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gatk_best_practices_RNA_seq_variant
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gatk_best_practices_RNA_seq_variant
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##Condition for each sample
for i in $(ls ./raw_reads | grep ^[^d]| rev | cut -c 10- | rev | uniq)
do
# Make direcotry for fastqc
mkdir -p ${i%}_fastqc
# Run fastqc
fastqc -o ./${i%}_fastqc -f fastq /home/suresh/rnaseq/raw_reads/${i%}_R1.fastq /home/suresh/rnaseq/raw_reads/${i%}_R2.fastq
# Make directory to store cutadapt results
mkdir -p cutadapt
#Run cutadapt
cutadapt --quality-cutoff=20 --format=fastq -o ./cutadapt/${i%}_R1.cutadapt.fastq -p ./cutadapt/${i%}_R2.cutadapt.fastq /home/suresh/rnaseq/raw_reads/${i%}_R1.fastq /home/suresh/rnaseq/raw_reads/${i%}_R2.fastq
# Run top hat and save the out with directory extension _tophatout
tophat2 --no-coverage-search -o ./${i%}_tophat_out -G ./reference/genes_chr12.gtf -p 2 ./reference/chr12 ./cutadapt/${i%}_R1.cutadapt.fastq ./cutadapt/${i%}_R2.cutadapt.fastq
# Collect the stats
samtools flagstat ./${i%}_tophat_out/accepted_hits.bam > ./${i%}_tophat_out/accepted_hits.flagstats
#Sort bam
samtools sort -T /tmp/align.bam ./${i%}_tophat_out/accepted_hits.bam -o ./${i%}_tophat_out/accepted_hits.pos.sorted.bam
# Index the bam
samtools index ./${i%}_tophat_out/accepted_hits.pos.sorted.bam
# picard mkdir
mkdir -p ${i%}_picard
# Dedup bam
java -jar /opt/picard-tools-1.119/MarkDuplicates.jar \
METRICS_FILE=${i%}_picard/${i%}.dedup.metrics \
REMOVE_DUPLICATES=true ASSUME_SORTED=true \
VALIDATION_STRINGENCY=LENIENT \
CREATE_INDEX=true \
I=./${i%}_tophat_out/accepted_hits.pos.sorted.bam \
O=./${i%}_picard/q20.cutadapt.sorted.dedup.bam
# Add read groups
java -jar /opt/picard-tools-1.119/AddOrReplaceReadGroups.jar \
I=./${i%}_picard/q20.cutadapt.sorted.dedup.bam \
O=./${i%}_picard/q20.cutadapt.sorted.dedup.rg.bam \
CREATE_INDEX=true SO=coordinate RGID=${i%%_*} RGLB=${i%} RGPL=ILLUMINA RGSM=${i%} RGPU=GCCAAT
# mkdir gatk
mkdir -p ./${i%}_gatk
# Split N cigar reads
java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
-T SplitNCigarReads \
-R /home/suresh/rnaseq/reference/chr12.fa \
-I ./${i%}_picard/q20.cutadapt.sorted.dedup.rg.bam \
-o ./${i%}_gatk/q20.cutadapt.sorted.dedup.snc.bam \
-U ALLOW_N_CIGAR_READS
#Sort bam
samtools sort -T /tmp/align.bam ./${i%}_gatk/q20.cutadapt.sorted.dedup.snc.bam -o ./${i%}_gatk/q20.cutadapt.sorted.dedup.snc.sorted.bam
# Index the bam
samtools index ./${i%}_gatk/q20.cutadapt.sorted.dedup.snc.sorted.bam
# Create intervals
java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
-T RealignerTargetCreator \
-R /home/suresh/rnaseq/reference/chr12.fa \
-I ./${i%}_gatk/q20.cutadapt.sorted.dedup.snc.sorted.bam \
--known /home/suresh/ngs_exercise/data/hg19/Mills_and_1000G_gold_standard.indels.hg19.chr12.vcf \
-L chr12 -o ./${i%}_gatk/forIndelRealigner.intervals
# Realign bam files
java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
-T IndelRealigner \
-R /home/suresh/rnaseq/reference/chr12.fa \
-I ./${i%}_gatk/q20.cutadapt.sorted.dedup.snc.sorted.bam \
-known /home/suresh/ngs_exercise/data/hg19/Mills_and_1000G_gold_standard.indels.hg19.chr12.vcf \
-L chr12 \
-targetIntervals ./${i%}_gatk/forIndelRealigner.intervals \
-o ./${i%}_gatk/q20.cutadapt.sorted.dedup.snc.sorted.realigned.bam
# Base recalibration First pass
java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
-T BaseRecalibrator \
-R /home/suresh/rnaseq/reference/chr12.fa \
-I ./${i%}_gatk/q20.cutadapt.sorted.dedup.snc.sorted.realigned.bam \
-knownSites /home/suresh/rnaseq/reference/chr12.dbsnp.b141.b37.hg19.tidy.vcf \
-o ./${i%}_gatk/recal_data.table
# Base recalibration second pass
java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
-T BaseRecalibrator \
