ChIP-Seq workflow

I have developed a general framework to handel ChIP-seq data, including download .sra data, fastq file extract, quality filter, reads mapping, uniqe mapping extract, PCR duplication removal, peak calling and peak visualization.

0. Working preparation

mkdir work_directory
cd work_directory
mkdir 1fastq 2fastqc 3filtered_fq 4sam 5uniq_sam 6sorted_bam 7dedup_bam 8macs2

1.Download data from sra list file (download.txt);

awk 'type=substr($0,1,3){pre_id=substr($0,1,6)}split($1,m,"."){print "/sra/sra-instant/reads/ByRun/sra/"type"/"pre_id"/"m[1]"/"$1".sra"}' SraAccList.txt > download.txt 
for m in $(cat download.txt); do if [ ! -e $(basename $m) ]; then echo $m; ascp -i ~/.aspera/connect/etc/asperaweb_id_dsa.openssh -k 1 -QT -l 200m anonftp@ftp-trace.ncbi.nlm.nih.gov:$m .; fi; done

2.Convert sra data to fastq format;

for sra in $(ls *.sra); do fastq-dump $m --split-files -O 1fastq;done   #used for paired-end sequenced data
for sra in $(ls *.sra); do fastq-dump $m -O 1fastq;done                 #used for single-end sequenced data

3.fastqc inspceting

cd 1fastq
for m in $(ls *.fastq); do echo $m; fastqc $m  -o ../2fastqc; done

4 Reads filter

(1) Trimmomatic

(1.1) paired-end

cd ../1fastq 
for m in $(ls *.fastq ); do java -jar ~/mysoft/Trimmomatic-0.36/trimmomatic-0.36.jar PE -threads 7 $m_1.fastq $m_2.fastq  ../3filtered_fq/$m_1.paired.fastq ../3filtered_fq/$m_1.unpaired.fastq ../3filtered_fq/$m_2.paired.fastq ../3filtered_fq/$m_2.unpaired.fastq ILLUMINACLIP:~/mysoft/Trimmomatic-0.36/adapters/my_adapter.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:100

(1.2) single-end

for m in $(ls *.fastq ); do java -jar ~/mysoft/Trimmomatic-0.36/trimmomatic-0.36.jar SE -threads 7 $m  ../3filtered_fq/$m_filtered.fastq  ILLUMINACLIP:/home/wuzefeng/mysoft/Trimmomatic-0.36/adapters/TruSeq9-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:100

5.bowtie2 mapping

cd ../3filtered_fq/

(single-end)

for m in $(ls *.filtered.fastq); do bowtie2 -x ~/genome.index -1 $m_1.filtered.fastq -2  $m_2.filtered.fastq  -S ../4sam/$m.sam --no-mixed  --no-discordant --no-unal -p 7 ;done

paired-end

for m in $(ls *.filtered.fastq); do bowtie2 -x ~/gebnome.index -U $m  -S ../4sam/$m.sam  -p 7  --no-unal; done

6.Get unique mapped reads

cd ../4sam/
for m in $(ls *.sam); do samtools view -Sh $m | grep -e "^@" -e "XM:i:[012][^0-9]" | grep -v "XS:i:" > ../5uniq_sam/$m.uniq.sam;done

7.uniq_sam to sorted_bam

cd ../5uniq_sam
for  m in $(ls *.sam); do  picard-tools SortSam  INPUT=$m OUTPUT=../6sorted_bam/$m.sorted.bam SO=coordinate; done

8.Remove duplications

cd ../6sorted_bam
for m in $(ls *.bam); do samtools rmdup -s $m ../7dedup_bam/$m.dedup.bam ; done

9.Peak calling

cd ../7dedup_bam
macs2 callpeak -t H3K4me3.bam -c input.bam -f BAM -g 1.2e8 -n H3K4me3 -B --SPMR -q  0.01 --outdir ../8macs2/narrow/  # narrow peak
macs2 callpeak -t H3K4me3.bam -c input.bam -f BAM -g 1.2e8 -n H3K4me3.bam -B --SPMR  --broad --broad-cutoff 0.1  --outdir ../8macs2/broad  # broad peaks

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