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README.md

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Here's a README.md draft for your GitHub repository that describes the analysis pipeline for PTA and WGS data:

PTA-WGS Analysis Pipeline

This repository contains a workflow description and tools for analyzing Primary Template-directed Amplification (PTA) and Whole Genome Sequencing (WGS) data. The pipeline includes all steps necessary for high-quality variant detection, including mapping, duplicate marking, realignment, variant calling, and filtration.

Overview

This pipeline processes WGS reads mapped against the GRCh38 reference genome, focusing on:

  • Handling PTA-specific artifacts using advanced machine learning models (PTATO Random Forest Model).
  • High-quality variant calling with GATK.
  • Comprehensive variant filtration for somatic and germline mutation detection.

Key Features

  • Support for both FASTQ and CRAM inputs.
  • Modular WDL workflow for running on Terra or locally.
  • Incorporation of existing GATK tools and custom PTA-specific artifact handling.

Workflow Steps

1. Input Files

  • FASTQ or CRAM files: Raw sequencing data.
  • Reference Genome: GRCh38.
  • Additional inputs: BED files, BAM indices, and genome-specific annotations.

2. Pipeline Steps

Step 1: Mapping Reads (if FASTQ provided)

  • Tool: BWA mem
  • Output: SAM/BAM files.

Step 2: Processing CRAM/BAM Files

  • Duplicate marking using Sambamba.
  • Realignment with GATK BaseRecalibrator.

Step 3: Variant Calling

  • Tool: GATK HaplotypeCaller
  • Multi-sample mode with EMIT_ALL_CONFIDENT_SITES.

Step 4: Variant Filtration

  • Tool: GATK VariantFiltration
  • Filters:
    • QD < 2.0
    • MQ < 40.0
    • FS > 60.0
    • HaplotypeScore > 13.0
    • SOR > 4.0 and more.

Step 5: PTA Artifact Filtering

  • PTATO Random Forest Model trained with 26 genomic features:
    • Allelic imbalance (most important).
    • DNA replication timing.
    • Distance to the nearest gene.
    • Repeat regions and sequence context.

Installation and Dependencies

Requirements

  • Docker
  • Cromwell/WDL runtime (for Terra)
  • Tools included:
    • BWA (v0.7.17)
    • Sambamba (v0.6.8)
    • GATK (v4.1.3.0)
    • Samtools (v1.10)

Installation

  1. Clone the repository:

    git clone [email protected]:broadinstitute/PTA_Analysis.git
cd PTA_Analysis

  2. Install dependencies:

    • Use Docker containers for all tools.

  3. Prepare input files (FASTQ or CRAM, Reference Genome).

Running the Pipeline

On Terra

  1. Import the WDL workflows into Terra.
  2. Set up input JSON files for each step.

Locally

Run the pipeline using Cromwell:

java -jar cromwell.jar run main.wdl -i inputs.json

Repository Contents

  • workflows/: WDL workflows for each pipeline step.
  • tasks/: Individual WDL tasks for mapping, duplicate marking, realignment, and variant calling.
  • docker/: Dockerfiles for each tool used in the pipeline.
  • examples/: Example input files and configuration JSONs.

Resources