BS-seq分析流程（一）

短序列的质控都可以使用trimmomatic，这里不多做介绍，得到的clean data可做下面分析
一、比对和甲基化位点提取（Bismark）
Bismark安装及使用

git clone https://github.com/FelixKrueger/Bismark.git
#conda install -c bioconda bismark
#Genome Preparation
/opt/biosoft/Bismark-0.23.0/bismark_genome_preparation --parallel 40 --verbose ./
#bismark alignment
for i in `cat ../samples.txt`
do
    echo "/opt/biosoft/Bismark-0.23.0/bismark --parallel 40 --genome ./ --phred33-quals -1 ../$i.1.fastq -2 ../$i.2.fastq"
done > bismark.list
ParaFly -c bismark.list -CPU 4
#deduplicate 
for i in `cat ../samples.txt`
do
    echo "/opt/biosoft/Bismark-0.23.0/deduplicate_bismark --bam $i.1_bismark_bt2_pe.bam"
done > deduplicate_bismark.list
ParaFly -c deduplicate_bismark.list -CPU 9
#bismark_methylation_extractor提取甲基化信息
for i in `ls *deduplicated.bam`
do
    echo "/opt/biosoft/Bismark-0.23.0/bismark_methylation_extractor $i -p --genome ./ --gzip --bedGraph --cytosine_report --CX --buffer_size 20G --output ./"
done > methylation_extractor.list
ParaFly -c methylation_extractor.list -CPU 9
/opt/biosoft/Bismark-0.23.0/bismark2report
/opt/biosoft/Bismark-0.23.0/bismark2summary
for i in `cat ../samples.txt`
do
    echo "/opt/biosoft/Bismark-0.23.0/coverage2cytosine $i.1_bismark_bt2_pe.deduplicated.bismark.cov.gz --merge_CpG --genome ./ -o $i.CpG.output"
done > coverage2cytosine.list
ParaFly -c coverage2cytosine.list -CPU 9
/opt/biosoft/Bismark-0.23.0/bam2nuc --genome_folder ./ --genomic_composition_only
for i in `cat ../samples.txt`
do
    echo "/opt/biosoft/Bismark-0.23.0/bam2nuc --genome_folder ./ $i.1_bismark_bt2_pe.deduplicated.bam"
done > bam2nuc.list
ParaFly -c bam2nuc.list -CPU 9



/opt/biosoft/Bismark-0.23.0/bismark2bedGraph -o buffer_CpG.bedGraph --buffer 5G CpG_*