Answers.Body: <p>You should have also received the following Nextflow warning:</p>
  <pre><code>WARN: Input tuple does not match tuple declaration in process `samtools_index`
  </code></pre>
  <p>Specifically, the problem is that the output of your <em>convert_sam_to_bam</em> process is:</p>
  <pre><code>tuple val(replicateId), path(&quot;${replicateId}.Aligned.bam&quot;), path(&quot;${replicateId}.Aligned.sorted.bam&quot;)
  </code></pre>
  <p>But the <em>samtools_index</em> process is expecting:</p>
  <pre><code>tuple val(replicateId), path(&quot;${replicateId}.Aligned.sorted.bam&quot;)
  </code></pre>
  <hr />
  <p>If you would like to declare multiple output channels, you can use the <code>emit</code> keyword (this is documented under <a href="https://www.nextflow.io/docs/latest/process.html#additional-options" rel="nofollow noreferrer">additional options</a>) to define names for the output channel(s). For example:</p>
  <pre><code>process convert_sam_to_bam {
  
      input:
      tuple val(replicateId), path(&quot;${replicateId}.Aligned.sam&quot;)
  
      output:
      tuple val(replicateId), path(&quot;<span class="math-container">${replicateId}.Aligned.bam"), emit: aligned_bam
      tuple val(replicateId), path("$</span>{replicateId}.Aligned.sorted.bam&quot;), emit: sorted_bam
  
      script:
      &quot;&quot;&quot;
      samtools view \\
          -b \\
          -o &quot;<span class="math-container">${replicateId}.Aligned.bam" \\
          "$</span>{replicateId}.Aligned.sam&quot;
  
      samtools sort \\
          -o &quot;<span class="math-container">${replicateId}.Aligned.sorted.bam" \\
          "$</span>{replicateId}.Aligned.bam&quot;
      &quot;&quot;&quot;
  }
  </code></pre>
  <pre><code>workflow {
  
      ...
  
      samtools_index( convert_sam_to_bam.out.sorted_bam )
  }
  </code></pre>
  <hr />
  <p>Otherwise, either change the <em>convert_sam_to_bam</em> output declaration to:</p>
  <pre><code>tuple val(replicateId), path(&quot;${replicateId}.Aligned.sorted.bam&quot;)
  </code></pre>
  <p>Or use the <a href="https://www.nextflow.io/docs/latest/operator.html#map" rel="nofollow noreferrer">map</a> operator to get what you want, for example:</p>
  <pre><code>workflow {
  
      ...
  
      samtools_index(
          convert_sam_to_bam.out.map { rep, aligned, sorted -&gt; tuple( rep, sorted ) }
      )
  }
  </code></pre>
  <hr />
  <p>Note that a separate indexing process can be avoided using newer versions of <a href="https://www.htslib.org/doc/samtools.html" rel="nofollow noreferrer">samtools</a> with the <code>--write-index</code> option:</p>
  <blockquote>
  <p>To convert a SAM to a compressed BAM using BAI indexing: <code>samtools view --write-index in.sam -o out.bam##idx##out.bam.bai</code></p>
  </blockquote>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2024-09-17T20:38:01.537Z
Answers.Text: Thanks a lot! This was helpful.
Answers.UserDisplayName: siddhartha das
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2024-09-15T15:00:55.843Z
Answers.LastActivityDate: 2024-09-15T15:00:55.843Z
Answers.UserDisplayName: Steve
Body: <p>I am a beginner to Nextflow  and using it to setup Chipseq workflow and in the workflow below the samtools_index process throws the following error:</p>
  <pre><code>[E::hts_idx_push] Chromosome blocks not continuous
    [E::sam_index] Read 'FCC1L20ACXX:4:1101:1645:2230#0/1' with ref_name='chr17', ref_length=83257441, flags=16, pos=58851543 cannot be indexed
    samtools index: failed to create index for &quot;TCDD_plus_E2_ERa_Rep_1_ChIPSeq_first_1000.Aligned.sorted.bam&quot;: Inappropriate ioctl for device
  </code></pre>
  <p>The command executed is:</p>
  <pre><code>/Users/siddhaduio.no/Desktop/All_omics_tools/samtools-1.14/samtools index TCDD_plus_E2_ERa_Rep_1_ChIPSeq_first_1000.Aligned.sorted.bam
  </code></pre>
  <p>However, when I index the BAM file outside of nextflow, it runs without any problems. I was wondering what might be the issue here?</p>
  <pre><code>#!/usr/bin/env nextflow
  
  nextflow.enable.dsl = 2
  
  params.genome = '/Users/siddhaduio.no/Desktop/All_omics_tools/bowtie2-2.4.4-macos-x86_64/hg38_index/human_hg38_index.*.bt2'
  params.reads = '/Volumes/matthews/AHR_ER_project_files/FASTQ/TCDD_plus_E2_ERa_Rep_{1,2,4}_ChIPSeq_first_1000.fastq'
  params.results = '/Volumes/matthews/AHR_ER_project_files/FASTQ/results'
  
  
  
