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GATK Somatic SNV+Indel+CNV+SV

最近准备为sliverworkspace 图形化生信平台开发报告设计器,需要一个较为复杂的pipeline作为测试数据,就想起来把之前的 [[满分室间质评之GATK Somatic SNV+Indel+CNV+SV(下)性能优化]]翻出来用一下。跑了一遍发现还是各种问题,于是想把pipeline改造成免部署、首次运行初始化环境的版本,以便需要时候能够直接运行起来,于是有了本文。

一句话描述就是:开箱即用的pipeline,能够根据样本version_reference自动选择参考基因组版本,根据project_bed文件选择项目bed,自动初始化环境、安装所需软件、下载ref文件和数据库的版本

为了让pipeline能够直接运行,无需部署,这里使用docker容器保证运行环境的一致性:见文章:基于docker的生信基础环境镜像构建,这里采用的方案是带ssh服务的docker+conda环境,整个pipeline在一个通用容器中运行。

本文代码较长,可能略复杂,想直接运行的可以下载 workflow文件,导入sliverworkspace图形化生信平台直接运行,获取并查看图形化分析报告

相关代码和workflow文件可以访问笔者github项目地址

导入workflow操作

导入workflow操作

分析流程整体概览

概览图

docker 镜像拉取及部署配置

bash
# 拉取docker镜像
docker     pull     doujiangbaozi/sliverworkspace:1.10

docker-compose.yml配置文件

yaml
version: "3"
services:
  GATK:
    image: doujiangbaozi/sliverworkspace:1.10
    container_name: GATK
    volumes:
      - /home/sliver/Data/data:/opt/data:rw                                #挂载输入数据目录
      - /home/sliver/Manufacture/gatk/envs:/root/mambaforge-pypy3/envs     #挂载envs目录
      - /home/sliver/Manufacture/sliver/ref:/opt/ref:rw                    #挂载reference目录
      - /home/sliver/Manufacture/gatk/result:/opt/result:rw                #挂载中间文件和结果目录
    environment:
      - TZ=Asia/Shanghai                                                   #设置时区
      - PS=20191124                                                        #设置ssh密码
      - PT=9024                                                            #设置ssh连接端口

docker 镜像部署运行

bash
# 在docker-compose.yml文件同级目录下运行
docker-compose up -d

# 或者docker-compose -f docker-compose.yml所在目录
docker-compose -f somedir/docker-compose.yml up -d

# 可以通过ssh连接到docker 运行pipeline命令了,连接端口和密码见docker-compose.yml配置文件相关字段
ssh root@127.0.0.1 -p9024

测试数据

测试数据来自2017年卫计委室间质评提供的bed文件(pipeline会自动下载)和测试数据,修改命名以匹配pipeline输入端,也可以替换为自己的数据文件,因为室间质评目前参考基因组还停留在hg19版本,所以本流程仍然使用hg19(GRCH37),如果要切换到hg38,可以将version_reference变量值设置为hg38,project_bed设置为Illumina_pt2_hg38.bed。pipeline会使用hg38(GRCH38)版本和对应的bed文件。

文件名(按照需要有调整)文件大小MD5
B1701_R1.fq.gz4.85G07d3cdccee41dbb3adf5d2e04ab28e5b
B1701_R2.fq.gz4.77Gc2aa4a8ab784c77423e821b9f7fb00a7
B1701NC_R1.fq.gz3.04G4fc21ad05f9ca8dc93d2749b8369891b
B1701NC_R2.fq.gz3.11Gbc64784f2591a27ceede1727136888b9

变量名称

bash
# 变量初始化赋值
  sn=1701                               #样本编号
  pn=GS03                               #pipeline 代号
  data=/opt/data                        #数据输入目录
  result=/opt/result                    #数据输出、中间文件目录
  project_bed=Illumina_pt2.bed          #参考基因组hg19下的bed文件
  version_reference=hg19                #人参考基因组版本hg19或者hg38
  version_fastqc=0.12.1                 #fastqc 版本
  version_multiqc=1.21                  #multiqc 版本
  version_cnvkit=0.9.10                 #cnvkit 版本
  version_manta=1.6.0                   #manta 版本
  version_gatk=4.3.0.0                  #gatk 版本                                 
  version_sambamba=1.0.1                #sambamba 版本                             
  version_samtools=1.17                 #samtools 版本                             
  version_bwa=0.7.17                    #bwa 版本                                  
  version_fastp=0.23.2                  #fastp 版本                                
  version_vep=108.2                     #vep软件版本
  version_vep_cache=108                 #vep-cache数据库版本                               
  envs=/root/mambaforge-pypy3/envs	    #mamba envs 目录                           
  threads=32                         	#最大线程数                                   
  memory=32G                        	#内存占用                                    
  scatter=8                          	#Mutect2 分拆并行运行interval list 份数          
  event=2                          	    #gatk FilterMutectCalls --max-events-in-region 数值
  snv_tlod=16.00                      	#snv 过滤参数 tload 值                        
  snv_vaf=0.01                       	#snv 过滤参数 丰度/突变频率                        
  snv_depth=500                        	#snv 过滤参数 支持reads数/depth 测序深度            
  cnv_dep=1000                       	#cnv 过滤参数 支持reads数/depth 测序深度            
  cnv_min=-0.5                       	#cnv 过滤参数 log2最小值                        
  cnv_max=0.5                        	#cnv 过滤参数 log2 最大值                       
  sv_score=200                        	#sv  过滤参数 score                           

## 以上变量个可以具体根据需求调整

表格:

变量名变量值备注
sn1701样本编号
pnGS03pipeline 代号 GATK Somatic 03版本
project_bedIllumina_pt2.bed参考基因组hg19下的bed文件
version_referencehg19人参考基因组版本hg19 / hg38
version_fastqc0.12.1fastqc 版本
version_multiqc1.21multiqc 版本
version_cnvkit0.9.10cnvkit 版本
version_manta1.6.0manta版本
version_gatk4.3.0.0gatk 版本
version_sambamba1.0.1sambamba 版本
version_samtools1.17samtools 版本
version_bwa0.7.17bwa 版本
version_fastp0.23.2fastp 版本
version_vep108.2vep软件版本
version_vep_cache108vep cache 版本
envs/root/mambaforge-pypy3/envsmamba envs 目录
threads32最大线程数
memory32G内存占用
scatter8Mutect2 分拆并行运行interval list 份数
event2gatk FilterMutectCalls --max-events-in-region 数值
snv_tlod16.00snv 过滤参数 tload 值
snv_vaf0.01snv 过滤参数 丰度/突变频率
snv_depth500snv 过滤参数 支持reads数/depth 测序深度
cnv_dep1000cnv 过滤参数 支持reads数/depth 测序深度
cnv_min-0.5cnv 过滤参数 log2最小值
cnv_max0.5cnv 过滤参数 log2 最大值
sv_score200sv 过滤参数 score

Pipeline/workflow 具体步骤:

1. fastp 默认参数过滤,看下原始数据质量,clean data

bash
#conda检测环境是否存在,首次运行不存在创建该环境并安装软件
if [ ! -d "${envs}/${pn}.fastp" ]; then
    echo "Creating the environment ${pn}.fastp"
    mamba create -n ${pn}.fastp -y fastp=${version_fastp} fastqc=${version_fastqc} multiqc=${version_multiqc}
fi

mamba	activate ${pn}.fastp

mkdir -p ${result}/${sn}/trimmed
mkdir -p ${result}/${sn}/qc

fastqc -t ${threads}\
	${data}/GS03/${sn}_tumor_R1.fq.gz \
	${data}/GS03/${sn}_tumor_R2.fq.gz \
	-o ${result}/${sn}/qc &

fastqc -t ${threads}\
	${data}/GS03/${sn}_normal_R1.fq.gz \
	${data}/GS03/${sn}_normal_R2.fq.gz \
	-o ${result}/${sn}/qc &

fastp -w 16 \
    -i ${data}/GS03/${sn}_tumor_R1.fq.gz  \
    -I ${data}/GS03/${sn}_tumor_R2.fq.gz  \
    -o ${result}/${sn}/trimmed/${sn}_tumor_R1_trimmed.fq.gz \
    -O ${result}/${sn}/trimmed/${sn}_tumor_R2_trimmed.fq.gz \
    -h ${result}/${sn}/trimmed/${sn}_tumor_fastp.html \
    -j ${result}/${sn}/trimmed/${sn}_tumor_fastp.json &

