This pipeline is designed for processing of paired-end ATAC-seq libraries. The pipeline was developed to be run on an HPC cluster with the slurm job submission engine, but most of the scripts can be run locally. Beginning from raw FASTQ files, the pipeline calls peaks and generates signal tracks. An accessome is created from all samples, which is used to compute read counts to calculate differential accessibility. The pipeline integrates ATAC-seq and RNA-seq data by annotating peaks to nearby genomic elements, and merging differentially expressed genes with differentially accessible peaks. It further characterizes differentially accessible elements by performing Gene Set Enrichment Analysis and motif enrichment.

The shell scripts (.s) are intended for submission using slurm, and may require modification before they can be run with other job submission managers. rscript.s is a wrapper script for submitting R scripts as batch jobs. All R scripts (.R) can instead be run interactively with Rscript. Job scripts accepting an array of file indices will require the user to implement iteration before they can be run locally.

Tools required: bamutils, bedtools, Bowtie2, deeptools, gcc>=6.3, gsl>=2.3, htseq, kent, macs2, picard, R>=3.4.2

R packages required: biomaRt, circlize, chromVAR, ComplexHeatmap, DBI, DESeq2, edgeR, fgsea, GenomicFeatures, GenomicRanges, ggplot2, lattice, latticeExtra, motifmatchr, optparse, reshape2, RSQLite, rtracklayer, TFBSTools, UpSetR, VennDiagram

Genome data

Usage:

get KH2013 transcript model from GHOST

wget -U firefox http://ghost.zool.kyoto-u.ac.jp/datas/KH.KHGene.2013.gff3.zip
unzip KH.KHGene.2013.gff3.zip

install BSgenomes.Cintestinalis.KH.JoinedScaffold

cd BSgenome.Cintestinalis.KH.JoinedScaffold
./forgeGenome.sh

align ATACseq reads

sbatch -a1-52 bowtie.s

perform QC, generate bigWig files for IGV plots

sbatch -a1-52 seqstats.s

call peaks

sbatch -a1-52 macs2.s

merge peaks into accessome

sbatch peakome.s

get read counts for peaks in accessome

sbatch -a1-52 counts.s

create STAR index

sbatch staridx.s

align RNAseq reads and get counts

sbatch -a1-6 star.s

annotate peaks and initialize database

./rscript.s writeDB.R

calculate differential expression & add to database

./rscript.s rnaseqFoxf.R
./rscript.s rnaseqMAPK.R

calculate differential accessibility & add to database

./rscript.s atacDESeq.R

perform GSEA on peak accessibility

./rscript.s runFgsea.R

#motif analysis of DA peaks add CIS-BP orthogs to database

wget -O KH-ENS.blast.zip https://www.aniseed.cnrs.fr/aniseed/download/?file=data%2Fci%2FKH-ENS.blast.zip
unzip KH-ENS.blast.zip
Rscript writeCISBPorthologs.R

run ChromVAR

./rscript.s runChromVar.R