开箱即用版本 满分室间质评之GATK Somatic SNV+Indel+CNV+SV（2024-04-30更新）

2024-05-03 21:21:57 浏览数 (1)

本次更新内容：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

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。

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项目地址导入操作

导入操作

分析流程整体概览

docker 镜像拉取及部署配置

代码语言：javascript复制
 # 拉取docker镜像
 docker     pull     doujiangbaozi/sliverworkspace:1.10

docker-compose.yml配置文件

代码语言：javascript复制
 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

代码语言：javascript复制
 # 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.gz

4.85G

07d3cdccee41dbb3adf5d2e04ab28e5b

B1701_R2.fq.gz

4.77G

c2aa4a8ab784c77423e821b9f7fb00a7

B1701NC_R1.fq.gz

3.04G

4fc21ad05f9ca8dc93d2749b8369891b

B1701NC_R2.fq.gz

3.11G

bc64784f2591a27ceede1727136888b9

变量名称：

变量名

变量值

备注

sn

1701

样本编号

pn

GS03

pipeline 代号 GATK Somatic 03版本

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 / 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

vep 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

Pipeline/workflow 具体步骤：

1. fastp 默认参数过滤，看下原始数据质量，clean data

代码语言：javascript复制
 #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

代码语言：javascript复制
 #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

代码语言：javascript复制
 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)

代码语言：javascript复制
 #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

代码语言：javascript复制
 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。

代码语言：javascript复制
 #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

代码语言：javascript复制
 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

代码语言：javascript复制
 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

代码语言：javascript复制
 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

代码语言：javascript复制
 #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}_GRCh37" ]; then
         aria2c -x 16 -s 48 https://ftp.ensembl.org/pub/release-${version_vep}/variation/indexed_vep_cache/homo_sapiens_vep_${version_vep}_GRCh37.tar.gz -d /opt/ref/
         tar -zxvf /opt/ref/homo_sapiens_vep_${version_vep}_GRCh37.tar.gz -C /opt/ref/vep-cache/
     elif [ -f "/opt/ref/homo_sapiens_vep_${version_vep}_GRCh37.tar.gz.aria2" ]; then
         aria2c -x 16 -s 48 https://ftp.ensembl.org/pub/release-${version_vep}/variation/indexed_vep_cache/homo_sapiens_vep_${version_vep}_GRCh37.tar.gz -c -d /opt/ref/
         tar -zxvf /opt/ref/homo_sapiens_vep_${version_vep}_GRCh37.tar.gz -C /opt/ref/vep-cache/
     fi
 ​
     if [ ! -d "/opt/ref/vep-cache/homo_sapiens_refseq/${version_vep}_GRCh37" ]; then
         aria2c -x 16 -s 48 http://ftp.ensembl.org/pub/release-${version_vep}/variation/vep/homo_sapiens_refseq_vep_${version_vep}_GRCh37.tar.gz -d /opt/ref/
         tar -zxvf /opt/ref/homo_sapiens_refseq_vep_${version_vep}_GRCh37.tar.gz -C /opt/ref/vep-cache/
     elif [ -f "/opt/ref/homo_sapiens_refseq_vep_${version_vep}_GRCh37.tar.gz.aria2" ]; then
         aria2c -x 16 -s 48 http://ftp.ensembl.org/pub/release-${version_vep}/variation/vep/homo_sapiens_refseq_vep_${version_vep}_GRCh37.tar.gz -c -d /opt/ref/
         tar -zxvf /opt/ref/homo_sapiens_refseq_vep_${version_vep}_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 ${version_vep} 
         --everything 
         --dir_cache /opt/ref/vep-cache/ 
         --dir_plugins /opt/ref/vep-cache/Plugins 
         --species homo_sapiens 
         --assembly GRCh37 
         --fasta /opt/ref/${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}_GRCh38" ]; then
         aria2c -x 16 -s 48 https://ftp.ensembl.org/pub/release-${version_vep}/variation/indexed_vep_cache/homo_sapiens_vep_${version_vep}_GRCh38.tar.gz -d /opt/ref/
         tar -zxvf /opt/ref/homo_sapiens_vep_${version_vep}_GRCh38.tar.gz -C /opt/ref/vep-cache/
     elif [ -f "/opt/ref/homo_sapiens_vep_${version_vep}_GRCh38.tar.gz.aria2" ]; then
         aria2c -x 16 -s 48 https://ftp.ensembl.org/pub/release-${version_vep}/variation/indexed_vep_cache/homo_sapiens_vep_${version_vep}_GRCh38.tar.gz -c -d /opt/ref/
         tar -zxvf /opt/ref/homo_sapiens_vep_${version_vep}_GRCh38.tar.gz -C /opt/ref/vep-cache/
     fi
 ​
     if [ ! -d "/opt/ref/vep-cache/homo_sapiens_refseq/${version_vep}_GRCh38" ]; then
         aria2c -x 16 -s 48 http://ftp.ensembl.org/pub/release-${version_vep}/variation/vep/homo_sapiens_refseq_vep_${version_vep}_GRCh38.tar.gz -d /opt/ref/
         tar -zxvf /opt/ref/homo_sapiens_refseq_vep_${version_vep}_GRCh38.tar.gz -C /opt/ref/vep-cache/
     elif [ -f "/opt/ref/homo_sapiens_refseq_vep_${version_vep}_GRCh38.tar.gz.aria2" ]; then
         aria2c -x 16 -s 48 http://ftp.ensembl.org/pub/release-${version_vep}/variation/vep/homo_sapiens_refseq_vep_${version_vep}_GRCh38.tar.gz -c -d /opt/ref/
         tar -zxvf /opt/ref/homo_sapiens_refseq_vep_${version_vep}_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 ${version_vep} 
         --everything 
         --dir_cache /opt/ref/vep-cache/ 
         --dir_plugins /opt/ref/vep-cache/Plugins 
         --species homo_sapiens 
         --assembly GRCh37 
         --fasta /opt/ref/${version_reference}/${version_reference}.fasta 
         --refseq 
         --force_overwrite 
         --format vcf 
         --tab 
         --shift_3prime 1  
         --offline
 ​
     mamba deactivate
 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

代码语言：javascript复制
 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

代码语言：javascript复制
 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

代码语言：javascript复制
 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

代码语言：javascript复制
 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

代码语言：javascript复制
 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

代码语言：javascript复制
 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.html

multiqc聚合报告

1701/1701.result.SNV.tsv

SNV最终突变结果

1701/1701/cnv/1701_cnv_heatmap.png

CNV结果热图

1701/cnv/1701_cnv_scatter.png

CNV结果分布图

1701/cnv/1701.result.CNV.tsv

CNV最终结果

1701.result.SV.tsv

SV最终结果

1701.result.QC.tsv

最终质控结果

1701/qc/multiqc_report.html

multiqc报告

生物基因生物信息学 GATK 分析流程 开箱即用

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