fastp -w 16 \
    -i ${data}/GS03/${sn}_normal_R1.fq.gz  \
    -I ${data}/GS03/${sn}_normal_R2.fq.gz  \
    -o ${result}/${sn}/trimmed/${sn}_normal_R1_trimmed.fq.gz \
    -O ${result}/${sn}/trimmed/${sn}_normal_R2_trimmed.fq.gz \
    -h ${result}/${sn}/trimmed/${sn}_normal_fastp.html \
    -j ${result}/${sn}/trimmed/${sn}_normal_fastp.json &
wait

fastqc -t ${threads}\
	${result}/${sn}/trimmed/${sn}_tumor_R1_trimmed.fq.gz \
	${result}/${sn}/trimmed/${sn}_tumor_R2_trimmed.fq.gz \
	-o ${result}/${sn}/qc &

fastqc -t ${threads}\
	${result}/${sn}/trimmed/${sn}_normal_R1_trimmed.fq.gz \
	${result}/${sn}/trimmed/${sn}_normal_R2_trimmed.fq.gz \
	-o ${result}/${sn}/qc &

wait

mamba	deactivate

2. normal文件fastq比对到参考基因组,sort 排序,mark duplicate 得到 marked.bam

bash
#conda检测环境是否存在,首次运行不存在创建该环境并安装软件
if [ ! -d "${envs}/${pn}.align" ]; then
    mamba create -n ${pn}.align -y bwa=${version_bwa} samtools=${version_samtools} 
fi

#从github下载sambamba static 比 mamba 安装的版本速度快1倍以上.这是个很诡异的地方
if [ ! -f "${envs}/${pn}.align/bin/sambamba" ]; then
    aria2c https://github.com/biod/sambamba/releases/download/v${version_sambamba}/sambamba-${version_sambamba}-linux-amd64-static.gz -d ${envs}/${pn}.align/bin
    gzip -cdf ${envs}/${pn}.align/bin/sambamba-${version_sambamba}-linux-amd64-static.gz  >  ${envs}/${pn}.align/bin/sambamba 
    chmod a+x ${envs}/${pn}.align/bin/sambamba
fi

mamba	activate ${pn}.align

if [ "${version_reference}" == hg19 ]; then
    echo	"USE reference Version : ${version_reference}"
    mkdir	-p /opt/ref/hg19
    #如果没有检测到参考基因组序列,则下载序列并使用bwa创建索引
    if [ ! -f "/opt/ref/hg19/hg19.fasta" ]; then
    	aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg19/ucsc.hg19.fasta.gz -d /opt/ref/hg19 -o hg19.fasta.gz
    	cd /opt/ref/hg19 && gzip -d /opt/ref/hg19/hg19.fasta.gz
	else
        if [ -f "/opt/ref/hg19/ucsc.hg19.fasta.gz.aria2" ]; then
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg19/ucsc.hg19.fasta.gz -c -d /opt/ref/hg19 -o hg19.fasta.gz
            cd /opt/ref/hg19 && gzip -d /opt/ref/hg19/hg19.fasta.gz
        fi
	fi
	
    if  [ ! -f /opt/ref/hg19/hg19.fasta.amb ] ||
        [ ! -f /opt/ref/hg19/hg19.fasta.ann ] ||
        [ ! -f /opt/ref/hg19/hg19.fasta.bwt ] ||
        [ ! -f /opt/ref/hg19/hg19.fasta.pac ] ||
        [ ! -f /opt/ref/hg19/hg19.fasta.sa ]; then
        bwa index /opt/ref/hg19/hg19.fasta
    fi
    #检测samtools索引是否存在,如不存在则使用samtools创建参考基因组索引
    if [ ! -f "/opt/ref/hg19/hg19.fasta.fai" ]; then
    	samtools faidx /opt/ref/hg19/hg19.fasta
    fi
elif [ "${version_reference}" == "hg38" ]; then
    echo	"USE reference Version : ${version_reference}"
    mkdir	-p /opt/ref/hg38
#如果没有检测到参考基因组序列,则下载序列并使用bwa创建索引
    if [ ! -f "/opt/ref/hg38/hg38.fasta" ]; then
    	aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/Homo_sapiens_assembly38.fasta.gz -d /opt/ref/hg38 -o hg38.fasta.gz
        cd /opt/ref/hg38 && gzip -d /opt/ref/hg38/hg38.fasta.gz
	else
        if [ -f "/opt/ref/hg38/hg38.fasta.gz.aria2" ]; then
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/Homo_sapiens_assembly38.fasta.gz -d /opt/ref/hg38 -d /opt/ref/hg38 -o hg38.fasta.gz
            cd /opt/ref/hg38 && gzip -d /opt/ref/hg38/hg38.fasta.gz
        fi
    fi
    
    if  [ ! -f /opt/ref/hg38/hg38.fasta.amb ] ||
        [ ! -f /opt/ref/hg38/hg38.fasta.ann ] ||
        [ ! -f /opt/ref/hg38/hg38.fasta.bwt ] ||
        [ ! -f /opt/ref/hg38/hg38.fasta.pac ] ||
        [ ! -f /opt/ref/hg38/hg38.fasta.sa ]; then
        bwa index /opt/ref/hg38/hg38.fasta
    fi
    #检测samtools索引是否存在,如不存在则使用samtools创建参考基因组索引
    if [ ! -f "/opt/ref/hg38/hg38.fasta.fai" ]; then
    	samtools faidx /opt/ref/hg38/hg38.fasta
    fi
fi




mkdir -p ${result}/${sn}/aligned
#比对基因组管道输出成bam文件,管道输出排序
bwa mem \
    -t ${threads} -M \
    -R "@RG\\tID:${sn}_normal\\tLB:${sn}_normal\\tPL:Illumina\\tPU:Miseq\\tSM:${sn}_normal" \
    /opt/ref/${version_reference}/${version_reference}.fasta  ${result}/${sn}/trimmed/${sn}_normal_R1_trimmed.fq.gz ${result}/${sn}/trimmed/${sn}_normal_R2_trimmed.fq.gz \
    | sambamba view -S -f bam -l 0 /dev/stdin \
    | sambamba sort -t ${threads} -m 2G --tmpdir=${result}/${sn}/aligned -o ${result}/${sn}/aligned/${sn}_normal_sorted.bam /dev/stdin

#防止linux打开文件句柄数超过限制,报错退出
ulimit -n 10240

#使用sambamba对sorted bam文件标记重复
sambamba markdup \
    --tmpdir ${result}/${sn}/aligned \
    -t ${threads} ${result}/${sn}/aligned/${sn}_normal_sorted.bam ${result}/${sn}/aligned/${sn}_normal_marked.bam 

#修改marked bam文件索引名,gatk和sambamba索引文件名需要保持一致
mv  ${result}/${sn}/aligned/${sn}_normal_marked.bam.bai ${result}/${sn}/aligned/${sn}_normal_marked.bai
#删除sorted bam文件
rm -f ${result}/${sn}/aligned/${sn}_normal_sorted.bam*

mamba	deactivate

3. tumor文件fastq比对到参考基因组,排序,mark duplicate 得到 marked.bam,与第2步类似

bash
if [ ! -d "${envs}/${pn}.align" ]; then
	mamba create -n ${pn}.align -y bwa=${version_bwa} samtools=${version_samtools} 
fi