  /*
  * Process to align chip seq reads to genome with bowtie2
  */
  
  
  process mapping {
  
  publishDir '/Volumes/matthews/AHR_ER_project_files/FASTQ/results', mode: 'copy'
  
  input:
  
  path genomeDir
  
  tuple val(replicateId), path(reads)
  
  
  
  output:
  
  tuple val(replicateId), path(&quot;${replicateId}.Aligned.sam&quot;)
  
  
  script:
  
  def btw2_index = genomeDir[0].simpleName
  
  &quot;&quot;&quot;
  /Users/siddhaduio.no/Desktop/All_omics_tools/bowtie2-2.4.4-macos-x86_64/bowtie2 -p 64 -q -x <span class="math-container">${btw2_index} -U $</span>{reads.join(',')} -S <span class="math-container">${replicateId}.Aligned.sam 2&gt; $</span>{replicateId}.bowtie.log
  
  
  &quot;&quot;&quot;
  
  
  }
  
  process convert_sam_to_bam {
  
  
  
  publishDir '/Volumes/matthews/AHR_ER_project_files/FASTQ/results', mode: 'copy'
  
  input:
  
  
  tuple val(replicateId), path(&quot;${replicateId}.Aligned.sam&quot;)
  
  
  
  output:
  
  tuple val(replicateId), path(&quot;<span class="math-container">${replicateId}.Aligned.bam"), path("$</span>{replicateId}.Aligned.sorted.bam&quot;)
  
  
  script:
  
  &quot;&quot;&quot;
  
  /Users/siddhaduio.no/Desktop/All_omics_tools/samtools-1.14/samtools view -S -b -o <span class="math-container">${replicateId}.Aligned.bam $</span>{replicateId}.Aligned.sam 
  /Users/siddhaduio.no/Desktop/All_omics_tools/samtools-1.14/samtools sort --output-fmt BAM -o <span class="math-container">${replicateId}.Aligned.sorted.bam $</span>{replicateId}.Aligned.bam
  rm ${replicateId}.Aligned.sam
  
  
  &quot;&quot;&quot;
  
  
  }
  
  
  process samtools_index {
  
  
  
  publishDir '/Volumes/matthews/AHR_ER_project_files/FASTQ/results', mode: 'copy'
  
  input:
  
  
  tuple val(replicateId), path(&quot;${replicateId}.Aligned.sorted.bam&quot;)
  
  
  
  output:
  
  tuple val(replicateId), path(&quot;${replicateId}.Aligned.sorted.bam.bai&quot;)
  
  
  script:
  
  &quot;&quot;&quot;
  
  /Users/siddhaduio.no/Desktop/All_omics_tools/samtools-1.14/samtools index ${replicateId}.Aligned.sorted.bam
  
  
  &quot;&quot;&quot;
  
  
  }
  
  
  workflow{
  
  reads_ch= Channel.fromPath(params.reads).map { fastq -&gt; tuple(fastq.simpleName, fastq)}
  index_ch= files(params.genome)
  reads_ch
  index_ch
  mapping(index_ch,reads_ch)
  convert_sam_to_bam(mapping.out)
  samtools_index(convert_sam_to_bam.out)
  
  
  }
  
  <span class="math-container">```</span>
  </code></pre>
  
Tags: <nextflow>
Title: Samtools index fails within nextflow workflow but not outside of it
ThreadText: # Samtools index fails within nextflow workflow but not outside of it
  
  (Asked by: username_0 on 2024-09-14)
  
  I am a beginner to Nextflow and using it to setup Chipseq workflow and in the workflow below the samtools\_index process throws the following error:
  
  ```
  [E::hts_idx_push] Chromosome blocks not continuous
    [E::sam_index] Read 'FCC1L20ACXX:4:1101:1645:2230#0/1' with ref_name='chr17', ref_length=83257441, flags=16, pos=58851543 cannot be indexed
    samtools index: failed to create index for "TCDD_plus_E2_ERa_Rep_1_ChIPSeq_first_1000.Aligned.sorted.bam": Inappropriate ioctl for device
  
  ```
  
  The command executed is:
  
  ```
  /Users/siddhaduio.no/Desktop/All_omics_tools/samtools-1.14/samtools index TCDD_plus_E2_ERa_Rep_1_ChIPSeq_first_1000.Aligned.sorted.bam
  
  ```
  
  However, when I index the BAM file outside of nextflow, it runs without any problems. I was wondering what might be the issue here?
  
