ATAC-seq peak calling with MACS2

2 minute read


ATAC-seq (Assay for Transposase Accessible Chromatin with high-throughput Sequencing) is a next-generation sequencing approach for the analysis of open chromatin regions to assess the genome-wise chromatin accessibility.

ATAC-seq achieves this by simultaneously fragmenting and tagging genomic DNA with sequencing adapters using the hyperactive Tn5 transposase enzyme 1. Other global chromatin accessibility methods include FAIRE-seq and DNase-seq. This document aims to provide accessibility.

Pre-processing of raw sequencing reads – before mapping the raw reads to the genome, trim the adapter sequences. Poor read quality or sequencing errors often lead to low mapping rate.

Mapping/alignment of sequencing reads to a reference genome – use Burrows-Wheeler Aligner (BWA) for mapping of sequencing reads. The output alignment file will be saved as a sequence alignment/map (SAM) format or binary version of SAM called BAM. Mark the duplicate reads using Picard 2 and exclude reads mapping to mitochondrial DNA and other chromosomes from analysis together with low quality reads (MAPQ<10 and reads in Encode black list regions) using SAMtools 3.

Filtering and shifting of the mapped reads - shift the read position +4 and -5 bp in the BAM file before peak calling adjust the reads alignment. When the Tn5 transposase cuts open chromatin regions, it introduces two cuts that are separated by 9 bp. Therefore, ATAC-seq reads aligning to the positive and negative strands need to be adjusted by +4 bp and -5 bp respectively to represent the center of the transposase binding site. Picard CollectInsertSizeMetrics will be used to compute the fragment sizes on alignment shifted BAM files.

Identification and visualization of the ATAC-seq peaks – use MACS2 for peak calling with the parameters nomodel or BAMPE 4 and identify the differentially enriched peaks using the MACS2 bdgdiff module. Individual peaks separated by <100 bp will be join together. For peak annotation and functional analysis use the R package ChIPpeakAnno or HOMER 5,6. First, ATAC-seq peaks will be categorized into different groups based on the nearest RefSeq gene i.e. promoter, untranslated regions (UTRs), intron and exon. Second, peaks that are within 5 kb upstream and 3 kb downstream of the Transcription Start Site (TSS) are associated to the nearest genes. Finally, these genes are then analyzed for over-represented gene ontology (GO) terms and KEGG pathways using ChIPpeakAnno. Visualize all sequencing tracks using the Integrated Genomic Viewer (IGV) 7.

Scripts are available for HPC Cluster.
For further reading: ATAC-seq-data-analysis-from-FASTQ-to-peaks

  1. ATAC-seq - Nature Methods. 2013; 10:1213–1218. 

  2. PICARD 

  3. HTSLIB 

  4. MACS 

  5. ChIPpeakAnno 

  6. HOMER 

  7. IGV