#从github下载sambamba static 比 mamba 安装的版本速度快1倍以上.
if [ ! -f "${envs}/${pn}.align/bin/sambamba" ]; then
    aria2c https://github.com/biod/sambamba/releases/download/v${version_sambamba}/sambamba-${version_sambamba}-linux-amd64-static.gz -d ${envs}/${pn}.align/bin
    gzip -cdf ${envs}/${pn}.align/bin/sambamba-${version_sambamba}-linux-amd64-static.gz  >  ${envs}/${pn}.align/bin/sambamba 
    chmod a+x ${envs}/${pn}.align/bin/sambamba
fi

mamba	activate ${pn}.align

if [ "${version_reference}" == hg19 ]; then
    echo	"USE reference Version : ${version_reference}"
    mkdir	-p /opt/ref/hg19
	#如果没有检测到参考基因组序列,则下载序列并使用bwa创建索引
	if [ ! -f "/opt/ref/hg19/hg19.fasta" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg19/ucsc.hg19.fasta.gz -d /opt/ref/hg19 -o hg19.fasta.gz
        cd /opt/ref/hg19 && gzip -d /opt/ref/hg19/hg19.fasta.gz
    else
        if [ -f "/opt/ref/hg19/ucsc.hg19.fasta.gz.aria2" ]; then
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg19/ucsc.hg19.fasta.gz -c -d /opt/ref/hg19 -o hg19.fasta.gz
            cd /opt/ref/hg19 && gzip -d /opt/ref/hg19/hg19.fasta.gz
        fi
	fi
	
    if  [ ! -f /opt/ref/hg19/hg19.fasta.amb ] ||
        [ ! -f /opt/ref/hg19/hg19.fasta.ann ] ||
        [ ! -f /opt/ref/hg19/hg19.fasta.bwt ] ||
        [ ! -f /opt/ref/hg19/hg19.fasta.pac ] ||
        [ ! -f /opt/ref/hg19/hg19.fasta.sa ]; then
        bwa index /opt/ref/hg19/hg19.fasta
    fi
    #检测samtools索引是否存在,如不存在则使用samtools创建参考基因组索引
    if [ ! -f "/opt/ref/hg19/hg19.fasta.fai" ]; then
    	samtools faidx /opt/ref/hg19/hg19.fasta
    fi
elif [ "${version_reference}" == "hg38" ]; then
    echo	"USE reference Version : ${version_reference}"
    mkdir	-p /opt/ref/hg38
    #如果没有检测到参考基因组序列,则下载序列并使用bwa创建索引
    if [ ! -f "/opt/ref/hg38/hg38.fasta" ]; then
    	aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/Homo_sapiens_assembly38.fasta.gz -d /opt/ref/hg38 -o hg38.fasta.gz
        cd /opt/ref/hg38 && gzip -d /opt/ref/hg38/hg38.fasta.gz
	else
        if [ -f "/opt/ref/hg38/hg38.fasta.gz.aria2" ]; then
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/Homo_sapiens_assembly38.fasta.gz -d /opt/ref/hg38 -d /opt/ref/hg38 -o hg38.fasta.gz
            cd /opt/ref/hg38 && gzip -d /opt/ref/hg38/hg38.fasta.gz
        fi
    fi
    
    if  [ ! -f /opt/ref/hg38/hg38.fasta.amb ] ||
        [ ! -f /opt/ref/hg38/hg38.fasta.ann ] ||
        [ ! -f /opt/ref/hg38/hg38.fasta.bwt ] ||
        [ ! -f /opt/ref/hg38/hg38.fasta.pac ] ||
        [ ! -f /opt/ref/hg38/hg38.fasta.sa ]; then
        bwa index /opt/ref/hg38/hg38.fasta
    fi
    #检测samtools索引是否存在,如不存在则使用samtools创建参考基因组索引
    if [ ! -f "/opt/ref/hg38/hg38.fasta.fai" ]; then
    	samtools faidx /opt/ref/hg38/hg38.fasta
    fi
fi

mkdir	-p ${result}/${sn}/aligned

bwa	mem \
    -t ${threads} -M \
    -R "@RG\\tID:${sn}_tumor\\tLB:${sn}_tumor\\tPL:Illumina\\tPU:Miseq\\tSM:${sn}_tumor" \
    /opt/ref/${version_reference}/${version_reference}.fasta  ${result}/${sn}/trimmed/${sn}_tumor_R1_trimmed.fq.gz ${result}/${sn}/trimmed/${sn}_tumor_R2_trimmed.fq.gz \
    | sambamba view -S -f bam -l 0 /dev/stdin \
    | sambamba sort -t ${threads} -m 2G --tmpdir=${result}/${sn}/aligned -o ${result}/${sn}/aligned/${sn}_tumor_sorted.bam /dev/stdin
ulimit -n 10240
sambamba  markdup \
    --tmpdir ${result}/${sn}/aligned \
    -t ${threads} ${result}/${sn}/aligned/${sn}_tumor_sorted.bam ${result}/${sn}/aligned/${sn}_tumor_marked.bam
    mv  ${result}/${sn}/aligned/${sn}_tumor_marked.bam.bai ${result}/${sn}/aligned/${sn}_tumor_marked.bai
    rm  -f ${result}/${sn}/aligned/${sn}_tumor_sorted.bam*

mamba	deactivate

4. 对上述bam文件生成重新校准表,为后续BQSR使用;Generates recalibration table for Base Quality Score Recalibration (BQSR)

bash
#conda检测环境是否存在,首次运行不存在创建该环境并安装软件
if [ ! -f "${envs}/${pn}.gatk/bin/gatk" ]; then
    mamba create -n ${pn}.gatk -y gatk4=${version_gatk} \
        r-base=3.6.2 \
        r-data.table=1.12.8 \
        r-dplyr=0.8.5 \
        r-getopt=1.20.3 \
        r-ggplot2=3.3.0 \
        r-gplots=3.0.3 \
        r-gsalib=2.1 \
        r-optparse=1.6.4 \
        r-backports=1.1.10 \
        tabix
fi

mamba activate ${pn}.gatk

if [ "${version_reference}" == hg19 ]; then
    echo	"USE reference Version : ${version_reference}"
    #这里有个巨坑,broadinstitute ftp站点bundle目录提供的hg19版本参考文件,默认格式运行会报错,提示没有索引,使用gatk创建索引仍然报错,其实是gz格式需要使用bgzip重新压缩并且使用tabix创建索引才行
    if [ ! -f "/opt/ref/hg19/dbsnp_138.hg19.vcf.gz" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg19/dbsnp_138.hg19.vcf.gz -d /opt/ref/hg19
    else 
        if [ -f "/opt/ref/hg19/dbsnp_138.hg19.vcf.gz.aria2" ]; then
            echo 'download continue...'
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg19/dbsnp_138.hg19.vcf.gz -c -d /opt/ref/hg19
        fi
    fi
    
    if [ ! -f "/opt/ref/hg19/dbsnp_138.hg19.vcf.gz.tbi" ]; then
        gzip -k -f -d /opt/ref/hg19/dbsnp_138.hg19.vcf.gz > /opt/ref/hg19/dbsnp_138.hg19.vcf
        bgzip -f --threads ${threads} /opt/ref/hg19/dbsnp_138.hg19.vcf > /opt/ref/hg19/dbsnp_138.hg19.vcf.gz
        tabix -f /opt/ref/hg19/dbsnp_138.hg19.vcf.gz
    fi

    if [ ! -f "/opt/ref/hg19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz -d /opt/ref/hg19
    else 
        if [ -f "/opt/ref/hg19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz.aria2" ]; then
            echo 'download continue...'
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz -c -d /opt/ref/hg19
        fi
    fi
    
    if [ ! -f "/opt/ref/hg19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz.tbi" ]; then
        gzip -k -f -d /opt/ref/hg19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz > /opt/ref/hg19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf
        bgzip -f --threads ${threads} /opt/ref/hg19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf > /opt/ref/hg19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz
        tabix -f /opt/ref/hg19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz
    fi

    if [ ! -f "/opt/ref/hg19/1000G_phase1.snps.high_confidence.hg19.sites.vcf.gz" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg19/1000G_phase1.snps.high_confidence.hg19.sites.vcf.gz -d /opt/ref/hg19
    else 
        if [ -f "/opt/ref/hg19/1000G_phase1.snps.high_confidence.hg19.sites.vcf.gz.aria2" ]; then
            echo 'download continue...'
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg19/1000G_phase1.snps.high_confidence.hg19.sites.vcf.gz -c -d /opt/ref/hg19
        fi
    fi
    
    if [ ! -f "/opt/ref/hg19/1000G_phase1.snps.high_confidence.hg19.sites.vcf.gz.tbi" ]; then
        gzip -k -f -d /opt/ref/hg19/1000G_phase1.snps.high_confidence.hg19.sites.vcf.gz > /opt/ref/hg19/1000G_phase1.snps.high_confidence.hg19.sites.vcf
        bgzip -f --threads ${threads} /opt/ref/hg19/1000G_phase1.snps.high_confidence.hg19.sites.vcf > /opt/ref/hg19/1000G_phase1.snps.high_confidence.hg19.sites.vcf.gz
        tabix -f /opt/ref/hg19/1000G_phase1.snps.high_confidence.hg19.sites.vcf.gz
    fi

