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commands.sh
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export PICARD_JAR=/usr/local/bin/picard.jar
export SNPEFF_HOME=/usr/local/src/snpEff/
export GATK_JAR=/usr/local/bin/GenomeAnalysisTK.jar
export BVATOOLS_JAR=/usr/local/bin/bvatools-1.6-full.jar
export TRIMMOMATIC_JAR=/usr/local/bin/trimmomatic-0.35.jar
export REF=/home/mBourgey/kyoto_workshop_WGS_2015/references/
cd $HOME
rsync -avP /home/reveleigh/cleanCopy/ $HOME/workshop
cd $HOME/workshop/
zless -S raw_reads/NA12878/runSRR_1/NA12878.SRR.33.pair1.fastq.gz
zcat raw_reads/NA12878/runSRR_1/NA12878.SRR.33.pair1.fastq.gz | head -n4
zcat raw_reads/NA12878/runSRR_1/NA12878.SRR.33.pair2.fastq.gz | head -n4
zgrep -c "^@SRR" raw_reads/NA12878/runSRR_1/NA12878.SRR.33.pair1.fastq.gz
zgrep -c "^@" raw_reads/NA12878/runSRR_1/NA12878.SRR.33.pair1.fastq.gz
mkdir originalQC/
java -Xmx1G -jar ${BVATOOLS_JAR} readsqc \
--read1 raw_reads/NA12878/runSRR_1/NA12878.SRR.33.pair1.fastq.gz \
--read2 raw_reads/NA12878/runSRR_1/NA12878.SRR.33.pair2.fastq.gz \
--threads 2 --regionName SRR --output originalQC/
java -Xmx1G -jar ${BVATOOLS_JAR} readsqc \
--read1 raw_reads/NA12878/runERR_1/NA12878.ERR.33.pair1.fastq.gz \
--read2 raw_reads/NA12878/runERR_1/NA12878.ERR.33.pair2.fastq.gz \
--threads 2 --regionName ERR --output originalQC/
scp -r <USER>@www.genome.med.kyoto-u.ac.jp:~/workshop/originalQC/ ./
cat adapters.fa
mkdir -p reads/NA12878/runSRR_1/
mkdir -p reads/NA12878/runERR_1/
java -Xmx2G -cp $TRIMMOMATIC_JAR org.usadellab.trimmomatic.TrimmomaticPE -threads 2 -phred33 \
raw_reads/NA12878/runERR_1/NA12878.ERR.33.pair1.fastq.gz \
raw_reads/NA12878/runERR_1/NA12878.ERR.33.pair2.fastq.gz \
reads/NA12878/runERR_1/NA12878.ERR.t20l32.pair1.fastq.gz \
reads/NA12878/runERR_1/NA12878.ERR.t20l32.single1.fastq.gz \
reads/NA12878/runERR_1/NA12878.ERR.t20l32.pair2.fastq.gz \
reads/NA12878/runERR_1/NA12878.ERR.t20l32.single2.fastq.gz \
ILLUMINACLIP:adapters.fa:2:30:15 TRAILING:20 MINLEN:32 \
2> reads/NA12878/runERR_1/NA12878.ERR.trim.out
java -Xmx2G -cp $TRIMMOMATIC_JAR org.usadellab.trimmomatic.TrimmomaticPE -threads 2 -phred33 \
raw_reads/NA12878/runSRR_1/NA12878.SRR.33.pair1.fastq.gz \
raw_reads/NA12878/runSRR_1/NA12878.SRR.33.pair2.fastq.gz \
reads/NA12878/runSRR_1/NA12878.SRR.t20l32.pair1.fastq.gz \
reads/NA12878/runSRR_1/NA12878.SRR.t20l32.single1.fastq.gz \
reads/NA12878/runSRR_1/NA12878.SRR.t20l32.pair2.fastq.gz \
reads/NA12878/runSRR_1/NA12878.SRR.t20l32.single2.fastq.gz \
