流程输入 | SRR22216743_1.fastq.gz SRR22216743_2.fastq.gz 测试数据下载 NYC SARS-CoV-2 genome sequencing from human nasopharyngeal swabs** 1 ILLUMINA (Illumina HiSeq 2000) run: 28,721 spots, 5.8M bases, 1.8Mb downloads 使用NCBI官方工具sra-toolkit拆分成fastq.gz文件 fastq-dump SRR22216743 --split-3 --gzip 得到SRR22216743_1.fastq.gz SRR22216743_2.fastq.gz 参考文件,默认路径/opt/ref下 Artic-ncov2019 artic-ncov2019 primer&参考序列 GCF_009858895.2_ASM985889v3_genomic.gff GFF文件 分析流程文件(可一键导入sliverworkspace运行)及报告文件,conda环境文件下载,导入操作 |
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运行环境 | docker image based on ubuntu21.04 Conda Mamba(默认使用清华源) ssh |
分析软件 | - fastp=0.23.2 - fastqc=0.11.9 - multiqc=1.13 - bwa=0.7.17 - minimap2=2.24 - samtools=1.16.1 - ivar=1.3.1 - quast=5.2.0 - pangolin=4.1.3 |
输出结果 | multiqc_report.html 测序数据trim前后质量数据 SRR22216743.samcov.tsv reads数量,覆盖度,测序深度,baseQ mapQ quast输出分析结果:alignment_viewer.html contig_size_viewer.html (拼接序列情况)report.html 按照序列一致性组装的新冠病毒序列 SRR22216743.consensus.fa Panglin 根据组装的序列分析得出病毒分型信息 lineage_report.csv samtools,ivar 根据primertrim.bam获的新冠病毒突变信息,过滤后得到 SRR22216743_variantsfiltered.tsv |
备注:docker运行的操作系统,推荐为Linux,windows,macOS系统改下docker可能部分功能(网络)不能正常运行
# 拉取docker镜像
docker pull doujiangbaozi/sliverworkspace:latest
# 查看docker 镜像
docker images
version: "3"
services:
SarsCov2:
image: doujiangbaozi/sliverworkspace:latest
container_name: SarsCov2
volumes:
- /media/sliver/Data/data:/opt/data:rw #挂载原始数据,放SC2目录下
- /media/sliver/Manufacture/SC2/envs:/root/mambaforge-pypy3/envs:rw #挂载envs conda环境目录
- /media/sliver/Manufacture/SC2/config:/opt/config:rw #挂载config conda配置文件目录
- /media/sliver/Manufacture/SC2/ref:/opt/ref:rw #挂载reference目录
- /media/sliver/Manufacture/SC2/result:/opt/result:rw #挂载中间文件和输出结果目录
ports:
- "9024:9024" #ssh连接端口可以按需修改
environment:
- TZ=Asia/Shanghai #设置时区
- PS=20191124 #修改默认ssh密码
- PT=9024 #修改默认ssh端口
基础环境运行
# docker-compose.yml 所在目录下运行
docker-compose up -d
# 或者
docker-compose up -d -f /路径/docker-compose.yaml
# 查看docker是否正常运行,docker-compose.yaml目录下运行
docker-compose ps
# 或者
docker ps
docker 容器使用,类似于登录远程服务器
# 登录docker,使用的是ssh服务,可以本地或者远程部署使用
ssh root@192.168.6.6 -p9024
# 看到如下,显示如下提示即正常登录
(base) root@SliverWorkstation:~#
#样本编号
export sn=SRR22216743
#数据输入目录
export data=/opt/data
#数据输出、中间文件目录
export result=/opt/result
#conda安装的环境目录
export envs=/root/mambaforge-pypy3/envs
#artic primer 版本V1,V2,V3,V4,V4.1
export artic_primer_version=V4.1
#设置可用线程数
export threads=8
#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}/qc" ]; then
mamba env create -f /opt/config/qc.yaml
fi
source activate qc
mkdir -p ${result}/${sn}/clean mkdir -p ${result}/${sn}/qc
fastqc ${data}/SC2/${sn}_1.fastq.gz ${data}/SC2/${sn}_2.fastq.gz -o ${result}/${sn}/qc
fastp -i ${data}/SC2/${sn}_1.fastq.gz -I ${data}/SC2/${sn}_2.fastq.gz \
-o ${result}/${sn}/clean/${sn}_1_clean.fastq.gz -O ${result}/${sn}/clean/${sn}_2_clean.fastq.gz \
-h ${result}/${sn}/qc/${sn}_fastp.html -j ${result}/${sn}/qc/${sn}_fastp.json
fastqc ${result}/${sn}/clean/${sn}_1_clean.fastq.gz ${result}/${sn}/clean/${sn}_2_clean.fastq.gz \
-o ${result}/${sn}/qc
#汇总一下之前结果,得到一个总体报告
multiqc ${result}/${sn}/qc/ -f -o ${result}/${sn}/qc
conda activate
mkdir -p ${result}/${sn}/aligned
if [ ! -d "/opt/ref/artic-ncov2019" ]; then
apt-get install -y git
git clone https://github.com/artic-network/artic-ncov2019.git "/opt/ref/artic-ncov2019"
fi
#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 "${envs}/SC2" ]; then
mamba env create -f /opt/config/SC2.yaml
fi
source activate SC2
#如果没有索引,创建索引
if [ ! -f /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta.amb ] ||
[ ! -f /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta.ann ] ||
[ ! -f /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta.bwt ] ||
[ ! -f /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta.pac ] ||
[ ! -f /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta.sa ]; then
if [ ! -f /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta ]; then
