ChIP-seq Analysis Pipeline

This project is a comprehensive pipeline for analyzing ChIP-seq sequencing data. The workflow includes downloading sequencing data, performing quality control, trimming, aligning reads to a reference genome, and calling peaks. The workflow is managed using a Makefile and requires certain software tools and modules to be available in your environment.

Project Structure

• WORKDIR: /shared/projects/2324_tp_m2_bsg/Chau/
  The working directory where the analysis will be conducted.

• INDEX: /shared/bank/homo_sapiens/hg38/bowtie2/hg38
  The reference genome index used for alignment with Bowtie2.

• GENOME_SIZE: 250e6
  The estimated size of the reference genome (adapt this value based on the genome you are working with).

Input Files

• sample_list.txt: A file containing sample identifiers and related metadata. Each line should contain two fields separated by a semicolon ;, representing paired samples (e.g., experimental and control).

Workflow Rules

1. Download

• Output: fastq/{smp}.fq
• Description: Downloads and decompresses sequencing data files from Zenodo.
• Command:

  wget https://zenodo.org/records/7646128/files/{wildcards.smp}.fq.gz -O fastq/{wildcards.smp}.fq.gz
  gunzip fastq/{wildcards.smp}.fq.gz

2. FastQC

• Output: fastqc/{smp}_fastqc.html
• Description: Performs quality control on the raw FASTQ files using FastQC.
• Command:

  fastqc -o fastqc {input}

3. Sickle

• Output: sickle/{smp}.fq.gz
• Description: Trims and filters the FASTQ files to remove low-quality bases and reads using Sickle.
• Command:

  sickle se -f {input} -o {output} -t sanger -g

4. Bowtie2 Alignment

• Output: bowtie/{smp}.bam
• Description: Aligns the trimmed reads to the reference genome using Bowtie2 and converts the output to BAM format.
• Command:

  bowtie2 -p 1 -U {input} -x {params.index} | samtools view -b | samtools sort > {output}

5. BAM Indexing

• Output: bowtie/{smp}.bam.bai
• Description: Indexes the BAM file using Samtools for downstream analysis.
• Command:

  samtools index {input}

6. BAM Coverage

• Output: bam_coverage/{smp}.bw
• Description: Generates coverage files in BigWig format using DeepTools.
• Command:

  bamCoverage -b {input.bam} -bs 25 -o {output} --region chr1

7. MACS2 Peak Calling

• Output: macs2/{smp}_peaks.narrowPeak
• Description: Calls peaks on aligned reads using MACS2 for detecting enriched regions.
• Command:

  macs2 callpeak -t {input.chip_bam} -c {input.input_bam} -n {wildcards.smp} -f BAM -g {params.genome_size} --outdir macs2

Setup Instructions

1. Modules Required:

   • FastQC 0.11.9
   • Sickle 1.33
   • Bowtie2 2.5.1
   • Samtools 1.15.1
   • DeepTools 3.5.0
   • MACS2 2.2.7.1

2. Run the Pipeline:

   • Ensure all necessary modules are loaded or installed in your environment.
   • Execute the Makefile using the following command:

     make

   • The pipeline will execute each rule in sequence and generate the corresponding output files in their respective directories.

3. File Organization:

   • fastq/: Contains downloaded and decompressed FASTQ files.
   • fastqc/: Contains FastQC reports.
   • sickle/: Contains trimmed FASTQ files.
   • bowtie/: Contains aligned BAM files and indices.
   • bam_coverage/: Contains BigWig coverage files.
   • macs2/: Contains peak-calling results from MACS2.

Notes

• Update the GENOME_SIZE and INDEX paths according to the genome you are analyzing.
• Ensure that the sample_list.txt file is properly formatted for accurate parsing and sample identification.

This README provides an overview of the workflow and guides you on setting up and executing the analysis pipeline efficiently.