http://www.htslib.org/workflow/#mapping_to_variant
分析流程：
step0—安装相关软件
step1—下载参考基因组数据和分析数据以及原始测序数据sra格式转换为fastq格式
sratoolkit ：https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=software
sratoolkit documentation：https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=toolkit_doc
如：
ls *.sra |while read id; do ~/biosoft/sratoolkit/sratoolkit.2.6.3-centos_linux64/bin/fastq-dump $id;done
rm *sra
step2—质控fastqc、去低值FASTX-Toolkit再质控
（1）
ls *.fastq | while read id ; do ~/biosoft/fastqc/FastQC/fastqc $id;done
Then we need to filter the bad quality reads according to the QC results.
##write a script by cat >filter.sh
（2）
ls *fastq |while read id
do
echo $id
## you need to adjust the parameter by yourself according to the QC results.
~/biosoft/fastx_toolkit_0.0.13/bin/fastq_quality_filter -v -q 20 -p 80 -Q33 -i $id -o tmp ;
~/biosoft/fastx_toolkit_0.0.13/bin/fastx_trimmer -v -f 1 -l 27 -i tmp -Q33 -z -o ${id%%.*}_clean.fq.gz ;
done
rm tmp
（3）
ls *_clean.fq.gz | while read id ; do ~/biosoft/fastqc/FastQC/fastqc $id;done
step3—alignment（bowtie2）
（1）build index
~/biosoft/bowtie/bowtie2-2.2.9/bowtie2-build ~/biosoft/bowtie/hg19_index /hg19.fa ~/biosoft/bowtie/hg19_index/hg19
（2）alignment
ls *.fastq | while read id ;
do
echo $id
~/biosoft/bowtie/bowtie2-2.2.9/bowtie2 -p 20 -x ~/biosoft/bowtie/hg19_index/hg19 -U $id -S ${id%%.*}.sam 2>${id%%.*}.align.log;
（3）## *.sam to *.bam
samtools view -bhS -q 30 ${id%%.*}.sam > ${id%%.*}.highQuality.bam
(http://www.htslib.org/doc/samtools.html)
## -F 1548 https://broadinstitute.github.io/picard/explain-flags.html
samtools sort ${id%%.*}.highQuality.bam ${id%%.*}.highQuality.sorted
samtools index ${id%%.*}.highQuality.sorted.bam
（4）unique mapping reads
grep -v "XS:i:" ${id%%.*}.sam |samtools view -bhS - >${id%%.*}.unique.bam
amtools sort ${id%%.*}.unique.bam ${id%%.*}.unique.sorted
samtools index ${id%%.*}.unique.sorted.bam
done
step4—peaks-calling-by-macs2
https://pypi.python.org/pypi/MACS2
Example for regular peak calling:
macs2 callpeak -t ChIP.bam -c Control.bam -f BAM -g hs -n test -B -q 0.01
如：
nohup time ~/.local/bin/macs2 callpeak -c SRR1042594.sorted.bam -t SRR1042593.sorted.bam -f BAM -B -g hs -n Xu_MUT_rep1 2>Xu_MUT_rep1.masc2.log
~/biosoft/PeakRanger/bin/peakranger ccat --format bam SRR1042594.sorted.bam SRR1042593.sorted.bam
Xu_MUT_rep1_ccat_report --report --gene_annot_file hg19refGene.txt -q 0.05 -t 4
step5—
step6—CEAS
http://liulab.dfci.harvard.edu/CEAS/download.html
wget http://liulab.dfci.harvard.edu/CEAS/src/CEAS-Package-1.0.2.tar.gz
wget http://liulab.dfci.harvard.edu/CEAS/src/hg19.refGene.gz ; gunzip hg19.refGene.gz
~/.local/bin/ceas --name=H3K36me3_ceas --pf-res=20 --gn-group-names='Top 10%,Bottom 10%'
| -g -g ~/biosoft/CEAS/annotation/hg19.refGene -b ../paper_results/GSM1278641_Xu_MUT_rep1_BAF155_MUT.peaks.bed -w ../rawData/SRR1042593.wig |