  ```
  #!/usr/bin/env nextflow
  
  nextflow.enable.dsl = 2
  
  params.genome = '/Users/siddhaduio.no/Desktop/All_omics_tools/bowtie2-2.4.4-macos-x86_64/hg38_index/human_hg38_index.*.bt2'
  params.reads = '/Volumes/matthews/AHR_ER_project_files/FASTQ/TCDD_plus_E2_ERa_Rep_{1,2,4}_ChIPSeq_first_1000.fastq'
  params.results = '/Volumes/matthews/AHR_ER_project_files/FASTQ/results'
  
  /*
  * Process to align chip seq reads to genome with bowtie2
  */
  
  process mapping {
  
  publishDir '/Volumes/matthews/AHR_ER_project_files/FASTQ/results', mode: 'copy'
  
  input:
  
  path genomeDir
  
  tuple val(replicateId), path(reads)
  
  output:
  
  tuple val(replicateId), path("${replicateId}.Aligned.sam")
  
  script:
  
  def btw2_index = genomeDir[0].simpleName
  
  """
  /Users/siddhaduio.no/Desktop/All_omics_tools/bowtie2-2.4.4-macos-x86_64/bowtie2 -p 64 -q -x ${btw2_index} -U ${reads.join(',')} -S ${replicateId}.Aligned.sam 2> ${replicateId}.bowtie.log
  
  """
  
  }
  
  process convert_sam_to_bam {
  
  publishDir '/Volumes/matthews/AHR_ER_project_files/FASTQ/results', mode: 'copy'
  
  input:
  
  tuple val(replicateId), path("${replicateId}.Aligned.sam")
  
  output:
  
  tuple val(replicateId), path("${replicateId}.Aligned.bam"), path("${replicateId}.Aligned.sorted.bam")
  
  script:
  
  """
  
  /Users/siddhaduio.no/Desktop/All_omics_tools/samtools-1.14/samtools view -S -b -o ${replicateId}.Aligned.bam ${replicateId}.Aligned.sam 
  /Users/siddhaduio.no/Desktop/All_omics_tools/samtools-1.14/samtools sort --output-fmt BAM -o ${replicateId}.Aligned.sorted.bam ${replicateId}.Aligned.bam
  rm ${replicateId}.Aligned.sam
  
  """
  
  }
  
  process samtools_index {
  
  publishDir '/Volumes/matthews/AHR_ER_project_files/FASTQ/results', mode: 'copy'
  
  input:
  
  tuple val(replicateId), path("${replicateId}.Aligned.sorted.bam")
  
  output:
  
  tuple val(replicateId), path("${replicateId}.Aligned.sorted.bam.bai")
  
  script:
  
  """
  
  /Users/siddhaduio.no/Desktop/All_omics_tools/samtools-1.14/samtools index ${replicateId}.Aligned.sorted.bam
  
  """
  
  }
  
  workflow{
  
  reads_ch= Channel.fromPath(params.reads).map { fastq -> tuple(fastq.simpleName, fastq)}
  index_ch= files(params.genome)
  reads_ch
  index_ch
  mapping(index_ch,reads_ch)
  convert_sam_to_bam(mapping.out)
  samtools_index(convert_sam_to_bam.out)
  
  }
  
  ```
  
  ```
  
  **Tags:** nextflow
  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2024-09-15. Score: 2 | Accepted answer
  
  You should have also received the following Nextflow warning:
  
  ```
  WARN: Input tuple does not match tuple declaration in process `samtools_index`
  
  ```
  
  Specifically, the problem is that the output of your *convert\_sam\_to\_bam* process is:
  
  ```
  tuple val(replicateId), path("${replicateId}.Aligned.bam"), path("${replicateId}.Aligned.sorted.bam")
  
  ```
  
  But the *samtools\_index* process is expecting:
  
  ```
  tuple val(replicateId), path("${replicateId}.Aligned.sorted.bam")
  
  ```
  
  ---
  
  If you would like to declare multiple output channels, you can use the `emit` keyword (this is documented under [additional options](https://www.nextflow.io/docs/latest/process.html#additional-options)) to define names for the output channel(s). For example:
  
  ```
  process convert_sam_to_bam {
  
      input:
      tuple val(replicateId), path("${replicateId}.Aligned.sam")
  
      output:
      tuple val(replicateId), path("${replicateId}.Aligned.bam"), emit: aligned_bam
      tuple val(replicateId), path("${replicateId}.Aligned.sorted.bam"), emit: sorted_bam
  
      script:
      """
      samtools view \\
          -b \\
          -o "${replicateId}.Aligned.bam" \\
          "${replicateId}.Aligned.sam"
  
      samtools sort \\
          -o "${replicateId}.Aligned.sorted.bam" \\
          "${replicateId}.Aligned.bam"
      """
  }
  
  ```
  
  ```
  workflow {
  
      ...
  
      samtools_index( convert_sam_to_bam.out.sorted_bam )
  }
  
  ```
  
  ---
  
  Otherwise, either change the *convert\_sam\_to\_bam* output declaration to:
  
  ```
  tuple val(replicateId), path("${replicateId}.Aligned.sorted.bam")
  
  ```
  
  Or use the [map](https://www.nextflow.io/docs/latest/operator.html#map) operator to get what you want, for example:
  
  ```
  workflow {
  
      ...
  
      samtools_index(
          convert_sam_to_bam.out.map { rep, aligned, sorted -> tuple( rep, sorted ) }
      )
  }
  
  ```
  
  ---
  
  Note that a separate indexing process can be avoided using newer versions of [samtools](https://www.htslib.org/doc/samtools.html) with the `--write-index` option:
  
  > To convert a SAM to a compressed BAM using BAI indexing: `samtools view --write-index in.sam -o out.bam##idx##out.bam.bai`
  
  > **Comments:**
  > **username_0**: Thanks a lot! This was helpful.  
  
  ---
  
  