wiki:PipelineCommands

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Version 2 (modified by laurent, 14 years ago) (diff)
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Below is the current GoNL pipeline commands along with rough execution times when available on the cluster as of January 25th 2011:

This analysis pipeline consists of two parts. The first one is based on lane analysis. Afterwards lanes are merged and analysis per lane is conducted.

Lane analysis

L1. Fastq to check read quality

/tools/FastQC/fastqc /data/filename_1.fq.gz \ -Dfastqc.output_dir=/output \ -Dfastqc.unzip=false

/tools/FastQC/fastqc /data/filename_2.fq.gz \ -Dfastqc.output_dir=/output \ -Dfastqc.unzip=false

L2. Align both pairs of FASTQ files using BWA (~6.5h)

/tools/bwa-0.5.8c_patched/bwa aln \ /resources//hg19/indices/b37_1kg.fa \ /data/filename_1.fq.gz -t 4 \ -f /output/filename.b37_1kg.1.sai

/tools/bwa-0.5.8c_patched/bwa aln \ /resources//hg19/indices/b37_1kg.fa \ /data/filename_2.fq.gz -t 4 \ -f /output/filename.b37_1kg.2.sai

L3. Create SAM file from both .sai files using BWA sampe (~3.5h)

/tools/bwa_45_patched/bwa sampe -P -p illumina -i lane -m sample_id -l library \ /resources//hg19/indices/b37_1kg.fa \ /output/filename.b37_1kg.1.sai /output/filename.b37_1kg.2.sai \ /data/filename_1.fq.gz /data/filename_2.fq.gz \ -f /output/filename.b37_1kg.sam

L4. Convert SAM to BAM file using Picard (~0.5h)

java -jar -Xmx3g /tools/picard-tools-1.32/SamFormatConverter.jar \ INPUT=/output/filename.b37_1kg.sam \ OUTPUT=/output/filename.b37_1kg.bam \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

L5. Sort BAM file and create corresponding index (~5h)

java -jar -Xmx3g /tools/picard-tools-1.32/SortSam.jar \ INPUT=/output/filename.b37_1kg.bam \ OUTPUT=/output/filename.b37_1kg.sorted.bam \ SORT_ORDER=coordinate \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

java -jar -Xmx3g /tools/picard-tools-1.32/BuildBamIndex.jar \ INPUT=/output/filename.b37_1kg.sorted.bam \ OUTPUT=/output/filename.b37_1kg.sorted.bam.bai \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

L6. Calculate QC metrics on alignment using Picard (~3.5h, excluding coverage - currently not working)

This Step is currently updated as:

Picard CalculateHsMetrics should be replaced by GATK DepthOfCoverage?
MeanQualityByCycle and QualityScoreDistribution should be moved after the recalibration step

java -jar -Xmx4g /tools/picard-tools-1.32/CollectAlignmentSummaryMetrics.jar \ I=/output/filename.b37_1kg.sorted.bam \ O=/output/filename.b37_1kg.AlignmentSummaryMetrics \ R=/resources//hg19/indices/b37_1kg.fa \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

java -jar /tools/picard-tools-1.32/CollectGcBiasMetrics.jar \ R=/resources//hg19/indices/b37_1kg.fa \ I=/output/filename.b37_1kg.sorted.bam \ O=/output/filename.b37_1kg.GcBiasMetrics \ CHART=/output/filename.b37_1kg.GcBiasMetrics.pdf \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

java -jar /tools/picard-tools-1.32/CollectInsertSizeMetrics.jar \ I=/output/filename.b37_1kg.sorted.bam \ O=/output/filename.b37_1kg.CollectInsertSizeMetrics \ H=/output/filename.b37_1kg.CollectInsertSizeMetrics.pdf \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

java -jar /tools/picard-tools-1.32/MeanQualityByCycle.jar \ I=/output/filename.b37_1kg.sorted.bam \ O=/output/filename.b37_1kg.MeanQualityByCycle \ CHART=/output/filename.b37_1kg.MeanQualityByCycle.pdf \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local java -jar /tools/picard-tools-1.32/QualityScoreDistribution.jar \ I=/output/filename.b37_1kg.sorted.bam \ O=/output/filename.b37_1kg.[wiki:QualityScoreDistribution] \ CHART=/output/filename.b37_1kg.[wiki:QualityScoreDistribution].pdf \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

java -jar /tools/picard-tools-1.32/BamIndexStats.jar \ INPUT=/output/filename.b37_1kg.sorted.bam \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

java -jar -Xmx3g /tools/picard-tools-1.32/CalculateHsMetricsWholeGenome.jar \ INPUT=/output/filename.b37_1kg.sorted.bam \ OUTPUT=/output/filename.b37_1kg.HsMetrics \ BAIT_INTERVALS=/resources//hg19/intervals/GoNL.interval_list \ TARGET_INTERVALS=/resources//hg19/intervals/GoNL.interval_list \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