    if [ ! -f "/opt/ref/hg19/1000G_phase1.indels.hg19.sites.vcf.gz" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg19/1000G_phase1.indels.hg19.sites.vcf.gz -d /opt/ref/
    else 
        if [ -f "/opt/ref/hg19/1000G_phase1.indels.hg19.sites.vcf.gz.aria2" ]; then
            echo 'download continue...'
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg19/1000G_phase1.indels.hg19.sites.vcf.gz -c -d /opt/ref/hg19
        fi
    fi
    
    if [ ! -f "/opt/ref/hg19/1000G_phase1.indels.hg19.sites.vcf.gz.tbi" ]; then
        gzip -k -f -d /opt/ref/hg19/1000G_phase1.indels.hg19.sites.vcf.gz > /opt/ref/hg19/1000G_phase1.indels.hg19.sites.vcf
        bgzip -f --threads ${threads} /opt/ref/hg19/1000G_phase1.indels.hg19.sites.vcf > /opt/ref/hg19/1000G_phase1.indels.hg19.sites.vcf.gz
        tabix -f /opt/ref/hg19/1000G_phase1.indels.hg19.sites.vcf.gz
    fi
    #创建参考序列hg19的dict字典文件
    if [ ! -f "/opt/ref/hg19/hg19.dict" ]; then
        gatk CreateSequenceDictionary -R /opt/ref/hg19/hg19.fasta -O /opt/ref/hg19/hg19.dict
    fi
    
    if [ ! -f "/opt/ref/${version_reference}/${project_bed}" ]; then
        #根据下载的Illumina_pt2.bed 文件创建interval list文件,坐标转换,起始坐标0修改为1
        #sed 's/chr//; s/\t/ /g' /opt/ref/hg19/Illumina_pt2.bed > /opt/ref/hg19/Illumina_pt2.processed.bed
        mkdir  -p /opt/ref/${version_reference}
		aria2c https://raw.githubusercontent.com/doujiangbaozi/sliverworkspace-util/main/somatic/projects/${project_bed} -d /opt/ref/hg19
        if [ ! -f "/opt/ref/hg19/${project_bed}.interval_list" ]; then
            gatk BedToIntervalList \
                -I	/opt/ref/hg19/${project_bed} \
                -SD	/opt/ref/hg19/hg19.dict \
                -O	/opt/ref/hg19/${project_bed}.interval_list
        fi
    fi
    


    mkdir -p ${result}/${sn}/recal

    gatk BaseRecalibrator \
            --known-sites /opt/ref/hg19/dbsnp_138.hg19.vcf.gz \
            --known-sites /opt/ref/hg19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz \
            --known-sites /opt/ref/hg19/1000G_phase1.indels.hg19.sites.vcf.gz \
            -L /opt/ref/hg19/${project_bed}.interval_list \
            -R /opt/ref/hg19/hg19.fasta \
            -I ${result}/${sn}/aligned/${sn}_tumor_marked.bam \
            -O ${result}/${sn}/recal/${sn}_tumor_recal.table &

    gatk BaseRecalibrator \
            --known-sites /opt/ref/hg19/dbsnp_138.hg19.vcf.gz \
            --known-sites /opt/ref/hg19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz \
            --known-sites /opt/ref/hg19/1000G_phase1.indels.hg19.sites.vcf.gz \
            -L /opt/ref/hg19/${project_bed}.interval_list \
            -R /opt/ref/hg19/hg19.fasta \
            -I ${result}/${sn}/aligned/${sn}_normal_marked.bam \
            -O ${result}/${sn}/recal/${sn}_normal_recal.table &

    wait
elif [ "${version_reference}" == "hg38" ]; then
    echo	"USE reference Version : ${version_reference}"
    if [ ! -f "/opt/ref/hg38/dbsnp_146.hg38.vcf.gz" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/dbsnp_146.hg38.vcf.gz -d /opt/ref/hg38
    else 
        if [ -f "/opt/ref/hg38/dbsnp_146.hg38.vcf.gz.aria2" ]; then
            echo 'download continue...'
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/dbsnp_146.hg38.vcf.gz -c -d /opt/ref/hg38
        fi
    fi
    if [ ! -f "/opt/ref/hg38/dbsnp_146.hg38.vcf.gz.tbi" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/dbsnp_146.hg38.vcf.gz.tbi -d /opt/ref/hg38
    else 
        if [ -f "/opt/ref/hg38/dbsnp_146.hg38.vcf.gz.tbi.aria2" ]; then
            echo 'download continue...'
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/dbsnp_146.hg38.vcf.gz.tbi -c -d /opt/ref/hg38
        fi
    fi

    if [ ! -f "/opt/ref/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz -d /opt/ref/hg38
    else 
        if [ -f "/opt/ref/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz.aria2" ]; then
            echo 'download continue...'
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz -c -d /opt/ref/hg38
        fi
    fi
    if [ ! -f "/opt/ref/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz.tbi" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz.tbi -d /opt/ref/hg38
    else 
        if [ -f "/opt/ref/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz.tbi.aria2" ]; then
            echo 'download continue...'
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz.tbi -c -d /opt/ref/hg38
        fi
    fi

    if [ ! -f "/opt/ref/hg38/1000G_phase1.snps.high_confidence.hg38.vcf.gz" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/1000G_phase1.snps.high_confidence.hg38.vcf.gz -d /opt/ref/hg38
    else 
        if [ -f "/opt/ref/hg38/1000G_phase1.snps.high_confidence.hg38.vcf.gz.aria2" ]; then
            echo 'download continue...'
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/1000G_phase1.snps.high_confidence.hg38.vcf.gz -c -d /opt/ref/hg38
        fi
    fi
    if [ ! -f "/opt/ref/hg38/1000G_phase1.snps.high_confidence.hg38.vcf.gz.tbi" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/1000G_phase1.snps.high_confidence.hg38.vcf.gz.tbi -d /opt/ref/hg38
    else 
        if [ -f "/opt/ref/hg38/1000G_phase1.snps.high_confidence.hg38.vcf.gz.tbi.aria2" ]; then
            echo 'download continue...'
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/hg38/1000G_phase1.snps.high_confidence.hg38.vcf.gz.tbi -c -d /opt/ref/hg38
        fi
    fi
    #创建参考序列hg38的dict字典文件
    if [ ! -f "/opt/ref/hg38/hg38.dict" ]; then
        gatk CreateSequenceDictionary -R /opt/ref/hg38/hg38.fasta -O /opt/ref/hg38/hg38.dict
    fi
    
    if [ ! -f "/opt/ref/${version_reference}/${project_bed}" ]; then
        #根据下载的Illumina_pt2.bed 文件创建interval list文件,坐标转换,起始坐标0修改为1
        #sed 's/chr//; s/\t/ /g' /opt/ref/hg38/Illumina_pt2.bed > /opt/ref/hg38/Illumina_pt2.processed.bed
        mkdir  -p /opt/ref/hg38
        aria2c https://raw.githubusercontent.com/doujiangbaozi/sliverworkspace-util/main/somatic/projects/${project_bed} -d /opt/ref/hg38 -o ${project_bed}
        if [ ! -f "/opt/ref/hg38/${project_bed}.interval_list" ]; then
            gatk BedToIntervalList \
                -I	/opt/ref/hg38/${project_bed} \
                -SD	/opt/ref/hg38/hg38.dict \
                -O	/opt/ref/hg38/${project_bed}.interval_list
        fi
    fi
    
    mkdir -p ${result}/${sn}/recal

    gatk BaseRecalibrator \
            --known-sites /opt/ref/hg38/dbsnp_146.hg38.vcf.gz \
            --known-sites /opt/ref/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz \
            --known-sites /opt/ref/hg38/1000G_phase1.snps.high_confidence.hg38.vcf.gz \
            -L /opt/ref/hg38/${project_bed}.interval_list \
            -R /opt/ref/hg38/hg38.fasta \
            -I ${result}/${sn}/aligned/${sn}_tumor_marked.bam \
            -O ${result}/${sn}/recal/${sn}_tumor_recal.table &

