<p>I have a problem with my nextflow pipeline.</p>
<p>My workflow looks like this:</p>
<ol>
<li><p>Uncompress the hisat2 genotpye index grch38_genome.tar.gz from <a href="https://genome-idx.s3.amazonaws.com/hisat/grch38_genome.tar.gz" rel="nofollow noreferrer">https://genome-idx.s3.amazonaws.com/hisat/grch38_genome.tar.gz</a></p>
</li>
<li><p>Align .fastq.gz files from /https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR1806626 and <a href="https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR1806627" rel="nofollow noreferrer">https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR1806627</a></p>
</li>
</ol>
<p>However, in process HISAT2 I get command exit status 255:</p>
<blockquote>
<p>Command error: &quot;grch38&quot; does not exist</p>
</blockquote>
<p>The command executed is:</p>
<blockquote>
<p>hisat2 --very-sensitive --no-spliced-alignment -x grch38 -U
SRR1806626.fastq.gz &gt; SRR1806626.fastq.sam</p>
</blockquote>
<p>Can someone please explain to me what I'm doing wrong?</p>
<p>My script.nf file for reproducing the error looks like this:</p>
<pre><code>#!/usr/bin/env nextflow

nextflow.enable.dsl=2

params.reads = &quot;<span class="math-container">$baseDir/data/*{1,2}*.fastq.gz" //https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR1806626, https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR1806627
params.hisat2_index = "$</span>baseDir/data/*_genome.tar.gz&quot; // https://genome-idx.s3.amazonaws.com/hisat/grch38_genome.tar.gz

log.info &quot;&quot;&quot;\
         reads:        <span class="math-container">${params.reads}
         hisat2_index: $</span>{params.hisat2_index}
         &quot;&quot;&quot;
         .stripIndent()

process UNCOMPRESS_GENOTYPE_INDEX {

  tag &quot;$hisat2_index_ch.baseName&quot;

  input:
  path(hisat2_index_ch)

  output:
  path('*')

  script:
  &quot;&quot;&quot;
  tar xvzf $hisat2_index_ch
  &quot;&quot;&quot;

}

process HISAT2 {

  tag &quot;$reads.baseName&quot;

  input:
  path(hisat2_indices)
  path(reads)

  output:
  path('*.sam')

  script:
  &quot;&quot;&quot;
  hisat2 --very-sensitive \
         --no-spliced-alignment \
         -x <span class="math-container">$hisat2_indices \
         -U $</span>reads \
         &gt; ${reads.baseName}.sam
  &quot;&quot;&quot;

}

workflow {

  Channel
    .fromPath( params.reads )
    .ifEmpty { exit 1, &quot;reads was empty - no input files supplied&quot; }
    .set { reads_ch }

  Channel.fromPath( params.hisat2_index )
    .ifEmpty { exit 1, &quot;hisat2_index was empty - no input file supplied&quot; }
    .set { hisat2_index_ch }

  UNCOMPRESS_GENOTYPE_INDEX(hisat2_index_ch)

  HISAT2(UNCOMPRESS_GENOTYPE_INDEX.out, reads_ch)

  //Command error:
  //(ERR): &quot;grch38&quot; does not exist
  //Exiting now ...

}
</code></pre>
<p>Kind regards,
Thomas</p>

CC BY-SA 4.0
2021-12-30T11:38:38.573Z
Hi @ATpoint, thank you for your answer. You are correct. I changed  `-x $hisat2_indices` to `-x "${hisat2_indices}/genome"` and then it worked.
Thomas Müller
<rna-seq><nextflow><rna-alignment><errors>
Nextflow HISAT2 command exit status 255 with prebuild index
# Nextflow HISAT2 command exit status 255 with prebuild index

(Asked by: username_0 on 2021-12-29)

I have a problem with my nextflow pipeline.
My workflow looks like this:

Uncompress the hisat2 genotpye index grch38_genome.tar.gz from https://genome-idx.s3.amazonaws.com/hisat/grch38_genome.tar.gz

Align .fastq.gz files from /https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR1806626 and https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR1806627


However, in process HISAT2 I get command exit status 255:

Command error: "grch38" does not exist

The command executed is:

hisat2 --very-sensitive --no-spliced-alignment -x grch38 -U
SRR1806626.fastq.gz > SRR1806626.fastq.sam

Can someone please explain to me what I'm doing wrong?
My script.nf file for reproducing the error looks like this:
#!/usr/bin/env nextflow

nextflow.enable.dsl=2

params.reads = "$baseDir/data/*{1,2}*.fastq.gz" //https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR1806626, https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR1806627
params.hisat2_index = "$baseDir/data/*_genome.tar.gz" // https://genome-idx.s3.amazonaws.com/hisat/grch38_genome.tar.gz

log.info """\
         reads:        ${params.reads}
         hisat2_index: ${params.hisat2_index}
         """
         .stripIndent()

process UNCOMPRESS_GENOTYPE_INDEX {

  tag "$hisat2_index_ch.baseName"

  input:
  path(hisat2_index_ch)

  output:
  path('*')

  script:
  """
  tar xvzf $hisat2_index_ch
  """

}

process HISAT2 {

  tag "$reads.baseName"

  input:
  path(hisat2_indices)
  path(reads)

  output:
  path('*.sam')

  script:
  """
  hisat2 --very-sensitive \
         --no-spliced-alignment \
         -x $hisat2_indices \
         -U $reads \
         > ${reads.baseName}.sam
  """

}

workflow {

  Channel
    .fromPath( params.reads )
    .ifEmpty { exit 1, "reads was empty - no input files supplied" }
    .set { reads_ch }

  Channel.fromPath( params.hisat2_index )
    .ifEmpty { exit 1, "hisat2_index was empty - no input file supplied" }
    .set { hisat2_index_ch }

  UNCOMPRESS_GENOTYPE_INDEX(hisat2_index_ch)

  HISAT2(UNCOMPRESS_GENOTYPE_INDEX.out, reads_ch)

  //Command error:
  //(ERR): "grch38" does not exist
  //Exiting now ...

}

Kind regards,
Thomas

**Tags:** rna-seq, nextflow, rna-alignment, errors
> **Comments:**
> **username_0**: Hi @ATpoint, thank you for your answer. You are correct. I changed  `-x $hisat2_indices` to `-x "${hisat2_indices}/genome"` and then it worked.  

---

## 0 answers