-R /home/suresh/rnaseq/reference/chr12.fa \
-I ./${i%}_gatk/q20.cutadapt.sorted.dedup.snc.sorted.realigned.bam \
-knownSites /home/suresh/rnaseq/reference/chr12.dbsnp.b141.b37.hg19.tidy.vcf \
-L chr12 \
-knownSites /home/suresh/ngs_exercise/data/hg19/Mills_and_1000G_gold_standard.indels.hg19.chr12.vcf \
-BQSR ./${i%}_gatk/recal_data.table \
-o ./${i%}_gatk/post_recal_data.table
# Create plots
java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
-T AnalyzeCovariates \
-R /home/suresh/rnaseq/reference/chr12.fa \
-L chr12 \
-before ./${i%}_gatk/recal_data.table \
-after ./${i%}_gatk/post_recal_data.table \
-plots ./${i%}_gatk/recalibration_plots.pdf
# Apply BQSR to sequence
java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
-T PrintReads \
-R /home/suresh/rnaseq/reference/chr12.fa \
-I ./${i%}_gatk/q20.cutadapt.sorted.dedup.snc.sorted.realigned.bam \
-L chr12 \
-BQSR ./${i%}_gatk/recal_data.table \
-o ./${i%}_gatk/q20.cutadapt.sorted.dedup.snc.sorted.realigned.recal.bam
# Variant calling
java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
-T HaplotypeCaller \
-R /home/suresh/rnaseq/reference/chr12.fa \
-I ./MeOH_REP1_gatk/q20.cutadapt.sorted.dedup.snc.sorted.realigned.recal.bam \
-I ./MeOH_REP2_gatk/q20.cutadapt.sorted.dedup.snc.sorted.realigned.recal.bam \
-I ./MeOH_REP3_gatk/q20.cutadapt.sorted.dedup.snc.sorted.realigned.recal.bam \
--dbsnp /home/suresh/rnaseq/reference/chr12.dbsnp.b141.b37.hg19.tidy.vcf \
-dontUseSoftClippedBases \
-stand_call_conf 20 \
-stand_emit_conf 20 \
-o ./meoh_output.raw.snps.indels.vcf
java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
-T HaplotypeCaller \
-R /home/suresh/rnaseq/reference/chr12.fa \
-I ./R3G_REP1_gatk/q20.cutadapt.sorted.dedup.snc.sorted.realigned.recal.bam \
-I ./R3G_REP2_gatk/q20.cutadapt.sorted.dedup.snc.sorted.realigned.recal.bam \
-I ./R3G_REP3_gatk/q20.cutadapt.sorted.dedup.snc.sorted.realigned.recal.bam \
--dbsnp /home/suresh/rnaseq/reference/chr12.dbsnp.b141.b37.hg19.tidy.vcf \
-dontUseSoftClippedBases \
-stand_call_conf 20 \
-stand_emit_conf 20 \
-o ./r3g_output.raw.snps.indels.vcf
# Discordant calls
#java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
# -T SelectVariants \
# -R /home/suresh/rnaseq/reference/chr12.fa \
# -V ./meoh_output.raw.snps.indels.vcf \
# --discordance ./r3g_output.raw.snps.indels.vcf \
# -o doutput.vcf \
# Concordant calls
#java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
# -T SelectVariants \
# -R /home/suresh/rnaseq/reference/chr12.fa \
# -V ./meoh_output.raw.snps.indels.vcf \
# --concordance ./r3g_output.raw.snps.indels.vcf \
# -o coutput.vcf \
# Variant filtering
java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
-T VariantFiltration \
-R /home/suresh/rnaseq/reference/chr12.fa \
-V ./${i%}_gatk/output.raw.snps.indels.vcf \
-window 35 \
-cluster 3 \
-filterName FS \
-filter "FS > 30.0" \
-filterName QD \
-filter "QD < 2.0" \
-o ./${i%}_gatk/output.raw.snps.indels.filtered.vcf
#GenotypeGVCFs MeOH
#java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
# -T GenotypeGVCFs \
# -R /home/suresh/rnaseq/reference/chr12.fa \
# -V ./MeOH_REP1_gatk/output.raw.snps.indels.g.vcf \
# -V ./MeOH_REP2_gatk/output.raw.snps.indels.g.vcf \
# -V ./MeOH_REP3_gatk/output.raw.snps.indels.g.vcf \
# -o meoh_output.vcf
#GenotypeGVCFs R3G
#java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
# -T GenotypeGVCFs \
# -R /home/suresh/rnaseq/reference/chr12.fa \
# -V ./R3G_REP1_gatk/output.raw.snps.indels.g.vcf \
# -V ./R3G_REP2_gatk/output.raw.snps.indels.g.vcf \
# -V ./R3G_REP3_gatk/output.raw.snps.indels.g.vcf \
# -o r3g_output.vcf
#GenotypeGVCFs combine R3G Meoh
#java -jar /opt/gatk-3.4-46/GenomeAnalysisTK.jar \
# -T CombineGVCFs \
# -R /home/suresh/rnaseq/reference/chr12.fa \
# -V r3g_output.vcf \
# -V meoh_output.vcf \
# -o meoh_r3g_output.vcf
done