    gatk BaseRecalibrator \
            --known-sites /opt/ref/hg38/dbsnp_146.hg38.vcf.gz \
            --known-sites /opt/ref/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz \
            --known-sites /opt/ref/hg38/1000G_phase1.snps.high_confidence.hg38.vcf.gz \
            -L /opt/ref/hg38/${project_bed}.interval_list \
            -R /opt/ref/hg38/hg38.fasta \
            -I ${result}/${sn}/aligned/${sn}_normal_marked.bam \
            -O ${result}/${sn}/recal/${sn}_normal_recal.table &

    wait
fi


    
mamba deactivate

5. 使用校准表对bam碱基质量校准,因为这一步gatk效率感人,所以同时计算insertsize,拆分interval list(后续mutect2并行运行需要),运行cnvkit batch,运行samtools depth计算测序深度,samtools flagstat 统计mapping比例及质量

bash
mkdir -p ${result}/${sn}/bqsr
mkdir -p ${result}/${sn}/stat
mkdir -p ${result}/${sn}/cnv
mkdir -p ${result}/${sn}/interval

mamba activate ${pn}.gatk

gatk ApplyBQSR \
    --bqsr-recal-file ${result}/${sn}/recal/${sn}_tumor_recal.table \
    -L /opt/ref/${version_reference}/${project_bed}.interval_list \
    -R /opt/ref/${version_reference}/${version_reference}.fasta \
    -I ${result}/${sn}/aligned/${sn}_tumor_marked.bam \
    -O ${result}/${sn}/bqsr/${sn}_tumor_bqsr.bam &


    
gatk ApplyBQSR \
    --bqsr-recal-file ${result}/${sn}/recal/${sn}_normal_recal.table \
    -L /opt/ref/${version_reference}/${project_bed}.interval_list \
    -R /opt/ref/${version_reference}/${version_reference}.fasta \
    -I ${result}/${sn}/aligned/${sn}_normal_marked.bam \
    -O ${result}/${sn}/bqsr/${sn}_normal_bqsr.bam &


    
gatk CollectInsertSizeMetrics \
    -I ${result}/${sn}/aligned/${sn}_tumor_marked.bam \
    -O ${result}/${sn}/stat/${sn}_tumor_insertsize_metrics.txt \
    -H ${result}/${sn}/stat/${sn}_tumor_insertsize_histogram.pdf &


    
gatk CollectInsertSizeMetrics \
    -I ${result}/${sn}/aligned/${sn}_normal_marked.bam \
    -O ${result}/${sn}/stat/${sn}_normal_insertsize_metrics.txt \
    -H ${result}/${sn}/stat/${sn}_normal_insertsize_histogram.pdf &

rm -f ${result}/${sn}/interval/*.interval_list
gatk SplitIntervals \
    -L /opt/ref/${version_reference}/${project_bed}.interval_list \
    -R /opt/ref/${version_reference}/${version_reference}.fasta \
    -O ${result}/${sn}/interval \
    --scatter-count ${scatter} &

mamba deactivate

if [ ! -d "${envs}/${pn}.cnvkit" ]; then
    mamba create -n ${pn}.cnvkit -y cnvkit=${version_cnvkit}
fi

if [ ! -f "/opt/ref/${version_reference}/refFlat.txt" ]; then
    aria2c -x 16 -s 16 http://hgdownload.soe.ucsc.edu/goldenPath/${version_reference}/database/refFlat.txt.gz -d /opt/ref/${version_reference}
    cd /opt/ref/${version_reference} && gzip -d refFlat.txt.gz
fi

mamba activate ${pn}.cnvkit

rm -f ${result}/${sn}/cnv/${sn}_reference.cnn

cnvkit.py batch \
    ${result}/${sn}/aligned/${sn}_tumor_marked.bam \
    --normal ${result}/${sn}/aligned/${sn}_normal_marked.bam \
    --method hybrid \
    --fasta  /opt/ref/${version_reference}/${version_reference}.fasta \
    --targets /opt/ref/${version_reference}/${project_bed} \
    --annotate /opt/ref/${version_reference}/refFlat.txt \
    --output-reference ${result}/${sn}/cnv/${sn}_reference.cnn \
    --output-dir ${result}/${sn}/cnv/ \
    --diagram \
    -p ${threads} &

mamba deactivate

mamba activate ${pn}.align

samtools depth -a -b /opt/ref/${version_reference}/Illumina_pt2.bed  --threads ${threads} \
    ${result}/${sn}/aligned/${sn}_tumor_marked.bam > \
    ${result}/${sn}/stat/${sn}_tumor_marked.depth  &

samtools depth -a -b /opt/ref/${version_reference}/Illumina_pt2.bed  --threads ${threads} \
    ${result}/${sn}/aligned/${sn}_normal_marked.bam > \
    ${result}/${sn}/stat/${sn}_normal_marked.depth &

samtools flagstat --threads ${threads} \
    ${result}/${sn}/aligned/${sn}_tumor_marked.bam  > \
    ${result}/${sn}/stat/${sn}_tumor_marked.flagstat   &

samtools flagstat --threads ${threads} \
    ${result}/${sn}/aligned/${sn}_normal_marked.bam > \
    ${result}/${sn}/stat/${sn}_normal_marked.flagstat &
    
mamba deactivate

wait

6. 计算堆叠数据( pileup metrics )以便后续评估污染,也可以根据拆分的interval list并行处理,处理之后合并。

bash
#官方巨坑,默认提供的small_exac_common_3_b37.vcf.gz默认染色体坐标不是以chr开头而是数字
mamba activate ${pn}.gatk

if [ "${version_reference}" == hg19 ]; then
    echo	"USE reference Version : ${version_reference}"
    #这里有个巨坑,从broadinstitute ftp 站点bundle Mutect2目录下载的参考文件,与同样下载的参考序列基因组坐标系不一致,参考基因组参考序列是chr1这种格式,这个af-only-gnomad是1,2,3这种格式,需要编写脚本处理
    if [ ! -f "/opt/ref/hg19/small_exac_common_3_b37.processed.vcf.gz" ]; then
        aria2c ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/Mutect2/GetPileupSummaries/small_exac_common_3_b37.vcf.gz -d /opt/ref/hg19
    else
        if [ -f "/opt/ref/hg19/small_exac_common_3_b37.vcf.gz.aria2" ]; then
            aria2c ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/Mutect2/GetPileupSummaries/small_exac_common_3_b37.vcf.gz -c -d /opt/ref/hg19
        fi
    fi
    if [ ! -f "/opt/ref/${version_reference}/small_exac_common_3_b37.processed.vcf.gz" ]; then
		if [ ! -f "${envs}/VcfProcessUtil.py" ]; then
			aria2c https://raw.fgit.cf/doujiangbaozi/sliverworkspace-util/main/somatic/VcfProcessUtil.py -d ${envs}/
			#aria2c https://raw.githubusercontent.com/doujiangbaozi/sliverworkspace-util/main/somatic/VcfProcessUtil.py -d ${envs}/
			chmod a+x ${envs}/VcfProcessUtil.py
		fi
		mamba activate ${pn}.cnvkit
		${envs}/VcfProcessUtil.py \
			-f /opt/ref/${version_reference}/small_exac_common_3_b37.vcf.gz \
			-o /opt/ref/${version_reference}/small_exac_common_3_b37.processed.vcf
		mamba deactivate
		mamba activate ${pn}.gatk
		cd /opt/ref/${version_reference}
		bgzip -f --threads ${threads} small_exac_common_3_b37.processed.vcf
		tabix -f small_exac_common_3_b37.processed.vcf.gz
		mamba deactivate
    fi
	mamba activate ${pn}.gatk
    for i in `ls ${result}/${sn}/interval/*.interval_list`;
    do
        echo $i
        gatk GetPileupSummaries \
            -R /opt/ref/${version_reference}/${version_reference}.fasta \
            -I ${result}/${sn}/bqsr/${sn}_tumor_bqsr.bam \
            -O ${i%.*}-tumor-pileups.table \
            -V /opt/ref/${version_reference}/small_exac_common_3_b37.processed.vcf.gz \
            -L $i \
            --interval-set-rule INTERSECTION &

        gatk GetPileupSummaries \
            -R /opt/ref/${version_reference}/${version_reference}.fasta \
            -I ${result}/${sn}/bqsr/${sn}_normal_bqsr.bam \
            -O ${i%.*}-normal-pileups.table \
            -V /opt/ref/${version_reference}/small_exac_common_3_b37.processed.vcf.gz \
            -L $i \
            --interval-set-rule INTERSECTION &

