Resources for Reusing Tools and Scripts

Overview

Teaching: 0 min
Exercises: 0 min

Questions

• How to find other solutions/CWL recipes for awkward problems?

Objectives

• Know good resources for finding solutions to common problems

Pre-written tool descriptions

When you start a CWL workflow, it is recommended to check if there is already a CWL document available for the tools you want to use. Bio-cwl-tools is a library of CWL documents for biology/life-sciences related tools.

The CWL documents of the previous steps were already provided for you, however, you can also find them in this library. In this episode you will use the bio-cwl-tools library to add the last step to the workflow.

Adding new step in workflow

The last step of our workflow is counting the RNA-seq reads for which we will use the featureCounts tool.

Exercise

Find the featureCounts tool in the bio-cwl-tools library. Have a look at the CWL document. Which inputs does this tool need? And what are the outputs of this tool?

Solution

The featureCounts CWL document can be found in the GitHub repo; it has 3 inputs: annotations and mapped_reads, both files, along with reads_are_paired (a boolean choice). These inputs can be found on lines 6, 9, and 12. The output of this tool is a file called featurecounts (line 27).

We need a local copy of featureCounts in order to use it in our workflow. We already imported this as a git submodule during setup, so the tool should be located at bio-cwl-tools/subread/featureCounts.cwl.

Exercise

Please copy the rna_seq_workflow_2.cwl file to create rna_seq_workflow_3.cwl. Add the featureCounts tool to the workflow. Similar to the STAR tool, this tool also needs more RAM than the default. To run the tool a minimum of 500 MiB of RAM is needed. Use a requirements entry with ResourceRequirement to allocate a ramMin of 500. Use the inputs and output of the previous exercise to connect this step to previous steps.

Solution

rna_seq_workflow_3.cwl

cwlVersion: v1.2
class: Workflow

inputs:
  rna_reads_fruitfly_forward:
    type: File
    format: http://edamontology.org/format_1930  # FASTQ
  rna_reads_fruitfly_reverse:
    type: File
    format: http://edamontology.org/format_1930  # FASTQ
  ref_fruitfly_genome: Directory
  fruitfly_gene_model: File

steps:
  quality_control_forward:
    run: bio-cwl-tools/fastqc/fastqc_2.cwl
    in:
      reads_file: rna_reads_fruitfly_forward
    out: [html_file]

  quality_control_reverse:
    run: bio-cwl-tools/fastqc/fastqc_2.cwl
    in:
      reads_file: rna_reads_fruitfly_reverse
    out: [html_file]

  trim_low_quality_bases:
    run: bio-cwl-tools/cutadapt/cutadapt-paired.cwl
    in:
      reads_1: rna_reads_fruitfly_forward
      reads_2: rna_reads_fruitfly_reverse
      minimum_length: { default: 20 }
      quality_cutoff: { default: 20 }
    out: [ trimmed_reads_1, trimmed_reads_2, report ]

  mapping_reads:
    requirements:
      ResourceRequirement:
        ramMin: 5120
    run: bio-cwl-tools/STAR/STAR-Align.cwl
    in:
      RunThreadN: {default: 4}
      GenomeDir: ref_fruitfly_genome
      ForwardReads: trim_low_quality_bases/trimmed_reads_1
      ReverseReads: trim_low_quality_bases/trimmed_reads_2
      OutSAMtype: {default: BAM}
      SortedByCoordinate: {default: true}
      OutSAMunmapped: {default: Within}
      Overhang: { default: 36 }  # the length of the reads - 1
      Gtf: fruitfly_gene_model
    out: [alignment]

  index_alignment:
    run: bio-cwl-tools/samtools/samtools_index.cwl
    in:
      bam_sorted: mapping_reads/alignment
    out: [bam_sorted_indexed]

  count_reads:
    requirements:
      ResourceRequirement:
        ramMin: 500
    run: bio-cwl-tools/subread/featureCounts.cwl
    in:
      mapped_reads: index_alignment/bam_sorted_indexed
      annotations: fruitfly_gene_model
      reads_are_paired: {default: true}
    out: [featurecounts]

outputs:
  quality_report_forward:
    type: File
    outputSource: quality_control_forward/html_file
  quality_report_reverse:
    type: File
    outputSource: quality_control_reverse/html_file
  bam_sorted_indexed:
    type: File
    outputSource: index_alignment/bam_sorted_indexed
  featurecounts:
    type: File
    outputSource: count_reads/featurecounts

The workflow is complete and we only need to complete the YAML input file. Please copy the workflow_input_2.yml file to workflow_input_3.yml, and add the last entry in the input file, which is the fruitfly_gene_model file.

workflow_input_3.yml

rna_reads_fruitfly_forward:
  class: File
  location: rnaseq/GSM461177_1_subsampled.fastqsanger
  format: http://edamontology.org/format_1930  # FASTQ
rna_reads_fruitfly_reverse:
  class: File
  location: rnaseq/GSM461177_2_subsampled.fastqsanger
  format: http://edamontology.org/format_1930  # FASTQ
ref_fruitfly_genome:
  class: Directory
  location: rnaseq/dm6-STAR-index
fruitfly_gene_model:
  class: File
  location: rnaseq/Drosophila_melanogaster.BDGP6.87.gtf
  format: http://edamontology.org/format_2306

You have finished the workflow and the input file and now you can run the whole workflow.

cwltool --cachedir cache rna_seq_workflow_3.cwl workflow_input_3.yml

Key Points

• bio-cwl-tools is a library of CWL documents for biology/life-sciences related tools