) has the <code>.1.bt2</code> suffix. We can then just strip this off to get the required prefix. For example:</p>
  <pre><code>process SAM_FOR_STRAND {
  
      input:
      path index_files, stageAs: &quot;bt2_index/*&quot;
      tuple val(pair_id), path(reads)
  
      output:
      path &quot;${pair_id}.sam&quot;
  
      script:
      def idx = index_files[0].getBaseName(2)    
  
      &quot;&quot;&quot;
      bowtie2 \\
          -x &quot;<span class="math-container">${idx}" \\
          -1 "$</span>{reads[0]}&quot; \\ 
          -2 &quot;<span class="math-container">${reads[1]}" \\ 
          -S "$</span>{pair_id}.sam&quot;
      &quot;&quot;&quot;
  }
  </code></pre>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2022-12-23T14:28:26.750Z
Answers.LastActivityDate: 2022-12-23T14:28:26.750Z
Answers.UserDisplayName: Steve
Body: <p>I'm using bowtie2 to build a small index. After building the index, I want to pass it to the bowtie2 alignment process. The issue is bowtie2-build outputs multiple index files with the .bt2 extension. When I try to provide these index files as an input to the Alignmnet process, it gives the following error:</p>
  <blockquote>
  <p>Error executing process &gt; 'SAM_FOR_STRAND (1)'</p>
  <p>Caused by:   Process <code>SAM_FOR_STRAND (1)</code> terminated with an error
  exit status (255)</p>
  <p>Command executed:</p>
  <p>bowtie2 -t -x index.1.bt2 index.2.bt2 index.3.bt2 index.4.bt2
  index.rev.1.bt2 index.rev.2.bt2 -1 SRR5655560_1.atria.fastq -2
  SRR5655560_2.atria.fastq -S &quot;SRR5655560.sam&quot;</p>
  <p>Command exit status:   255</p>
  <p>Command output:   (empty)</p>
  <p>Command error:   (ERR): &quot;index.1.bt2&quot; does not exist or is not a
  Bowtie 2 index   Exiting now ...</p>
  <p>executor &gt;  local (4) [71/c095e4] process &gt; INDEX              [100%]
  1 of 1 ✔ [1f/35a1e6] process &gt; GTF2BED            [100%] 1 of 1 ✔
  [fa/b1e933] process &gt; TRIM (SRR5655560)  [100%] 1 of 1 ✔ [d6/d47ead]
  process &gt; SAM_FOR_STRAND (1) [100%] 1 of 1, failed: 1 ✘ Error
  executing process &gt; 'SAM_FOR_STRAND (1)'</p>
  <p>Caused by:   Process <code>SAM_FOR_STRAND (1)</code> terminated with an error
  exit status (255)</p>
  <p>Command executed:</p>
  <p>bowtie2 -t -x index.1.bt2 index.2.bt2 index.3.bt2 index.4.bt2
  index.rev.1.bt2 index.rev.2.bt2 -1 SRR5655560_1.atria.fastq -2
  SRR5655560_2.atria.fastq -S &quot;SRR5655560.sam&quot;</p>
  </blockquote>
  <p>The following is simplifies version of the pipeline:</p>
  <pre><code>// Declare syntax version
  nextflow.enable.dsl=2
  
  /* 
   * pipeline input parameters 
   */
  
  
  params.reads = &quot;<span class="math-container">$projectDir/data/*_{1,2}.fastq"
  params.ref_genome = "$</span>projectDir/data/Glycine_max.Glycine_max_v2.1.dna.toplevel.fa&quot;
  params.gtf_file = &quot;$projectDir/data/Glycine_max.Glycine_max_v2.1.55.gtf&quot;
  params.outdir = &quot;results&quot;
  
  
  
  log.info &quot;&quot;&quot;\
  
          R N A S E Q - N F   P I P E L I N E    
          ===================================
          reads            : <span class="math-container">${params.reads}
          reference genome : $</span>{params.ref_genome}
          gtf              : <span class="math-container">${params.gtf_file}
          outdir           : $</span>{params.outdir}
          &quot;&quot;&quot;
          .stripIndent()
  
  /*
   * define the `index` process that creates a binary index
   * given the reference genome file
   */
  
  process INDEX {
  
    input:
    path ref_genome
  
    output:
    path &quot;index.*&quot;
  
    script:
    &quot;&quot;&quot;
    bowtie2-build $ref_genome index
    &quot;&quot;&quot;
  }
  
  /*
   * define the `GTF2BED` process that creates a BED file
   * given the params.gtf_file as input. 
   */
   
  process GTF2BED {
  
      input:
      path gtf
  
      output:
      path &quot;${gtf.baseName}.bed&quot;
  
      script:
      &quot;&quot;&quot;
      gtf2bed --gtf &quot;<span class="math-container">${gtf}" --bed "$</span>{gtf.baseName}.bed&quot;
      &quot;&quot;&quot;
  }
  