    done
    wait
    mamba deactivate
elif [ "${version_reference}" == "hg38" ]; then
    echo	"USE reference Version : ${version_reference}"
    if [ ! -f "/opt/ref/${version_reference}/small_exac_common_3.hg38.vcf.gz" ]; then
        aria2c ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/Mutect2/GetPileupSummaries/small_exac_common_3.hg38.vcf.gz -d /opt/ref/${version_reference}
    else
        if [ -f "/opt/ref/hg19/small_exac_common_3_b37.vcf.gz.aria2" ]; then
            aria2c ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/Mutect2/GetPileupSummaries/small_exac_common_3.hg38.vcf.gz -c -d /opt/ref/${version_reference}
        fi
    fi
    if [ ! -f "/opt/ref/${version_reference}/small_exac_common_3.hg38.vcf.gz.tbi" ]; then
        aria2c ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/Mutect2/GetPileupSummaries/small_exac_common_3.hg38.vcf.gz.tbi -d /opt/ref/${version_reference}
    else
        if [ -f "/opt/ref/hg19/small_exac_common_3_b37.vcf.gz.tbi.aria2" ]; then
            aria2c ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/Mutect2/GetPileupSummaries/small_exac_common_3.hg38.vcf.gz.tbi -c -d /opt/ref/${version_reference}
        fi
    fi
    mamba activate ${pn}.gatk
    for i in `ls ${result}/${sn}/interval/*.interval_list`;
    do
        echo $i
        gatk GetPileupSummaries \
            -R /opt/ref/${version_reference}/${version_reference}.fasta \
            -I ${result}/${sn}/bqsr/${sn}_tumor_bqsr.bam \
            -O ${i%.*}-tumor-pileups.table \
            -V /opt/ref/${version_reference}/small_exac_common_3.hg38.vcf.gz \
            -L $i \
            --interval-set-rule INTERSECTION &

        gatk GetPileupSummaries \
            -R /opt/ref/${version_reference}/${version_reference}.fasta \
            -I ${result}/${sn}/bqsr/${sn}_normal_bqsr.bam \
            -O ${i%.*}-normal-pileups.table \
            -V /opt/ref/${version_reference}/small_exac_common_3.hg38.vcf.gz \
            -L $i \
            --interval-set-rule INTERSECTION &

    done
    wait
    mamba deactivate
fi


mamba activate ${pn}.gatk
tables=
for i in `ls ${result}/${sn}/interval/*-tumor-pileups.table`;
do
    tables="$tables -I $i"
done

gatk GatherPileupSummaries \
    --sequence-dictionary /opt/ref/${version_reference}/${version_reference}.dict \
    $tables \
    -O ${result}/${sn}/stat/${sn}_tumor_pileups.table

nctables=
for i in `ls ${result}/${sn}/interval/*-normal-pileups.table`;
do
    nctables="$nctables -I $i"
done

gatk GatherPileupSummaries \
    --sequence-dictionary /opt/ref/${version_reference}/${version_reference}.dict \
    $nctables \
    -O ${result}/${sn}/stat/${sn}_normal_pileups.table
    
mamba deactivate

7. 使用GetPileupSummaries计算结果评估跨样本污染,结果用于后面 FilterMutectCall 过滤Mutect2输出结果

bash
mamba activate ${pn}.gatk

gatk CalculateContamination \
    -matched ${result}/${sn}/stat/${sn}_normal_pileups.table \
    -I ${result}/${sn}/stat/${sn}_tumor_pileups.table \
    -O ${result}/${sn}/stat/${sn}_contamination.table
    
mamba deactivate

8. Mutect2 call 突变,使用拆分的interval list,结束后将结果合并;同时并行运行manta call sv突变

bash
mkdir -p ${result}/${sn}/sv
mkdir -p ${result}/${sn}/snv

mamba activate ${pn}.gatk

if [ "${version_reference}" == hg19 ]; then
    echo	"USE reference Version : ${version_reference}"
    #这里有个巨坑,从broadinstitute ftp 站点bundle Mutect2目录下载的参考文件,与同样下载的参考序列基因组坐标系不一致,参考基因组参考序列是chr1这种格式,这个af-only-gnomad是1,2,3这种格式,需要编写脚本处理;hg38貌似没有这个问题,hg19的数据都不维护了么?
    if [ ! -f "/opt/ref/hg19/af-only-gnomad.raw.sites.b37.vcf.gz" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/Mutect2/af-only-gnomad.raw.sites.b37.vcf.gz -d /opt/ref/hg19
    else
        if [ -f "/opt/ref/hg19/af-only-gnomad.raw.sites.b37.vcf.gz.aria2" ]; then
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/Mutect2/af-only-gnomad.raw.sites.b37.vcf.gz -c -d /opt/ref/hg19
        fi
        if [ ! -f "${envs}/VcfProcessUtil.py" ]; then
            aria2c https://raw.fgit.cf/doujiangbaozi/sliverworkspace-util/main/somatic/VcfProcessUtil.py -d ${envs}/
            #aria2c https://raw.githubusercontent.com/doujiangbaozi/sliverworkspace-util/main/somatic/VcfProcessUtil.py -d ${envs}/
            chmod a+x ${envs}/VcfProcessUtil.py
        fi
        mamba activate ${pn}.cnvkit
        ${envs}/VcfProcessUtil.py \
            -f /opt/ref/hg19/af-only-gnomad.raw.sites.b37.vcf.gz \
            -o /opt/ref/hg19/af-only-gnomad.raw.sites.b37.processed.vcf
        mamba deactivate
        mamba activate ${pn}.gatk
        cd /opt/ref/hg19
        bgzip -f --threads ${threads} af-only-gnomad.raw.sites.b37.processed.vcf
        tabix -f af-only-gnomad.raw.sites.b37.processed.vcf.gz
        mamba deactivate
    fi
elif [ "${version_reference}" == "hg38" ]; then
    echo	"USE reference Version : ${version_reference}"
    if [ ! -f "/opt/ref/${version_reference}/af-only-gnomad.hg38.vcf.gz" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/Mutect2/af-only-gnomad.hg38.vcf.gz -d /opt/ref/${version_reference}
    else
        if [ -f "/opt/ref/${version_reference}/af-only-gnomad.hg38.vcf.gz.aria2" ]; then
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/Mutect2/af-only-gnomad.hg38.vcf.gz -c -d /opt/ref/${version_reference}
        fi
    fi
    if [ ! -f "/opt/ref/${version_reference}/af-only-gnomad.hg38.vcf.gz.tbi" ]; then
        aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/Mutect2/af-only-gnomad.hg38.vcf.gz.tbi -d /opt/ref/${version_reference}
    else
        if [ -f "/opt/ref/${version_reference}/af-only-gnomad.hg38.vcf.gz.tbi.aria2" ]; then
            aria2c -x 16 -s 32 ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/Mutect2/af-only-gnomad.hg38.vcf.gz.tbi -c -d /opt/ref/${version_reference}
        fi
    fi
fi


    
mamba activate ${pn}.gatk
if [ ! -f "/opt/ref/${version_reference}/${project_bed}.gz" ]; then
    bgzip -f -c /opt/ref/${version_reference}/${project_bed} > /opt/ref/hg19/${project_bed}.gz
    tabix -f -p bed /opt/ref/${version_reference}/${project_bed}.gz
else
    if [ ! -f "/opt/ref/${version_reference}/${project_bed}.gz.tbi" ]; then
        tabix -f -p bed /opt/ref/${version_reference}/${project_bed}.gz
    fi
fi
mamba deactivate

if [ ! -d "${envs}/${pn}.manta" ]; then
    mamba create -n ${pn}.manta -y manta=1.6.0
fi

mamba activate ${pn}.manta

rm -f ${result}/${sn}/sv/runWorkflow.py*
configManta.py  \
    --normalBam ${result}/${sn}/bqsr/${sn}_normal_bqsr.bam \
    --tumorBam  ${result}/${sn}/bqsr/${sn}_tumor_bqsr.bam \
    --referenceFasta /opt/ref/${version_reference}/${version_reference}.fasta \
    --exome \
    --callRegions /opt/ref/${version_reference}/${project_bed}.gz \
    --runDir ${result}/${sn}/sv

rm -rf ${result}/${sn}/sv/workspace
python ${result}/${sn}/sv/runWorkflow.py -m local -j ${threads} &

