2020-07-17靶向捕获测序数据分析记录4

作者: 程凉皮儿 | 来源:发表于2020-07-17 21:16 被阅读0次

2020-07-17靶向捕获测序数据分析记录4
NGS011 靶向测序方法分类
2020-07-14 靶向捕获测序数据分析记录2
2020-07-16 靶向捕获测序数据分析记录3
2020-07-13 靶向捕获测序数据分析记录1
2020-07-23 靶向捕获测序数据分析记录5
bamdst安装及使用
肿瘤外显子全流程notes
外显子测序简介及分析流程
NGS021 CRISPR靶向捕获测序

查看下后台运行的程序情况：

(base) root@1100150:~# ll ~/project/2.gatk/|grep '_bqsr.bam'|wc
    231    2079   12613
(base) root@1100150:~# ll ~/project/0.bwa/|grep 'ok'|wc
    250    2250   17146

已经成功转换的bam文件有250个，其中231个已经完成碱基质量值矫正，
下一步则是HaplotypeCaller，参考之前的练习https://www.jianshu.com/p/6ae5af0794a7
还是先写脚本calling.sh：

## start the gatk analysis
GATK=~/biosoft/gatk-4.1.7.0/gatk
#references
ref=~/reference/genome/Homo_sapiens_assembly38.fasta
cat config  | while read id
do
    if [ ! -f ~/project/2.gatk/gvcf/${id}.HC.g.vcf.gz]
    then
        time $samtools index ~/project/2.gatk/${id}_bqsr.bam 
        echo "** ${id}.sorted.marked.fixed.bqsr.bam index done **"
        
        time $GATK --java-options "-Xmx20G -Djava.io.tmpdir=./tmp" HaplotypeCaller \
        --emit-ref-confidence GVCF \
        -R $ref  \
        -I ~/project/2.gatk/${id}_bqsr.bam \
        -O ~/project/2.gatk/gvcf/${id}.HC.g.vcf.gz \
        1>~/project/2.gatk/${id}_log.HC 2>&1
        
        echo "** GVCF ${id}.HC.g.vcf.gz done **"
    fi
done

同样nohup bash calling.sh &提交到后台。
下一步则是分步call SNP,Indel:脚本预计内容如下

# VQSR
# first SNP mode 分别评估SNP和INDEL突变位点的质量
# SNP mode
samtools=samtools
GATK=~/biosoft/gatk-4.1.7.0/gatk

#references
ref=~/reference/genome/Homo_sapiens_assembly38.fasta
gatk_ref=~/reference/genome/Homo_sapiens_assembly38.fasta
gatk_bundle=~/annotation/variation/GATK

dbsnp=$gatk_bundle/dbsnp_146.hg38.vcf.gz
indel=$gatk_bundle/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz
G1000=$gatk_bundle/1000G_phase1.snps.high_confidence.hg38.vcf.gz
hapmap=$gatk_bundle/hapmap_3.3.hg38.vcf.gz
omini=$gatk_bundle/1000G_omni2.5.hg38.vcf.gz
mills=$gatk_bundle/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz
cat config  | while read id
do
    if [ ! -f ~/project/2.gatk/gvcf/${id}.HC.snps.recal]
    then
        time $GATK VariantRecalibrator \
        -R $ref \
        -V ~/project/2.gatk/gvcf/${id}.HC.g.vcf.gz \
        --max-gaussians 4 \
        -resource:hapmap,known=false,training=true,truth=true,prior=15.0 $hapmap \
        -resource:omini,known=false,training=true,truth=false,prior=12.0 $omini \
        -resource:1000G,known=false,training=true,truth=false,prior=10.0 $G1000 \
        -resource:snp,known=true,training=false,truth=false,prior=10.0 $dbsnp \
        -an DP -an QD -an SOR -an ReadPosRankSum -an MQRankSum \
        -mode SNP \
        --rscript-file ~/project/2.gatk/gvcf/${id}.HC.snps.plots.R \
        --tranches-file ~/project/2.gatk/gvcf/${id}.HC.snps.tranches \
        -O ~/project/2.gatk/gvcf/${id}.HC.snps.recal

        time $GATK ApplyVQSR \
        -R $ref \
        -V ~/project/2.gatk/gvcf/${id}.HC.g.vcf.gz \
        --truth-sensitivity-filter-level 99.0 \
        --tranches-file ~/project/2.gatk/gvcf/${id}.HC.snps.tranches \
        --recal-file ~/project/2.gatk/gvcf/${id}.HC.snps.recal \
        -mode SNP \
        -O ~/project/2.gatk/gvcf/${id}.HC.snps.VQSR.vcf.gz && echo "** SNPs VQSR done **"

## Indel mode
        time $GATK VariantRecalibrator \
        -R $ref \
        -V ~/project/2.gatk/gvcf/${id}.HC.g.vcf.gz \
        --max-gaussians 6 \
        -resource:mills,known=false,training=true,truth=true,prior=15.0 $mills \
        -an QD -an MQ -an MQRankSum -an ReadPosRankSum -an FS -an SOR \
        -mode INDEL \
        --rscript-file ~/project/2.gatk/gvcf/${id}.HC.snps.indels.plots.R \
        --tranches-file ~/project/2.gatk/gvcf/${id}.HC.snps.indels.tranches \
        -O ~/project/2.gatk/gvcf/${id}.HC.snps.indels.recal

        time $GATK ApplyVQSR \
        -R $ref \
        -V ~/project/2.gatk/gvcf/${id}.HC.snps.VQSR.vcf.gz \
        --truth-sensitivity-filter-level 99.0 \
        --tranches-file ~/project/2.gatk/gvcf/${id}.HC.snps.indels.tranches \
        --recal-file ~/project/2.gatk/gvcf/${id}.HC.snps.indels.recal \
        -mode INDEL \
        -O ~/project/2.gatk/gvcf/${id}.HC.snps.indels.VQSR.vcf.gz && echo "** SNPs and Indels VQSR $sample done **"
    fi
done

不知道需要运行多久，先不提交，先看前一步的结果如何。
先记录下，这个时间2020-07-17 21：19分时的结果：

(base) root@1100150:~# ll ~/project/0.bwa/|grep 'ok'|wc
    252    2268   17284
(base) root@1100150:~# ll ~/project/2.gatk/|grep '_bqsr.bam'|wc
    231    2079   12613
(base) root@1100150:~# ll ~/project/2.gatk/gvcf/
total 44
drwxr-xr-x 2 root root    2 Jul 10 00:43 ./
drwxr-xr-x 3 root root 1725 Jul 17 12:22 ../

2020-07-19更新查看样本情况

(wes) root@1100150:~/project# l ~/project/2.gatk/gvcf/
(wes) root@1100150:~/project# ll ~/project/2.gatk/|grep '_bqsr.bam'|wc
    255    2295   13931
(wes) root@1100150:~/project# ll ~/project/0.bwa/|grep 'ok'|wc
    461    4149   31681
(wes) root@1100150:~/project# date
Sun Jul 19 11:23:23 UTC 2020