  
  /*
   * define the `TRIM` process that creates trimmed fastq files
   * given the reads fastq file. 
   */
   
  process TRIM {
  
      tag { pair_id }
  
      input:
      tuple val(pair_id), path(reads)
  
      output:
      tuple val(pair_id), path(trimmed_reads), emit: trimmed_fastqs
      path &quot;${pair_id}/*.atria.log*&quot;, emit: logs
  
      script:
      def (r1, r2) = reads
  
      trimmed_reads = reads.collect {
          &quot;<span class="math-container">${pair_id}/$</span>{it.baseName}.atria.fastq&quot;
      }
  
      &quot;&quot;&quot;
      atria \\
          -r &quot;<span class="math-container">${r1}" \\
          -R "$</span>{r2}&quot; \\
          -o &quot;${pair_id}&quot; \\
          -t 1 \\
          -l 12 \\
          -q 30
      &quot;&quot;&quot;
  }
  
  /*
   * define the `SAM_FOR_STRAND` process that creates a SAM file
   * to be used by `STRAND_CHECK` process to determine 
   * the strandedness of each fastq file pair. 
   */
   
  process SAM_FOR_STRAND {
  
      input:
      path index
      tuple val(pair_id), path(reads)
  
      output:
      path &quot;${pair_id}.sam&quot;
  
      script:
      &quot;&quot;&quot;
      bowtie2 -x &quot;<span class="math-container">${index.baseName}" -1 $</span>{reads[0]} -2 <span class="math-container">${reads[1]} -S $</span>{pair_id}.sam
      &quot;&quot;&quot;
  }
  
  
  workflow {
  
      index_ch = INDEX(params.ref_genome)
  
      gtf2bed_ch=GTF2BED(params.gtf_file)
  
      read_pairs_ch = Channel.fromFilePairs(params.reads, checkIfExists:true)
  
      trim_out = TRIM(read_pairs_ch)
  
      sam_for_strand_ch = SAM_FOR_STRAND(index_ch, TRIM.out.trimmed_fastqs)
  
  }
  </code></pre>
  <p>I've tried passing the <em>index</em> argument directly to the script in the SAM_FOR_STRAND Alignmnet process, it gave the  &quot;index.1.bt2&quot; does not exist error. Then I tried giving the basename of the index files (as shown in the below). It still does not work, even though the index files are being created properly.</p>
  <pre><code> /*
       * define the `SAM_FOR_STRAND` process that creates a SAM file
       * to be used by `STRAND_CHECK` process to determine 
       * the strandedness of each fastq file pair. 
       */
       
      process SAM_FOR_STRAND {
      
          input:
          path index
          tuple val(pair_id), path(reads)
      
          output:
          path &quot;${pair_id}.sam&quot;
      
          script:
          &quot;&quot;&quot;
          bowtie2 -x &quot;<span class="math-container">${index.baseName}" -1 $</span>{reads[0]} -2 <span class="math-container">${reads[1]} -S $</span>{pair_id}.sam
          &quot;&quot;&quot;
      }
  </code></pre>
  <p>Best wishes.</p>
  
Tags: <nextflow>
Title: Alignment process in Nextflow not picking up the index files from the index process
ThreadText: # Alignment process in Nextflow not picking up the index files from the index process
  
  (Asked by: username_0 on 2022-12-23)
  
  I'm using bowtie2 to build a small index. After building the index, I want to pass it to the bowtie2 alignment process. The issue is bowtie2-build outputs multiple index files with the .bt2 extension. When I try to provide these index files as an input to the Alignmnet process, it gives the following error:
  
  > Error executing process > 'SAM\_FOR\_STRAND (1)'
  >
  > Caused by: Process `SAM_FOR_STRAND (1)` terminated with an error
  > exit status (255)
  >
  > Command executed:
  >
  > bowtie2 -t -x index.1.bt2 index.2.bt2 index.3.bt2 index.4.bt2
  > index.rev.1.bt2 index.rev.2.bt2 -1 SRR5655560\_1.atria.fastq -2
  > SRR5655560\_2.atria.fastq -S "SRR5655560.sam"
  >
  > Command exit status: 255
  >
  > Command output: (empty)
  >
  > Command error: (ERR): "index.1.bt2" does not exist or is not a
  > Bowtie 2 index Exiting now ...
  >
  > executor > local (4) [71/c095e4] process > INDEX [100%]
  > 1 of 1 ✔ [1f/35a1e6] process > GTF2BED [100%] 1 of 1 ✔
  > [fa/b1e933] process > TRIM (SRR5655560) [100%] 1 of 1 ✔ [d6/d47ead]
  > process > SAM\_FOR\_STRAND (1) [100%] 1 of 1, failed: 1 ✘ Error
  > executing process > 'SAM\_FOR\_STRAND (1)'
  >
  > Caused by: Process `SAM_FOR_STRAND (1)` terminated with an error
  > exit status (255)
  >
  > Command executed:
  >
  > bowtie2 -t -x index.1.bt2 index.2.bt2 index.3.bt2 index.4.bt2
  > index.rev.1.bt2 index.rev.2.bt2 -1 SRR5655560\_1.atria.fastq -2
  > SRR5655560\_2.atria.fastq -S "SRR5655560.sam"
  
