ATAC-Seq Pipeline Installation

git clone https://github.com/tobiasrausch/ATACseq.git

cd ATACseq

make all

If one of the above commands fail your operating system probably lacks some build essentials. These are usually pre-installed but if you lack them you need to install these. For instance, for Ubuntu this would require:

apt-get install build-essential g++ git wget unzip

Building Promoter Regions for QC

cd bed/ && Rscript promoter.R

Running the ATAC-Seq analysis pipeline for a single sample

./src/atac.sh <hg19|mm10> <read1.fq.gz> <read2.fq.gz> <genome.fa> <output prefix>

Plotting the key ATAC-Seq Quality Control metrics

If you have multiple output folders (one for each ATAC-Seq sample) you can simply concatenate the QC metrics of each sample.

head -n 1 ./*/*.key.metrics | grep "TssEnrichment" | uniq > summary.tsv

cat ./*/*.key.metrics | grep -v "TssEnrichment" >> summary.tsv

To plot the distribution for all QC parameters.

Rscript R/metrics.R summary.tsv

Differential peak calling

Merge peaks across samples and create a raw count matrix.

ls ./Sample1/Sample1.peaks ./Sample2/Sample2.peaks ./SampleN/SampleN.peaks > peaks.lst

ls ./Sample1/Sample1.bam ./Sample2/Sample2.bam ./SampleN/SampleN.bam > bams.lst

./src/count.sh hg19 peaks.lst bams.lst <output prefix>

To call differential peaks on a count matrix for TSS peaks, called counts.tss.gz, using DESeq2 we first need to create a file with sample level information (sample.info). For instance, if you have 2 replicates per condition:

echo -e "name\tcondition" > sample.info

zcat counts.tss.gz | head -n 1 | cut -f 5- | tr '\t' '\n' | sed 's/.final$//' | awk '{print $0"\t"int((NR-1)/2);}' >> sample.info

Rscript R/dpeaks.R counts.tss.gz sample.info