BioWDL: Expression-Quantification

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This workflow can be used to perform expression quantification for multiple BAM files. Expression levels will be determined for each BAM file/sample and will be merged together into a single table including all samples.

Expression quantification will be performed using StringTie and HTSeq-Count.

This workflow is part of BioWDL developed by the SASC team.

Usage

This workflow can be run using Cromwell:

java -jar cromwell-<version>.jar run -i inputs.json multi-bam-quantify.wdl

Inputs

Inputs are provided through a JSON file. The minimally required inputs are described below and a template containing all possible inputs can be generated using Womtool as described in the WOMtool documentation. See this page for some additional general notes and information about pipeline inputs.

{
  "MultiBamExpressionQuantification.bams": "A list of BAM files and sample identifiers (see 'BAM file input' below)",
  "MultiBamExpressionQuantification.strandedness": "The strandedness of the samples: FR (forward-reverse), RF (reverse-forward) or None",
  "MultiBamExpressionQuantification.outputDir": "The path to the output directory.",
  "MultiBamExpressionQuantification.referenceGtfFile": "The path to the annotations GTF file. If not specified, Stringtie will be run unguided and the GTF file it produces will be used for HTSeq-Count",
}

BAM file input

BAM files need to be given as a list with one item per sample. Each of the items should be an object containing a "Left" element (the sample id) and a "Right" element (the BAM file and its index) following the structure as shown here:

{
  "Left": "Sample identifier",
  "Right": {
    "file": "The path to the sample's BAM file",
    "index": "The path to the index for the sample's BAM file"
  }
}

Example

{
  "MultiBamExpressionQuantification.bams": [
    {
      "Left": "s1",
      "Right": {
        "file": "/home/user/mapping/results/s1.bam",
        "index": "/home/user/mapping/results/s1.bai"
      }
    }, {
      "Left": "s2",
      "Right": {
        "file": "/home/user/mapping/results/s2.bam",
        "index": "/home/user/mapping/results/s2.bai"
      }
    }
  ],
  "MultiBamExpressionQuantification.strandedness": "FR",
  "MultiBamExpressionQuantification.outputDir": "/home/user/expression/results",
  "MultiBamExpressionQuantification.referenceGtfFile": "/home/user/genomes/human/features/ensembl87.gtf"
}

Tool versions

Included in the repository is an environment.yml file. This file includes all the tool version on which the workflow was tested. You can use conda and this file to create an environment with all the correct tools.

Output

The multi-bam-quantify workflow produces two directories:

• stringtie: Contains the Stringtie output. Includes two additional folder:
  • FPKM: Contains per sample FPKM counts, extracted from the Stringtie abundance output. Also contains a file called all_samples.FPKM, which contains the FPKM values for all samples.
  • TPM: Contains per sample TPM counts, extracted from the Stringtie abundance output. Also contains a file called all_samples.TPM, which contains the TPM values for all samples.
• fragments_per_gene: Contains the HTSeq-Count output. Also contains a file called all_samples.fragments_per_gene, which contains the counts for all samples.

Contact

For any question about running this workflow and feature requests, please use the github issue tracker or contact the SASC team directly at: sasc@lumc.nl.