mamba deactivate

mamba activate ${pn}.gatk

if [ "${version_reference}" == hg19 ]; then
    echo	"USE reference Version : ${version_reference}"
    rm -f ${result}/${sn}/snv/vcf-file.list
    touch ${result}/${sn}/snv/vcf-file.list
    for i in `ls ${result}/${sn}/interval/*.interval_list`;
    do
        rm -f ${i%.*}_bqsr.vcf.gz
        gatk Mutect2 \
            -R /opt/ref/${version_reference}/${version_reference}.fasta \
            -I ${result}/${sn}/bqsr/${sn}_tumor_bqsr.bam  -tumor  ${sn}_tumor  \
            -I ${result}/${sn}/bqsr/${sn}_normal_bqsr.bam -normal ${sn}_normal \
            -L $i \
            -O ${i%.*}_bqsr.vcf.gz \
            --max-mnp-distance 10 \
            --germline-resource /opt/ref/${version_reference}/af-only-gnomad.raw.sites.b37.processed.vcf.gz \
            --native-pair-hmm-threads ${threads} &
        echo ${i%.*}_bqsr.vcf.gz >> ${result}/${sn}/snv/vcf-file.list
    done
elif [ "${version_reference}" == "hg38" ]; then
    echo	"USE reference Version : ${version_reference}"
    rm -f ${result}/${sn}/snv/vcf-file.list
    touch ${result}/${sn}/snv/vcf-file.list
    for i in `ls ${result}/${sn}/interval/*.interval_list`;
    do
        rm -f ${i%.*}_bqsr.vcf.gz
        gatk Mutect2 \
            -R /opt/ref/${version_reference}/${version_reference}.fasta \
            -I ${result}/${sn}/bqsr/${sn}_tumor_bqsr.bam  -tumor  ${sn}_tumor  \
            -I ${result}/${sn}/bqsr/${sn}_normal_bqsr.bam -normal ${sn}_normal \
            -L $i \
            -O ${i%.*}_bqsr.vcf.gz \
            --max-mnp-distance 10 \
            --germline-resource /opt/ref/${version_reference}/af-only-gnomad.hg38.vcf.gz \
            --native-pair-hmm-threads ${threads} &
        echo ${i%.*}_bqsr.vcf.gz >> ${result}/${sn}/snv/vcf-file.list
    done
fi
wait

    
rm -f ${result}/${sn}/snv/${sn}_bqsr.vcf.gz.stats
stats=
for z in `ls ${result}/${sn}/interval/*_bqsr.vcf.gz.stats`;
do
    stats="$stats -stats $z"
done

gatk MergeMutectStats $stats \
    -O ${result}/${sn}/snv/${sn}_bqsr.vcf.gz.stats

gatk MergeVcfs \
    -I ${result}/${sn}/snv/vcf-file.list \
    -O ${result}/${sn}/snv/${sn}_bqsr.vcf.gz
    
mamba deactivate

9. FilterMutectCalls 对Mutect结果突变过滤

bash
mamba activate ${pn}.gatk

gatk FilterMutectCalls \
    --max-events-in-region ${event} \
    --contamination-table ${result}/${sn}/stat/${sn}_contamination.table \
    -R /opt/ref/${version_reference}/${version_reference}.fasta \
    -V ${result}/${sn}/snv/${sn}_bqsr.vcf.gz \
    -O ${result}/${sn}/snv/${sn}_filtered.vcf.gz
    
mamba deactivate

10. 使用Vep注释过滤结果

bash
#conda检测环境是否存在,首次运行不存在创建该环境并安装软件
if [ ! -d "${envs}/${pn}.vep" ]; then
    echo "Creating the environment ${pn}.vep"
    mamba create -n ${pn}.vep -y ensembl-vep=${version_vep}
fi

mkdir -p /opt/result/${sn}/vcf

if [ "${version_reference}" == hg19 ]; then
echo	"USE reference Version : ${version_reference}"
    #检测vep注释数据库是否存在如果不存在则先下载
    if [ ! -d "/opt/ref/vep-cache/homo_sapiens/${version_vep_cache}_GRCh37" ]; then
        aria2c -x 16 -s 48 https://ftp.ensembl.org/pub/release-${version_vep_cache}/variation/indexed_vep_cache/homo_sapiens_vep_${version_vep_cache}_GRCh37.tar.gz -d /opt/ref/
        tar -zxvf /opt/ref/homo_sapiens_vep_${version_vep_cache}_GRCh37.tar.gz -C /opt/ref/vep-cache/
    elif [ -f "/opt/ref/homo_sapiens_vep_${version_vep_cache}_GRCh37.tar.gz.aria2" ]; then
        aria2c -x 16 -s 48 https://ftp.ensembl.org/pub/release-${version_vep_cache}/variation/indexed_vep_cache/homo_sapiens_vep_${version_vep_cache}_GRCh37.tar.gz -c -d /opt/ref/
        tar -zxvf /opt/ref/homo_sapiens_vep_${version_vep_cache}_GRCh37.tar.gz -C /opt/ref/vep-cache/
    fi

    if [ ! -d "/opt/ref/vep-cache/homo_sapiens_refseq/${version_vep_cache}_GRCh37" ]; then
        aria2c -x 16 -s 48 http://ftp.ensembl.org/pub/release-${version_vep_cache}/variation/vep/homo_sapiens_refseq_vep_${version_vep_cache}_GRCh37.tar.gz -d /opt/ref/
        tar -zxvf /opt/ref/homo_sapiens_refseq_vep_${version_vep_cache}_GRCh37.tar.gz -C /opt/ref/vep-cache/
    elif [ -f "/opt/ref/homo_sapiens_refseq_vep_${version_vep_cache}_GRCh37.tar.gz.aria2" ]; then
        aria2c -x 16 -s 48 http://ftp.ensembl.org/pub/release-${version_vep_cache}/variation/vep/homo_sapiens_refseq_vep_${version_vep_cache}_GRCh37.tar.gz -c -d /opt/ref/
        tar -zxvf /opt/ref/homo_sapiens_refseq_vep_${version_vep_cache}_GRCh37.tar.gz -C /opt/ref/vep-cache/
    fi
    
    mamba activate ${pn}.vep

    mkdir -p ${result}/${sn}/annotation
    vep \
        -i ${result}/${sn}/snv/${sn}_filtered.vcf.gz  \
        -o ${result}/${sn}/annotation/${sn}_filtered_vep.tsv \
        --offline \
        --cache \
        --cache_version ${version_vep_cache} \
        --everything \
        --dir_cache /opt/ref/vep-cache/ \
        --dir_plugins /opt/ref/vep-cache/Plugins \
        --species homo_sapiens \
        --assembly GRCh37 \
        --fasta /opt/ref/${version_reference}/${version_reference}.fasta \
        --refseq \
        --force_overwrite \
        --format vcf \
        --tab \
        --shift_3prime 1  \
        --offline

    mamba deactivate
    
elif [ "${version_reference}" == "hg38" ]; then
    echo	"USE reference Version : ${version_reference}"
    #检测vep注释数据库是否存在如果不存在则先下载
    if [ ! -d "/opt/ref/vep-cache/homo_sapiens/${version_vep_cache}_GRCh38" ]; then
        aria2c -x 16 -s 48 https://ftp.ensembl.org/pub/release-${version_vep_cache}/variation/indexed_vep_cache/homo_sapiens_vep_${version_vep_cache}_GRCh38.tar.gz -d /opt/ref/
        tar -zxvf /opt/ref/homo_sapiens_vep_${version_vep_cache}_GRCh38.tar.gz -C /opt/ref/vep-cache/
    elif [ -f "/opt/ref/homo_sapiens_vep_${version_vep_cache}_GRCh38.tar.gz.aria2" ]; then
        aria2c -x 16 -s 48 https://ftp.ensembl.org/pub/release-${version_vep_cache}/variation/indexed_vep_cache/homo_sapiens_vep_${version_vep_cache}_GRCh38.tar.gz -c -d /opt/ref/
        tar -zxvf /opt/ref/homo_sapiens_vep_${version_vep_cache}_GRCh38.tar.gz -C /opt/ref/vep-cache/
    fi