ILLUMINACLIP:adapters.fa:2:30:15 TRAILING:20 MINLEN:32 \
2> reads/NA12878/runSRR_1/NA12878.SRR.trim.out
cat reads/NA12878/runERR_1/NA12878.ERR.trim.out reads/NA12878/runSRR_1/NA12878.SRR.trim.out
mkdir postTrimQC/
java -Xmx1G -jar ${BVATOOLS_JAR} readsqc \
--read1 reads/NA12878/runERR_1/NA12878.ERR.t20l32.pair1.fastq.gz \
--read2 reads/NA12878/runERR_1/NA12878.ERR.t20l32.pair2.fastq.gz \
--threads 2 --regionName ERR --output postTrimQC/
java -Xmx1G -jar ${BVATOOLS_JAR} readsqc \
--read1 reads/NA12878/runSRR_1/NA12878.SRR.t20l32.pair1.fastq.gz \
--read2 reads/NA12878/runSRR_1/NA12878.SRR.t20l32.pair2.fastq.gz \
--threads 2 --regionName SRR --output postTrimQC/
mkdir -p alignment/NA12878/runERR_1
mkdir -p alignment/NA12878/runSRR_1
bwa mem -M -t 2 \
-R '@RG\tID:ERR_ERR_1\tSM:NA12878\tLB:ERR\tPU:runERR_1\tCN:Broad Institute\tPL:ILLUMINA' \
${REF}/b37.fasta \
reads/NA12878/runERR_1/NA12878.ERR.t20l32.pair1.fastq.gz \
reads/NA12878/runERR_1/NA12878.ERR.t20l32.pair2.fastq.gz \
| java -Xmx2G -jar ${PICARD_JAR} SortSam \
INPUT=/dev/stdin \
OUTPUT=alignment/NA12878/runERR_1/NA12878.ERR.sorted.bam \
CREATE_INDEX=true VALIDATION_STRINGENCY=SILENT SORT_ORDER=coordinate MAX_RECORDS_IN_RAM=500000
bwa mem -M -t 2 \
-R '@RG\tID:SRR_SRR_1\tSM:NA12878\tLB:SRR\tPU:runSRR_1\tCN:Broad Institute\tPL:ILLUMINA' \
${REF}/b37.fasta \
reads/NA12878/runSRR_1/NA12878.SRR.t20l32.pair1.fastq.gz \
reads/NA12878/runSRR_1/NA12878.SRR.t20l32.pair2.fastq.gz \
| java -Xmx2G -jar ${PICARD_JAR} SortSam \
INPUT=/dev/stdin \
OUTPUT=alignment/NA12878/runSRR_1/NA12878.SRR.sorted.bam \
CREATE_INDEX=true VALIDATION_STRINGENCY=SILENT SORT_ORDER=coordinate MAX_RECORDS_IN_RAM=500000
java -Xmx2G -jar ${PICARD_JAR} MergeSamFiles \
INPUT=alignment/NA12878/runERR_1/NA12878.ERR.sorted.bam \
INPUT=alignment/NA12878/runSRR_1/NA12878.SRR.sorted.bam \
OUTPUT=alignment/NA12878/NA12878.sorted.bam \
VALIDATION_STRINGENCY=SILENT CREATE_INDEX=true
ls -l alignment/NA12878/
samtools view -H alignment/NA12878/NA12878.sorted.bam | grep "^@RG"
java -Xmx2G -jar ${GATK_JAR} \
-T RealignerTargetCreator \
-R ${REF}/b37.fasta \
-o alignment/NA12878/realign.intervals \
-I alignment/NA12878/NA12878.sorted.bam \
-L 1
java -Xmx2G -jar ${GATK_JAR} \
-T IndelRealigner \
-R ${REF}/b37.fasta \
-targetIntervals alignment/NA12878/realign.intervals \
-o alignment/NA12878/NA12878.realigned.sorted.bam \
-I alignment/NA12878/NA12878.sorted.bam
java -Xmx2G -jar ${PICARD_JAR} MarkDuplicates \
REMOVE_DUPLICATES=false CREATE_MD5_FILE=true VALIDATION_STRINGENCY=SILENT CREATE_INDEX=true \