L7. Mark duplicates after alignment (~2.5h)

java -Xmx4g -jar /tools/picard-tools-1.32/MarkDuplicates.jar \ INPUT=/output/filename.b37_1kg.sorted.bam \ OUTPUT=/output/filename.b37_1kg.dedup.bam \ METRICS_FILE=/output/filename.b37_1kg.dedup.metrics \ REMOVE_DUPLICATES=false ASSUME_SORTED=true VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

L8. Realignment using knowns only (~2.5h)

java -Djava.io.tmpdir=/local -Xmx8g -jar \ /tools/Sting/dist/GenomeAnalysisTK.jar -l INFO -T IndelRealigner \ -U ALLOW_UNINDEXED_BAM -I /output/filename.b37_1kg.dedup.bam \ -targetIntervals /resources//hg19/intervals/realign_intervals_hg19_b37_1kg.intervals \ -R /resources//hg19/indices/b37_1kg.fa \ -D /resources//hg19/dbsnp/dbsnp_129_b37_b37_1kg.rod \ -[B:indels,VCF B:indels,VCF] /resources//hg19/indels/1kg.pilot_release.merged.indels.sites./hg19.b37_1kg.vcf \ -o /output/filename.b37_1kg.realigned.bam -knownsOnly -LOD 0.4 -compress 0

L9. Fix mates after realignment (~2h)

java -jar -Xmx6g /tools/picard-tools-1.32/FixMateInformation.jar \ INPUT=/output/filename.b37_1kg.realigned.bam \ OUTPUT=/output/filename.b37_1kg.matefixed.bam \ SORT_ORDER=coordinate VALIDATION_STRINGENCY=SILENT TMP_DIR=/local

L10. Calculate covariates before realignment (~12h - Note that this should be improved using more cores. In test at the moment)

java -jar -Xmx2g /tools/Sting/dist/GenomeAnalysisTK.jar -l INFO \ -T CountCovariates -U ALLOW_UNINDEXED_BAM \ -R /resources//hg19/indices/b37_1kg.fa \ --DBSNP /resources//hg19/dbsnp/dbsnp_129_b37_b37_1kg.rod \ -I /output/filename.b37_1kg.matefixed.bam \ -cov ReadGroupcovariate -cov QualityScoreCovariate -cov CycleCovariate -cov DinucCovariate \ -recalFile /output/filename.b37_1kg.matefixed.covariate_table.csv

L11. Recalibrate mate fixed and realigned alignment (~5h)

java -jar -Xmx4g /tools/Sting/dist/GenomeAnalysisTK.jar -l INFO \ -T TableRecalibration -U ALLOW_UNINDEXED_BAM \ -R /resources//hg19/indices/b37_1kg.fa -I /output/filename.b37_1kg.matefixed.bam \ --recal_file /output/filename.b37_1kg.matefixed.covariate_table.csv \ --out /output/filename.b37_1kg.recal.bam

L12. Sort and index recalibrated alignment (~5h)

java -jar -Xmx3g /tools/picard-tools-1.32/SortSam.jar \ INPUT=/output/filename.b37_1kg.recal.bam \ OUTPUT=/output/filename.b37_1kg.recal.sorted.bam \ SORT_ORDER=coordinate \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

java -jar -Xmx3g /tools/picard-tools-1.32/BuildBamIndex.jar \ INPUT=/output/filename.b37_1kg.recal.sorted.bam \ OUTPUT=/output/filename.b37_1kg.recal.sorted.bam.bai \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

L13. Calculate covariates after realignment and recalibration (~12h Note that this should be improved using more cores. In test at the moment)

java -jar -Xmx2g /tools/Sting/dist/GenomeAnalysisTK.jar -l INFO \ -T CountCovariates -U ALLOW_UNINDEXED_BAM \ -R /resources//hg19/indices/b37_1kg.fa \ --DBSNP /resources//hg19/dbsnp/dbsnp_129_b37_b37_1kg.rod \ -I /output/filename.b37_1kg.recal.sorted.bam \ -cov ReadGroupcovariate -cov QualityScoreCovariate -cov CycleCovariate -cov DinucCovariate \ -recalFile /output/filename.b37_1kg.recal.covariate_table.csv

