ChiPSeqFPro (short from ChIP-Seq Full Processing) is a pipeline that will perform full processing of ChIPSeq data starting from the fastq.gz files. It performs fastqc quality control, mapping to the human genome hg19 or mouse mm10 using bwa, sam to bam conversion, peak calling with MACS, and finally creates bigwig files from bam files using bam2BigWig tool.
#Dependencies
Place fastqc.gz in a working folder
mkdir work.folder cp path-to-files/*fastq.gz work.folder
FastQC
Instalation (Linux), place FastQC folder in working directory:
cd work.folder wget http://www.bioinformatics.babraham.ac.uk/projects/fastqc/fastqc_v0.11.5.zip unzip fastqc_v0.11.5.zip chmod 755 ./FastQC/fastqc
Reference genome
Download the reference genome, in this example it is human hg19:
mkdir ~/reference_genomes
cd ~/reference_genomes
mkdir hg19
cd hg19
wget --timestamping
'ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips/hg19.2bit'
-O hg19.2bit
wget http://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/twoBitToFa
chmod 755 twoBitToFa
./twoBitToFa hg19.2bit hg19.fa
For the mouse genome:
mkdir mm10
cd mm10
wget --timestamping
'http://hgdownload.cse.ucsc.edu/goldenPath/mm10/bigZips/mm10.2bit'
-O mm10.2bit
./twoBitToFa mm10.2bit mm10.fa
Install BWA*
#Download to your ~ folder the latest version from http://sourceforge.net/projects/bio-bwa/files/ bunzip2 bwa-0.7.15.tar.bz2 tar xvf bwa-0.7.15.tar cd bwa-0.7.15 make #edit ~/.bashrc to add bwa to your PATH nano ~/.bashrc export PATH=$PATH:~/bwa-0.7.15 source ~/.bashrc #test the installation bwa
Indexing the reference genome with BWA
Use BWA to index the reference genome, use number of core on your machine, e.g. 64.
cd ~/reference_genomes #for human genome bwa index -a bwtsw hg19.fa #for mouse genome bwa index -a bwtsw mm10.fa
Install MACS2
Installing MACS through PyPI system.
pip install MACS2
Download bam2bigwig
For the human genome hg19:
wget https://raw.githubusercontent.com/milospjanic/bam2bigwig/master/bam2bigwig.sh chmod 775 bam2bigwig.sh
For the mouse genome mm10:
wget https://raw.githubusercontent.com/milospjanic/bam2bigwig/master/bam2bigwig.mm10.sh chmod 775 bam2bigwig.mm10.sh
Running
ChIPSeqFPro is composed of four pipelines that will run on either human genome hg19 or mouse genome mm10, using either paired-end (PE) or single-read (SR) sequences.
ChIPSeqFPro.PE.hg19.sh ChIPSeqFPro.PE.mm10.sh ChIPSeqFPro.SR.hg19.sh ChIPSeqFPro.SR.mm10.sh
After placing fastq.gz files in the working folder run the script that is suitable for your experiment, e.g:
chmod 755 ChIPSeqFPro.PE.hg19.sh ./ChIPSeqFPro.PE.hg19.sh 64
The number indicates number of cores you want to allocate for the analysis.
Don't forget to place the FastQC folder into the working folder! This is the only requirement necessary to be placed in the working directory, in addition to the fastqc.gz files and bam2bigwig.sh (bam2bigwig.mm10.sh).
Dont forget that reference genome needs to be in your ~/reference_genomes folder, in case you switch to another user account script may not work because it searches for ~/reference_genomes folder.