Changes between Initial Version and Version 1 of PipelineCommands


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Timestamp:
Jan 25, 2011 9:52:38 AM (15 years ago)
Author:
laurent
Comment:

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  • PipelineCommands

    v1 v1  
     1Below is the current GoNL pipeline commands along with rough execution times when available on the cluster as of January 25th 2011:
     2
     3This analysis pipeline consists of two parts. The first one is based on lane analysis. Afterwards lanes are merged and analysis per lane is conducted.
     4
     5= Lane analysis =
     6'''L1. Fastq to check read quality'''
     7
     8{{{
     9/tools/FastQC/fastqc /data/filename_1.fq.gz \ -Dfastqc.output_dir=/output \ -Dfastqc.unzip=false
     10
     11/tools/FastQC/fastqc /data/filename_2.fq.gz \ -Dfastqc.output_dir=/output \ -Dfastqc.unzip=false
     12}}}
     13'''L2. Align both pairs of FASTQ files using BWA '''(~6.5h)'''[[BR]] '''
     14
     15{{{
     16/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
     17
     18/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
     19}}}
     20'''L3. Create SAM file from both .sai files using BWA sampe '''(~3.5h)'''[[BR]] '''
     21
     22{{{
     23/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
     24}}}
     25'''L4. Convert SAM to BAM file using Picard '''(~0.5h)'''[[BR]] '''
     26
     27{{{
     28java -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
     29}}}
     30'''L5. Sort BAM file and create corresponding index '''(~5h)'''[[BR]] '''
     31
     32{{{
     33java -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
     34
     35java -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
     36}}}
     37'''L6. Calculate QC metrics on alignment using Picard''' (~3.5h)
     38
     39This Step is currently updated as:
     40
     41 * [http://www.bbmriwiki.nl/search/opensearch?q=wiki%3ACalculateHsMetrics Picard CalculateHsMetrics] should be replaced by GATK DepthOfCoverage
     42 * !MeanQualityByCycle and !QualityScoreDistribution should be moved after the recalibration step
     43
     44{{{
     45java -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
     46
     47java -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
     48
     49java -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
     50
     51java -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
     52
     53java -jar /tools/picard-tools-1.32/BamIndexStats.jar \ INPUT=/output/filename.b37_1kg.sorted.bam \ VALIDATION_STRINGENCY=LENIENT \ TMP_DIR=/local
     54
     55java -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
     56}}}
     57'''L7. Mark duplicates after alignment'''
     58
     59{{{
     60java -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
     61}}}
     62'''L8. Realignment using knowns only'''
     63
     64{{{
     65java -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
     66}}}
     67'''L9. Fix mates after realignment'''
     68
     69{{{
     70java -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
     71}}}
     72'''L10. Calculate covariates before realignment '''
     73
     74{{{
     75java -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
     76}}}
     77'''L11. Recalibrate mate fixed and realigned alignment'''
     78
     79{{{
     80java -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
     81}}}
     82'''L12. Sort and index recalibrated alignment'''
     83
     84{{{
     85java -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
     86
     87java -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
     88}}}
     89'''L13. Calculate covariates after realignment and recalibration'''
     90
     91{{{
     92java -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
     93}}}
     94'''L14. Analyze covariates before and after'''
     95
     96{{{
     97java -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
     98
     99java -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
     100}}}
     101= Sample analysis =
     102'''S1. Merging three lanes to one'''
     103
     104{{{
     105java -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
     106}}}
     107== To perform QC on initial data SNP calling is performed. This procedure consists of three steps. ==
     108'''S2. SNP calling using Unified Genotyper'''
     109
     110{{{
     111java -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
     112}}}
     113'''S3. Filter SNPs using variant filtration'''
     114
     115{{{
     116java -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"||
     117}}}
     118'''S4. Generate statistics on called SNPs using variant eval '''
     119
     120{{{
     121java -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
     122}}}
     123== After this QC check the indel and SNP calling takes place using the next commands. ==
     124'''S5. Create targets for realignment'''
     125
     126{{{
     127java -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
     128}}}
     129'''S6. Realignment using created targets, dbSNP and indels from 1kg project'''
     130
     131{{{
     132java -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
     133}}}
     134'''S7.  Fix mates after realignment and create corresponding BAM index'''
     135
     136{{{
     137java -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
     138
     139java -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
     140}}}
     141'''S8. Indel calling on realigned BAM file'''
     142
     143{{{
     144java -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
     145}}}
     146'''S9. Filter indels '''
     147
     148{{{
     149perl /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
     150}}}
     151'''S10. SNP calling on realigned BAM file'''
     152
     153{{{
     154java -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
     155}}}
     156'''S11. Create indel mask used in SNP calling python '''
     157
     158{{{
     159/tools/makeIndelMask.py \ /output/sample_id.b37_1kg.indels.filtered.bed 10 \ /output/sample_id.b37_1kg.indels.mask.bed
     160}}}
     161'''S12. Filter SNP calls using the indel mask, dbsnp and indels from 1kg project'''
     162
     163{{{
     164java -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"||
     165}}}
     166'''S13. Variant eval on detected SNPs'''
     167
     168{{{
     169java -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
     170}}}