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Old 10-09-2013, 03:25 AM   #14
choishingwan
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Location: Hong Kong

Join Date: Feb 2012
Posts: 21
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@vd4mindia: From your code, I guess you are running GATK version before 2.6 right? You are right about using the dbsnp137. From the look of it it seems correct. In case if you want to use the latest gatk, you can refer to the following site: http://gatkforums.broadinstitute.org...se-2-0-retired

Personally, I use the following code if it helps:

Quote:
samtools sort <sample>.bwa.bam <sample>.bwa.sort

#Index
samtools index <sample>.bwa.sort.bam

#mark duplicate
java -Xmx5g -jar MarkDuplicates.jar INPUT=<sample>.bwa.sort.bam OUTPUT=<sample>.bwa.sort.deduped.bam METRICS_FILE=<sample>.duplicates REMOVE_DUPLICATES=TRUE VALIDATION_STRINGENCY=LENIENT CREATE_INDEX=TRUE


#Realignment based on known insert sites (Using Java 1.7 from now on as required by GATK)
java -Xmx5g -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -R Reference.fa -I <sample>.bwa.sort.deduped.arg.bam -known 1000G_phase1.indels.hg19.vcf -known Mills_and_1000G_gold_standard.indels.hg19.vcf -o <sample>.realign.intervals -S LENIENT

java -Xmx5g -jar GenomeAnalysisTK.jar -T IndelRealigner -R Reference.fa -I <sample>.bwa.sort.deduped.arg.bam -targetIntervals <sample>.realign.intervals -known 1000G_phase1.indels.hg19.vcf -known Mills_and_1000G_gold_standard.indels.hg19.vcf -o <sample>.bwa.sort.deduped.arg.realigned.bam -S LENIENT

java -Xmx5g -jar GenomeAnalysisTK.jar -T BaseRecalibrator -R Reference.fa -l INFO -I <sample>.bwa.sort.deduped.arg.realigned.bam -knownSites 1000G_phase1.indels.hg19.vcf -knownSites Mills_and_1000G_gold_standard.indels.hg19.vcf -knownSites dbsnp_137.hg19.vcf -o <sample>.recalibration_report.grp -S LENIENT

java -Xmx5g -jar GenomeAnalysisTK.jar -T PrintReads -R Reference.fa -l INFO -I <sample>.bwa.sort.deduped.arg.realigned.bam -BQSR <sample>.recalibration_report.grp -o <sample>.bwa.sort.deduped.arg.realigned.recalibrated.bam -S LENIENT
This is how we do the analysis here in our lab, not necessarily the correct way. We did use the latest GATK though, so some of the code might differ from yours.
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