cp -f /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/SARS-CoV-2.reference.fasta \
/opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta
fi
bwa index /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta
fi
bwa mem -t ${threads} \
/opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta \
${result}/${sn}/clean/${sn}_1_clean.fastq.gz ${result}/${sn}/clean/${sn}_2_clean.fastq.gz | \
samtools sort | samtools view -F 4 -o ${result}/${sn}/aligned/${sn}.sorted.bam
samtools index ${result}/${sn}/aligned/${sn}.sorted.bam
mv ${result}/${sn}/aligned/${sn}.sorted.bam.bai ${result}/${sn}/aligned/${sn}.sorted.bai
conda deactivate
source activate SC2
if [ ! -f /opt/ref/artic-ncov2019/ARTIC-${artic_primer_version}.bed ]; then
if [ ! -f /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.scheme.bed ]; then
cp -r /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/SARS-CoV-2.scheme.bed \
/opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.scheme.bed
fi
perl -ne 'my @x=split m/\t/; print join("\t",@x[0..3], 60, $x[3]=~m/LEFT/?"+":"-"),"\n";' \
< /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.scheme.bed \
> /opt/ref/artic-ncov2019/ARTIC-${artic_primer_version}.bed
fi
ivar trim -e -i ${result}/${sn}/aligned/${sn}.sorted.bam \
-b /opt/ref/artic-ncov2019/ARTIC-${artic_primer_version}.bed \
-p ${result}/${sn}/aligned/${sn}.primertrim
conda deactivate
source activate SC2
samtools sort ${result}/${sn}/aligned/${sn}.primertrim.bam \
-o ${result}/${sn}/aligned/${sn}.primertrim.sorted.bam
conda deactivate
source activate SC2
samtools mpileup -A -d 1000 -B -Q 0 \
--reference /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta \
${result}/${sn}/aligned/${sn}.primertrim.sorted.bam | ivar consensus -p ${result}/${sn}/${sn}.consensus -n N -i ${sn}
grep -v ">" ${result}/${sn}/${sn}.consensus.fa | grep -o -E "C|A|T|G" | wc -l
conda deactivate
#conda检测环境是否存在,首次运行不存在创建该环境并安装软件
if [ ! -d "${envs}/pangolin" ]; then
mamba env create -f /opt/config/pangolin.yaml
fi
source activate pangolin
pangolin ${result}/${sn}/${sn}.consensus.fa --outdir ${result}/${sn}
conda deactivate
source activate SC2
if [ ! -f "/opt/ref/GCF_009858895.2_ASM985889v3_genomic.gff" ]; then
aria2c https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/009/858/895/GCF_009858895.2_ASM985889v3/GCF_009858895.2_ASM985889v3_genomic.gff.gz -d /opt/ref
gzip -f -d /opt/ref/GCF_009858895.2_ASM985889v3_genomic.gff.gz
fi
samtools mpileup -aa -A -d 0 -B -Q 0 \
--reference /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta \
${result}/${sn}/aligned/${sn}.primertrim.sorted.bam | \
ivar variants -p ${result}/${sn}/${sn}_variants -q 30 -t 0.01 -m 0 \
-r /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta \
-g /opt/ref/GCF_009858895.2_ASM985889v3_genomic.gff
ivar filtervariants -p ${result}/${sn}/${sn}_variantsfiltered ${result}/${sn}/${sn}_variants.tsv
conda deactivate
source activate SC2
if [ ! -f "/opt/ref/GCF_009858895.2_ASM985889v3_genomic.gff" ]; then
aria2c https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/009/858/895/GCF_009858895.2_ASM985889v3/GCF_009858895.2_ASM985889v3_genomic.gff.gz -d /opt/ref
gzip -f -d /opt/ref/GCF_009858895.2_ASM985889v3_genomic.gff.gz
fi
quast ${result}/${sn}/${sn}.consensus.fa \
-r /opt/ref/artic-ncov2019/primer_schemes/nCoV-2019/${artic_primer_version}/nCoV-2019.reference.fasta \
--features /opt/ref/GCF_009858895.2_ASM985889v3_genomic.gff \
--ref-bam ${result}/${sn}/aligned/${sn}.sorted.bam \
--output-dir ${result}/${sn}/quast
samtools coverage ${result}/${sn}/aligned/${sn}.sorted.bam -o ${result}/${sn}/aligned/${sn}.samcov.tsv
conda deactivate
https://github.com/artic-network/artic-ncov2019
https://github.com/CDCgov/SARS-CoV-2_Sequencing/tree/master/protocols/BFX-UT_ARTIC_Illumina
原创声明:本文系作者授权腾讯云开发者社区发表,未经许可,不得转载。
如有侵权,请联系 cloudcommunity@tencent.com 删除。
原创声明:本文系作者授权腾讯云开发者社区发表,未经许可,不得转载。
如有侵权,请联系 cloudcommunity@tencent.com 删除。