ATAC/ChIP-seq Overview

This section covers the analysis pipeline for ATAC-seq and ChIP-seq data, which are commonly used to study chromatin accessibility and protein-DNA interactions.

What is ATAC-seq?

ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) identifies regions of open chromatin across the genome.

What is ChIP-seq?

ChIP-seq (Chromatin Immunoprecipitation sequencing) identifies genome-wide binding sites of DNA-associated proteins such as transcription factors, histone modifications, and other chromatin-associated proteins.

Link to the ATAC/ChIP-seq pipeline:

Genomics_pipelines - ChIP/ATAC pipeline link

The README file in the repository provides detailed instructions on how to run the pipeline, including required software and dependencies.

Pipeline Steps

Quality Checks - FastQC analysis of raw reads
Trimming - Removing adapters and low-quality bases (fastp)
Alignment - Mapping reads to reference genome (using Bowtie2)
Post-alignment processing - Filtering, duplicate removal, BAM file processing (samtools)
Visualization - Creating bigWig files for genome browser visualization (bamCoverage)

Additional Steps:

Peak calling - Identifying enriched regions (Lanceotron)
Differential analysis - Comparing conditions (DiffBind)

Outputs

BAM files (aligned reads)
BigWig files (genome-wide coverage tracks)
Peak files (BED/narrowPeak format)
QC reports

How we run the pipeline

It is run on the CCB cluster using SLURM job scheduler.

sbatch ChIP_pipeline_Bowtie2_fast.sh <genome> 
#Where <genome> is e.g. hg38 or mm10

It requires a folder structure as follows:

mm10_mESC_Rad21_ChIP_WT_C3_Rep1/
    ├── ChIP_seq.yaml
    ├── LC-Rad21-WT-C3-Rep1-i15_S1_L001_R1_001.fastq.gz
    ├── LC-Rad21-WT-C3-Rep1-i15_S1_L001_R2_001.fastq.gz
    ├── LC-Rad21-WT-C3-Rep1-i15_S1_L002_R1_001.fastq.gz
    ├── LC-Rad21-WT-C3-Rep1-i15_S1_L002_R2_001.fastq.gz
    ├── LC-Rad21-WT-C3-Rep1-i15_S1_L003_R1_001.fastq.gz
    ├── LC-Rad21-WT-C3-Rep1-i15_S1_L003_R2_001.fastq.gz
    ├── LC-Rad21-WT-C3-Rep1-i15_S1_L004_R1_001.fastq.gz
    └── LC-Rad21-WT-C3-Rep1-i15_S1_L004_R2_001.fastq.gz

OR:

mm10_mESC_Rad21_ChIP_WT_C3_Rep2/
    ├── ChIP_seq.yaml
    ├── LC-Rad21-WT-C3-Rep2-i16_R1.fastq.gz
    └── LC-Rad21-WT-C3-Rep2-i16_R2.fastq.gz

Where the fastq.gz files are the raw sequencing data files, ideally as symlinks generated with ln -s from your raw data folder.

It will use the name of the parent folder as the sample name (here: mm10_mESC_Rad21_ChIP_WT_C3_Rep1) for all output files. So make sure the folder name is descriptive of the sample using the guidance from the Naming Conventions section:

<species>_<cell_type>_<assay>_<condition/genotype>_<replicate>

The following pages (STEP 3-5) will cover the individual steps of the ATAC/ChIP-seq pipeline in more detail. Additional steps are covered in later sections (STEP 6: Peak Calling and STEP 7: Differential Analysis).