INPUT=alignment/NA12878/NA12878.realigned.sorted.bam \
OUTPUT=alignment/NA12878/NA12878.sorted.dup.bam \
METRICS_FILE=alignment/NA12878/NA12878.sorted.dup.metrics
less alignment/NA12878/NA12878.sorted.dup.metrics
java -Xmx2G -jar ${GATK_JAR} \
-T BaseRecalibrator \
-nct 2 \
-R ${REF}/b37.fasta \
-knownSites ${REF}/dbSnp-137.vcf.gz \
-L 1:47000000-47171000 \
-o alignment/NA12878/NA12878.sorted.dup.recalibration_report.grp \
-I alignment/NA12878/NA12878.sorted.dup.bam
java -Xmx2G -jar ${GATK_JAR} \
-T PrintReads \
-nct 2 \
-R ${REF}/b37.fasta \
-BQSR alignment/NA12878/NA12878.sorted.dup.recalibration_report.grp \
-o alignment/NA12878/NA12878.sorted.dup.recal.bam \
-I alignment/NA12878/NA12878.sorted.dup.bam
java -Xmx2G -jar ${GATK_JAR} \
-T DepthOfCoverage \
--omitDepthOutputAtEachBase \
--summaryCoverageThreshold 10 \
--summaryCoverageThreshold 25 \
--summaryCoverageThreshold 50 \
--summaryCoverageThreshold 100 \
--start 1 --stop 500 --nBins 499 -dt NONE \
-R ${REF}/b37.fasta \
-o alignment/NA12878/NA12878.sorted.dup.recal.coverage \
-I alignment/NA12878/NA12878.sorted.dup.recal.bam \
-L 1:47000000-47171000
less -S alignment/NA12878/NA12878.sorted.dup.recal.coverage.sample_interval_summary
java -Xmx2G -jar ${PICARD_JAR} CollectInsertSizeMetrics \
VALIDATION_STRINGENCY=SILENT \
REFERENCE_SEQUENCE=${REF}/b37.fasta \
INPUT=alignment/NA12878/NA12878.sorted.dup.recal.bam \
OUTPUT=alignment/NA12878/NA12878.sorted.dup.recal.metric.insertSize.tsv \
HISTOGRAM_FILE=alignment/NA12878/NA12878.sorted.dup.recal.metric.insertSize.histo.pdf \
METRIC_ACCUMULATION_LEVEL=LIBRARY
less -S alignment/NA12878/NA12878.sorted.dup.recal.metric.insertSize.tsv
java -Xmx2G -jar ${PICARD_JAR} CollectAlignmentSummaryMetrics \
VALIDATION_STRINGENCY=SILENT \
REFERENCE_SEQUENCE=${REF}/b37.fasta \
INPUT=alignment/NA12878/NA12878.sorted.dup.recal.bam \
OUTPUT=alignment/NA12878/NA12878.sorted.dup.recal.metric.alignment.tsv \
METRIC_ACCUMULATION_LEVEL=LIBRARY
less -S alignment/NA12878/NA12878.sorted.dup.recal.metric.alignment.tsv
mkdir variants
samtools mpileup -L 1000 -B -q 1 -g \
-f ${REF}/b37.fasta \
-r 1:47000000-47171000 \
alignment/NA12878/NA12878.sorted.dup.recal.bam | bcftools view -vcg - \
> variants/mpileup.vcf
bgzip -c variants/mpileup.vcf > variants/mpileup.vcf.gz
tabix -p vcf variants/mpileup.vcf.gz
zless -S variants/mpileup.vcf.gz
####Add-on
samtools view alignment/NA12878/NA12878.sorted.bam | head –n4
samtools view -c -f4 alignment/NA12878/NA12878.sorted.bam
samtools view -c -F4 alignment/NA12878/NA12878.sorted.bam