L14. Analyze covariates before and after (Currently not working)

java -jar -Xmx4g /tools/Sting/dist/AnalyzeCovariates.jar -l INFO \ -resources /resources//hg19/indices/b37_1kg.fa \ --recal_file /output/filename.b37_1kg.matefixed.covariate_table.csv \ -outputDir /output/filename.b37_1kg.recal.stats_before/ \ -Rscript ${rscript} -ignoreQ 5

java -jar -Xmx4g /tools/Sting/dist/AnalyzeCovariates.jar -l INFO \ -resources /resources//hg19/indices/b37_1kg.fa \ --recal_file /output/filename.b37_1kg.recal.covariate_table.csv \ -outputDir /output/filename.b37_1kg.recal.stats_after/ \ -Rscript ${rscript} -ignoreQ 5

Sample analysis

S1. Merging three lanes to one

java -jar -Xmx4g /tools/picard-tools-1.32/MergeSamFiles.jar \ INPUT/output/filename.b37_1kg.recal.sorted.ba \ INPUT/output/filename2.b37_1kg.recal.sorted.ba \ INPUT/output/filename3.b37_1kg.recal.sorted.ba \ ASSUME_SORTED=true USE_THREADING=true \ TMP_DIR=/local MAX_RECORDS_IN_RAM=30000000 \ OUTPUT=/output/sample_id.b37_1kg.bam SORT_ORDER=coordinate \ VALIDATION_STRINGENCY=SILENT

To perform QC on initial data SNP calling is performed. This procedure consists of three steps.

S2. SNP calling using Unified Genotyper

java -Xmx4g -jar /tools/Sting/dist/GenomeAnalysisTK.jar \ -l INFO -T UnifiedGenotyper -I /output/sample_id.b37_1kg.bam \ --out /output/sample_id.b37_1kg.qc_check_snps.vcf \ -R /resources//hg19/indices/b37_1kg.fa \ -D /resources//hg19/dbsnp/dbsnp_129_b37_b37_1kg.rod \ -stand_call_conf 30.0 -stand_emit_conf 10.0

S3. Filter SNPs using variant filtration

java -jar -Xmx4g /tools/Sting/dist/GenomeAnalysisTK.jar \ -l INFO -T VariantFiltration \ -[B:variant,VCF B:variant,VCF] /output/sample_id.b37_1kg.qc_check_snps.vcf \ -R /resources//hg19/indices/b37_1kg.fa \ -D /resources//hg19/dbsnp/dbsnp_129_b37_b37_1kg.rod \ --out /output/sample_id.b37_1kg.qc_check_snps.filtered.vcf \ --maskName InDel --clusterWindowSize 10 \ --filterName GATK_standard \ --filterExpression "AB > 0.75 && DP > 40 || DP > 100 || MQ0 > 40 || SB > -0.10"||

S4. Generate statistics on called SNPs using variant eval

java -Xmx4g -jar /tools/Sting/dist/GenomeAnalysisTK.jar \ -T VariantEval -R /resources//hg19/indices/b37_1kg.fa \ -l INFO \ -[B:eval,VCF B:eval,VCF] /output/sample_id.b37_1kg.qc_check_snps.filtered.vcf \ -D /resources//hg19/dbsnp/dbsnp_129_b37_b37_1kg.rod \ -o /output/sample_id.b37_1kg.qc_check_snps.filtered.eval

After this QC check the indel and SNP calling takes place using the next commands.

S5. Create targets for realignment

java -Xmx4g -jar -Djava.io.tmpdir=/local /tools/Sting/dist/GenomeAnalysisTK.jar -l INFO \ -T RealignerTargetCreator \ -I /output/sample_id.b37_1kg.bam \ -R /resources//hg19/indices/b37_1kg.fa \ -D /resources//hg19/dbsnp/dbsnp_129_b37_b37_1kg.rod \ -[B:indels,VCF B:indels,VCF] /resources//hg19/indels/1kg.pilot_release.merged.indels.sites./hg19.b37_1kg.vcf \ -o /output/sample_id.b37_1kg.realign.intervals

