天真的我準備把全部流程遷移到GATK4

我在生信技能樹上面發佈的GATK4教程也有不少了 本着儘量使用最新版軟件的原則，也準備把之前的gatk對RNA-seq數據找變異的流程進行轉換：

$GATK  --java-options "-Xmx25G -Djava.io.tmpdir=./" AddOrReplaceReadGroups \
    -I $id -O ${sample}_right.bam -SO coordinate -ID ${sample}  -LB rna \
    -PL illumina -PU hiseq  -SM ${sample}

    $GATK  --java-options "-Xmx25G -Djava.io.tmpdir=./"  MarkDuplicates \
    -I ${sample}_right.bam -O ${sample}_marked.bam -M $sample.metrics --REMOVE_DUPLICATES TRUE

    $GATK  --java-options "-Xmx25G -Djava.io.tmpdir=./"  FixMateInformation \
    -I ${sample}_marked.bam -O ${sample}_marked_fixed.bam  -SO coordinate 

    $GATK  --java-options "-Xmx25G -Djava.io.tmpdir=./"  SplitNCigarReads \
    -R $GENOME  -I ${sample}_marked_fixed.bam  -O ${sample}_marked_fixed_split.bam \
    -rf ReassignOneMappingQuality -RMQF 255 -RMQT 60 -U ALLOW_N_CIGAR_READS

    #--fix_misencoded_quality_scores
    ## --fix_misencoded_quality_scores only if phred 64

但是走到了 SplitNCigarReads 才發現，這個命令當初學的太久了，忘記各個參數啥意思了，就想搜索看看如何轉換。

還真發現了有人問同樣的問題，GATK4: How to reassign STAR mapping quality from 255 to 60 with SplitNCigarReads ，而且GATK4開發團隊也回答了：EDIT: Geraldine responded here.

但是這是一個否定回答，開發團隊讓我們回去用GATK3來跑流程。

One risk that I see is that using the STAR --outSAMmapqUnique 60 option maybe fixes the issue with GATK, but that other downstream tools maybe still depend on the (still default) STAR mapping quality value of 255 (e.g. cufflinks).

The mapping quality MAPQ (column 5) is 255 for uniquely mapping reads, and int(-10*log10(1- 1/Nmap)) for multi-mapping reads. This scheme is same as the one used by TopHat and is com- patible with Cuffinks. The default MAPQ=255 for the unique mappers maybe changed with --outSAMmapqUnique parameter (integer 0 to 255) to ensure compatibility with downstream tools such as GATK.

https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf

好吧，回去就回去，gatk3代碼是：

module load java/1.8.0_91 
GATK=/home/jianmingzeng/biosoft/GATK/GenomeAnalysisTK.jar
PICARD=/home/jianmingzeng/biosoft/picardtools/2.9.2/picard.jar 
GENOME=/home/jianmingzeng/biosoft/GATK/resources/bundle/hg38/Homo_sapiens_assembly38.fasta
INDEX=/home/jianmingzeng/biosoft/GATK/resources/bundle/hg38/bwa_index/gatk_hg38 
DBSNP=/home/jianmingzeng/biosoft/GATK/resources/bundle/hg38/dbsnp_146.hg38.vcf.gz
kgSNP=/home/jianmingzeng/biosoft/GATK/resources/bundle/hg38/1000G_phase1.snps.high_confidence.hg38.vcf.gz
kgINDEL=/home/jianmingzeng/biosoft/GATK/resources/bundle/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz

TMPDIR=/home/jianmingzeng/tmp/software
## samtools and bwa are in the environment
## samtools Version: 1.3.1 (using htslib 1.3.1)
## bwa Version: 0.7.15-r1140
cat $1 |while read id
do
    echo $id
    file=$(basename $id )
    sample=${file%%_*}
    echo $sample
    java -Djava.io.tmpdir=$TMPDIR    -Xmx25g -jar $PICARD  AddOrReplaceReadGroups \
    I=$id O=${sample}.bam SO=coordinate RGID=${sample}  RGLB=rna \
    RGPL=illumina RGPU=hiseq  RGSM=${sample}
    java -Djava.io.tmpdir=$TMPDIR    -Xmx25g -jar $PICARD MarkDuplicates \
    INPUT=${sample}.bam OUTPUT=${sample}_marked.bam METRICS_FILE=$sample.metrics REMOVE_DUPLICATES=TRUE
    java -Djava.io.tmpdir=$TMPDIR    -Xmx25g -jar $PICARD FixMateInformation \
    INPUT=${sample}_marked.bam OUTPUT=${sample}_marked_fixed.bam SO=coordinate

    samtools index ${sample}_marked_fixed.bam
    java -Djava.io.tmpdir=$TMPDIR   -Xmx25g -jar $GATK -T SplitNCigarReads \
    -R $GENOME  -I ${sample}_marked_fixed.bam  -o ${sample}_marked_fixed_split.bam \
    -rf ReassignOneMappingQuality -RMQF 255 -RMQT 60 -U ALLOW_N_CIGAR_READS
    #--fix_misencoded_quality_scores
    ## --fix_misencoded_quality_scores only if phred 64
    java -Djava.io.tmpdir=$TMPDIR   -Xmx25g -jar $GATK -T HaplotypeCaller  \
    -R $GENOME -I ${sample}_marked_fixed_split.bam --dbsnp $DBSNP  \
    -stand_emit_conf 10 -o  ${sample}_raw.vcf
    rm ${sample}.bam ${sample}_marked.bam ${sample}_marked_fixed.bam ${sample}_marked_fixed_split.bam
done

純乾貨代碼，誰學誰獲益。