    if [ ! -d "/opt/ref/vep-cache/homo_sapiens_refseq/${version_vep_cache}_GRCh38" ]; then
        aria2c -x 16 -s 48 http://ftp.ensembl.org/pub/release-${version_vep_cache}/variation/vep/homo_sapiens_refseq_vep_${version_vep_cache}_GRCh38.tar.gz -d /opt/ref/
        tar -zxvf /opt/ref/homo_sapiens_refseq_vep_${version_vep_cache}_GRCh38.tar.gz -C /opt/ref/vep-cache/
    elif [ -f "/opt/ref/homo_sapiens_refseq_vep_${version_vep_cache}_GRCh38.tar.gz.aria2" ]; then
        aria2c -x 16 -s 48 http://ftp.ensembl.org/pub/release-${version_vep_cache}/variation/vep/homo_sapiens_refseq_vep_${version_vep_cache}_GRCh38.tar.gz -c -d /opt/ref/
        tar -zxvf /opt/ref/homo_sapiens_refseq_vep_${version_vep_cache}_GRCh38.tar.gz -C /opt/ref/vep-cache/
    fi
    
    mamba activate ${pn}.vep

    mkdir -p ${result}/${sn}/annotation
    vep \
        -i ${result}/${sn}/snv/${sn}_filtered.vcf.gz  \
        -o ${result}/${sn}/annotation/${sn}_filtered_vep.tsv \
        --offline \
        --cache \
        --cache_version ${version_vep_cache} \
        --everything \
        --dir_cache /opt/ref/vep-cache/ \
        --dir_plugins /opt/ref/vep-cache/Plugins \
        --species homo_sapiens \
        --assembly GRCh38 \
        --fasta /opt/ref/${version_reference}/${version_reference}.fasta \
        --refseq \
        --force_overwrite \
        --format vcf \
        --tab \
        --shift_3prime 1  \
        --offline

    mamba deactivate
fi

11. 使用脚本处理注释结果和过滤vcf结果,输出和室间质评要求格式的数据表格

bash
mamba activate ${pn}.cnvkit

if [ ! -f "${envs}/MatchedSnvVepAnnotationFilter.py" ]; then
    aria2c https://raw.githubusercontent.com/doujiangbaozi/sliverworkspace-util/main/somatic/MatchedSnvVepAnnotationFilter.py -d ${envs}/
    chmod a+x ${envs}/MatchedSnvVepAnnotationFilter.py
fi

${envs}/MatchedSnvVepAnnotationFilter.py \
    -e normal_artifact   \
    -e germline   \
    -i strand_bias   \
    -i clustered_events   \
    --min-vaf=${snv_vaf} \
    --min-tlod=${snv_tlod} \
    --min-depth=${snv_depth} \
    -v ${result}/${sn}/snv/${sn}_filtered.vcf.gz   \
    -a ${result}/${sn}/annotation/${sn}_filtered_vep.tsv   \
    -o ${result}/${sn}/annotation/${sn}.result.SNV.tsv

mamba deactivate

12. 使用cnvkit提供工具输出分布图和热图

bash
mamba activate ${pn}.cnvkit
    
    cnvkit.py scatter ${result}/${sn}/cnv/${sn}_tumor_marked.cnr \
    -s ${result}/${sn}/cnv/${sn}_tumor_marked.cns \
    -i ' ' \
    -n ${sn}_normal \
    -o ${result}/${sn}/cnv/${sn}_cnv_scatter.png -t  &&

cnvkit.py heatmap ${result}/${sn}/cnv/${sn}_tumor_marked.cns \
-o ${result}/${sn}/cnv/${sn}_cnv_heatmap.png

mamba deactivate

13. 使用cnvkit call 根据cnvkit batch输出结果推算拷贝数

bash
mamba activate ${pn}.cnvkit

cnvkit.py call ${result}/${sn}/cnv/${sn}_tumor_marked.cns \
	-o ${result}/${sn}/cnv/${sn}_tumor_marked.call.cns

mamba deactivate

14. 编写脚本处理cnvkit输出,计算cnv基因,exon位置,gain/lost,cn数

bash
mamba activate ${pn}.cnvkit

if [ ! -f "${envs}/CnvAnnotationFilter.py" ]; then
    aria2c https://raw.githubusercontent.com/doujiangbaozi/sliverworkspace-util/main/somatic/CnvAnnotationFilter.py -d ${envs}/
    chmod a+x ${envs}/CnvAnnotationFilter.py
fi

if [ ! -f "/opt/ref/${version_reference}/refGene.txt" ]; then
    aria2c -x 16 -s 16 http://hgdownload.cse.ucsc.edu/goldenPath/${version_reference}/database/refGene.txt.gz -d /opt/ref/${version_reference} -o refGene.txt.gz
    cd /opt/ref/${version_reference} && gzip -d refGene.txt.gz
fi

python ${envs}/CnvAnnotationFilter.py  \
    -r /opt/ref/${version_reference}/refGene.txt \
    -i ${cnv_min} \
    -x ${cnv_max} \
    -D ${cnv_dep} \
    -f ${result}/${sn}/cnv/${sn}_tumor_marked.call.cns \
    -o ${result}/${sn}/cnv/${sn}.result.CNV.tsv

mamba deactivate

15. 编写脚本处理manta的输出,获取最终sv输出结果,起始位置,基因、频率等

bash
mamba activate ${pn}.cnvkit

if [ ! -f "${envs}/SvAnnotationFilter.py" ]; then
    aria2c https://raw.githubusercontent.com/doujiangbaozi/sliverworkspace-util/main/somatic/SvAnnotationFilter.py -d ${envs}/
    chmod a+x ${envs}/SvAnnotationFilter.py
fi

if [ ! -f "/opt/ref/${version_reference}/refGene.txt" ]; then
    aria2c -x 16 -s 16 http://hgdownload.cse.ucsc.edu/goldenPath/${version_reference}/database/refGene.txt.gz -d /opt/ref/${version_reference} -o refGene.txt.gz
    cd /opt/ref/${version_reference} && gzip -d refGene.txt.gz
fi

${envs}/SvAnnotationFilter.py \
    -r /opt/ref/${version_reference}/refGene.txt \
    -s ${sv_score} \
    -f ${result}/${sn}/sv/results/variants/somaticSV.vcf.gz \
    -o ${result}/${sn}/sv/${sn}.result.SV.tsv

mamba deactivate

16. 根据之前fastp,samtools depth,samtools flagstat,gatk CollectInsertSizeMetrics等输出,给出综合 QC数据

bash
mamba activate ${pn}.cnvkit

if [ ! -f "${envs}/MatchedQcProcessor.py" ]; then
    aria2c https://raw.githubusercontent.com/doujiangbaozi/sliverworkspace-util/main/somatic/MatchedQcProcessor.py -d ${envs}/
    chmod a+x ${envs}/MatchedQcProcessor.py
fi

${envs}/MatchedQcProcessor.py  --bed /opt/ref/${version_reference}/${project_bed} \
    --out ${result}/${sn}/stat/${sn}.result.QC.tsv \
    --sample-fastp=${result}/${sn}/trimmed/${sn}_tumor_fastp.json \
    --sample-depth=${result}/${sn}/stat/${sn}_tumor_marked.depth \
    --sample-flagstat=${result}/${sn}/stat/${sn}_tumor_marked.flagstat \
    --sample-insertsize=${result}/${sn}/stat/${sn}_tumor_insertsize_metrics.txt \
    --normal-fastp=${result}/${sn}/trimmed/${sn}_normal_fastp.json \
    --normal-depth=${result}/${sn}/stat/${sn}_normal_marked.depth \
    --normal-flagstat=${result}/${sn}/stat/${sn}_normal_marked.flagstat  \
    --normal-insertsize=${result}/${sn}/stat/${sn}_normal_insertsize_metrics.txt

mamba deactivate

mamba activate ${pn}.fastp

multiqc ${result}/${sn}/ -f -o ${result}/${sn}/qc

mamba deactivate

最终输出

文件名备注
report.pdf由sliverworkspace设计的图形化分析报告打印输出为PDF
1701/qc/multiqc_report.htmlmultiqc聚合报告
1701/1701.result.SNV.tsvSNV最终突变结果
1701/1701/cnv/1701_cnv_heatmap.pngCNV结果热图
1701/cnv/1701_cnv_scatter.pngCNV结果分布图
1701/cnv/1701.result.CNV.tsvCNV最终结果
1701.result.SV.tsvSV最终结果
1701.result.QC.tsv最终质控结果
1701/qc/multiqc_report.htmlmultiqc报告