  The following is simplifies version of the pipeline:
  
  ```
  // Declare syntax version
  nextflow.enable.dsl=2
  
  /* 
   * pipeline input parameters 
   */
  
  params.reads = "$projectDir/data/*_{1,2}.fastq"
  params.ref_genome = "$projectDir/data/Glycine_max.Glycine_max_v2.1.dna.toplevel.fa"
  params.gtf_file = "$projectDir/data/Glycine_max.Glycine_max_v2.1.55.gtf"
  params.outdir = "results"
  
  log.info """\
  
          R N A S E Q - N F   P I P E L I N E    
          ===================================
          reads            : ${params.reads}
          reference genome : ${params.ref_genome}
          gtf              : ${params.gtf_file}
          outdir           : ${params.outdir}
          """
          .stripIndent()
  
  /*
   * define the `index` process that creates a binary index
   * given the reference genome file
   */
  
  process INDEX {
  
    input:
    path ref_genome
  
    output:
    path "index.*"
  
    script:
    """
    bowtie2-build $ref_genome index
    """
  }
  
  /*
   * define the `GTF2BED` process that creates a BED file
   * given the params.gtf_file as input. 
   */
   
  process GTF2BED {
  
      input:
      path gtf
  
      output:
      path "${gtf.baseName}.bed"
  
      script:
      """
      gtf2bed --gtf "${gtf}" --bed "${gtf.baseName}.bed"
      """
  }
  
  /*
   * define the `TRIM` process that creates trimmed fastq files
   * given the reads fastq file. 
   */
   
  process TRIM {
  
      tag { pair_id }
  
      input:
      tuple val(pair_id), path(reads)
  
      output:
      tuple val(pair_id), path(trimmed_reads), emit: trimmed_fastqs
      path "${pair_id}/*.atria.log*", emit: logs
  
      script:
      def (r1, r2) = reads
  
      trimmed_reads = reads.collect {
          "${pair_id}/${it.baseName}.atria.fastq"
      }
  
      """
      atria \\
          -r "${r1}" \\
          -R "${r2}" \\
          -o "${pair_id}" \\
          -t 1 \\
          -l 12 \\
          -q 30
      """
  }
  
  /*
   * define the `SAM_FOR_STRAND` process that creates a SAM file
   * to be used by `STRAND_CHECK` process to determine 
   * the strandedness of each fastq file pair. 
   */
   
  process SAM_FOR_STRAND {
  
      input:
      path index
      tuple val(pair_id), path(reads)
  
      output:
      path "${pair_id}.sam"
  
      script:
      """
      bowtie2 -x "${index.baseName}" -1 ${reads[0]} -2 ${reads[1]} -S ${pair_id}.sam
      """
  }
  
  workflow {
  
      index_ch = INDEX(params.ref_genome)
  
      gtf2bed_ch=GTF2BED(params.gtf_file)
  
      read_pairs_ch = Channel.fromFilePairs(params.reads, checkIfExists:true)
  
      trim_out = TRIM(read_pairs_ch)
  
      sam_for_strand_ch = SAM_FOR_STRAND(index_ch, TRIM.out.trimmed_fastqs)
  
  }
  
  ```
  
  I've tried passing the *index* argument directly to the script in the SAM\_FOR\_STRAND Alignmnet process, it gave the "index.1.bt2" does not exist error. Then I tried giving the basename of the index files (as shown in the below). It still does not work, even though the index files are being created properly.
  
  ```
   /*
       * define the `SAM_FOR_STRAND` process that creates a SAM file
       * to be used by `STRAND_CHECK` process to determine 
       * the strandedness of each fastq file pair. 
       */
       
      process SAM_FOR_STRAND {
      
          input:
          path index
          tuple val(pair_id), path(reads)
      
          output:
          path "${pair_id}.sam"
      
          script:
          """
          bowtie2 -x "${index.baseName}" -1 ${reads[0]} -2 ${reads[1]} -S ${pair_id}.sam
          """
      }
  
  ```
  
  Best wishes.
  
  **Tags:** nextflow
  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2022-12-23. Score: 1 | Accepted answer
  
  When we run Bowtie2, we just need to make sure we supply the index basename using `-x`. This is the index filename prefix minus the `.X.bt2` extension. A safe assumption is that the first file in the collection of output files (sorted in lexicographical order) has the `.1.bt2` suffix. We can then just strip this off to get the required prefix. For example:
  
  ```
  process SAM_FOR_STRAND {
  
      input:
      path index_files, stageAs: "bt2_index/*"
      tuple val(pair_id), path(reads)
  
      output:
      path "${pair_id}.sam"
  
      script:
      def idx = index_files[0].getBaseName(2)    
  
      """
      bowtie2 \\
          -x "${idx}" \\
          -1 "${reads[0]}" \\ 
          -2 "${reads[1]}" \\ 
          -S "${pair_id}.sam"
      """
  }
  
  ```
  
  
  ---
  
  