ww-saturation: Saturation Mutagenesis Workflow

Overview

This repository contains a WDL (Workflow Description Language) pipeline for performing saturation mutagenesis analysis on RNA-seq data. The workflow processes multiple samples in parallel, aligning paired-end sequencing reads to a reference genome using BWA, converting the resulting SAM files to BAM format, and performing saturation mutagenesis analysis using GATK's AnalyzeSaturationMutagenesis tool.

Workflow Steps

For each sample in parallel:

1. Alignment (BWA): Paired-end reads are aligned to a reference genome
2. SAM to BAM Conversion: The aligned reads are converted from SAM to BAM format
3. Saturation Mutagenesis Analysis: GATK's AnalyzeSaturationMutagenesis tool analyzes mutations across the specified ORF range

Files in this Repository

• ww-saturation.wdl: The main workflow definition file
• ww-saturation-inputs.json: Example input parameters for the workflow
• ww-saturation-options.json: Runtime configuration options for Cromwell

Requirements

• Cromwell or another WDL-compatible workflow execution engine
• Docker (all tools run in containers)
• Required Docker images:
  • getwilds/bwa:0.7.17
  • getwilds/samtools:1.11
  • getwilds/gatk:4.3.0.0

Usage

1. Prepare your input files:

   • Arrays of paired-end FASTQ files
   • Reference genome (FASTA, index, and dictionary files)
   • Define your ORF range
   • List of sample names

2. Modify the inputs JSON file:

   {
     "saturation_mutagenesis.fastq_1_array": ["/path/to/sample1_R1.fastq.gz", "/path/to/sample2_R1.fastq.gz"],
     "saturation_mutagenesis.fastq_2_array": ["/path/to/sample1_R2.fastq.gz", "/path/to/sample2_R2.fastq.gz"],
     "saturation_mutagenesis.sample_names": ["sample1", "sample2"],
     ...
   }

3. Run the workflow:

   java -jar cromwell.jar run ww-saturation.wdl -i ww-saturation-inputs.json -o ww-saturation-options.json

Input Parameters

Parameter	Description
fastq_1_array	Array of paths to first FASTQ files (R1) for each sample
fastq_2_array	Array of paths to second FASTQ files (R2) for each sample
sample_names	Array of sample identifiers used for output file naming
reference_fasta	Path to reference genome FASTA file
reference_fasta_index	Path to reference genome index (.fai)
reference_dict	Path to reference sequence dictionary (.dict)
orf_range	Open Reading Frame range (e.g., "122-781")

Resource Configuration

Task-specific resources can be configured in the inputs file:

• AlignReads.threads: Number of CPU threads for BWA alignment (default: 4)
• AlignReads.memory_gb: Memory allocation for alignment in GB (default: 8)
• SamToBam.threads: Number of CPU threads for SAM/BAM processing (default: 4)
• SamToBam.memory_gb: Memory allocation for SAM/BAM processing in GB (default: 8)
• AnalyzeSaturationMutagenesis.memory_gb: Memory allocation for GATK in GB (default: 16)

Outputs

The workflow produces arrays of output files:

• variant_counts: Counts of different variants observed at each position
• aa_counts: Amino acid counts at each position
• aa_fractions: Amino acid fractions (frequencies) at each position
• codon_counts: Counts of different codons at each position
• codon_fractions: Codon fractions (frequencies) at each position
• cov_length_counts: Coverage length distribution data
• read_counts: Read counts information
• ref_coverage: Reference coverage statistics

Each output file array contains one file per input sample.

Runtime Options

The ww-saturation-options.json file contains Cromwell runtime options:

• Continues execution of remaining tasks when possible if one task fails
• Enables workflow caching for faster reruns
• Configures task retry behavior
• Specifies output directory structure

Parallelization

The workflow:

• Processes multiple samples simultaneously using WDL's scatter-gather mechanism
• Returns arrays of output files
• Can significantly reduce total execution time when processing multiple samples

By default, Cromwell will execute the scattered tasks in parallel up to the limit of your execution backend. You can configure the maximum concurrent tasks in your Cromwell configuration.

Contributing

Contributions to improve the workflow are welcome. Please submit pull requests or open issues to suggest enhancements or report bugs.

License

This project is licensed under the MIT License - see the LICENSE file for details.