S6. Realignment using created targets, dbSNP and indels from 1kg project

java -Djava.io.tmpdir=/local –Xmx6g -jar \ /tools/Sting/dist/GenomeAnalysisTK.jar -l INFO -T IndelRealigner \ -I /output/sample_id.b37_1kg.bam \ -targetIntervals /output/sample_id.b37_1kg.realign.intervals \ -R /resources//hg19/indices/b37_1kg.fa \ -D /resources//hg19/dbsnp/dbsnp_129_b37_b37_1kg.rod \ -[B:indels,VCF B:indels,VCF] /resources//hg19/indels/1kg.pilot_release.merged.indels.sites./hg19.b37_1kg.vcf \ --out /output/sample_id.b37_1kg.realigned.bam \ -maxReads 500000

S7. Fix mates after realignment and create corresponding BAM index

java -jar -Xmx6g /tools/picard-tools-1.32/FixMateInformation.jar \ INPUT=/output/sample_id.b37_1kg.realigned.bam \ OUTPUT=/output/sample_id.b37_1kg.matesfixed.bam \ SORT_ORDER=coordinate VALIDATION_STRINGENCY=SILENT TMP_DIR=/local

java -jar -Xmx3g /tools/picard-tools-1.32/BuildBamIndex.jar \ INPUT=/output/sample_id.b37_1kg.matesfixed.bam \ OUTPUT=/output/sample_id.b37_1kg.matesfixed.bam.bai \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local

S8. Indel calling on realigned BAM file

java -Xmx4g -jar /tools/Sting/dist/GenomeAnalysisTK.jar -l INFO \ -T IndelGenotyperV2 -I /output/sample_id.b37_1kg.matesfixed.bam \ --out /output/sample_id.b37_1kg.indels.vcf \ --bedOutput /output/sample_id.b37_1kg.indels.bed \ -R /resources//hg19/indices/b37_1kg.fa \ -verbose /output/sample_id.b37_1kg.indels.verboseoutput.txt

S9. Filter indels

perl /tools/filterSingleSampleCalls.pl \ --calls /output/sample_id.b37_1kg.indels.bed > /output/sample_id.b37_1kg.indels.filtered.bed \ --max_cons_av_mm 3.0 --max_cons_nqs_av_mm 0.5 --mode ANNOTATE

S10. SNP calling on realigned BAM file

java -Xmx4g -jar /tools/Sting/dist/GenomeAnalysisTK.jar \ -l INFO -T UnifiedGenotyper -I /output/sample_id.b37_1kg.matesfixed.bam \ --out /output/sample_id.b37_1kg.snps.vcf \ -R /resources//hg19/indices/b37_1kg.fa \ -D /resources//hg19/dbsnp/dbsnp_129_b37_b37_1kg.rod \ -stand_call_conf 30.0 -stand_emit_conf 10.0

S11. Create indel mask used in SNP calling python

/tools/makeIndelMask.py \ /output/sample_id.b37_1kg.indels.filtered.bed 10 \ /output/sample_id.b37_1kg.indels.mask.bed

S12. Filter SNP calls using the indel mask, dbsnp and indels from 1kg project

java -jar -Xmx4g /tools/Sting/dist/GenomeAnalysisTK.jar \ -l INFO -T VariantFiltration \ -[B:variant,VCF B:variant,VCF] /output/sample_id.b37_1kg.snps.vcf \ -[B:mask,Bed B:mask,Bed] /output/sample_id.b37_1kg.indels.mask.bed \ -R /resources//hg19/indices/b37_1kg.fa \ -D /resources//hg19/dbsnp/dbsnp_129_b37_b37_1kg.rod \ --out /output/sample_id.b37_1kg.snps.filtered.vcf \ --maskName InDel --clusterWindowSize 10 \ --filterName GATK_standard \ --filterExpression "AB > 0.75 && DP > 40 || DP > 100 || MQ0 > 40 || SB > -0.10"||

S13. Variant eval on detected SNPs

java -Xmx4g -jar /tools/Sting/dist/GenomeAnalysisTK.jar \ -T VariantEval -R /resources//hg19/indices/b37_1kg.fa \ -l INFO \ -[B:eval,VCF B:eval,VCF] /output/sample_id.b37_1kg.snps.filtered.vcf \ -D /resources//hg19/dbsnp/dbsnp_129_b37_b37_1kg.rod \ -o /output/sample_id.b37_1kg.snps.filtered.eval

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