Changes between Version 16 and Version 17 of SOPs/variant_calling_GATK


Ignore:
Timestamp:
07/30/15 09:28:12 (9 years ago)
Author:
thiruvil
Comment:

--

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  • SOPs/variant_calling_GATK

    v16 v17  
    22== Using GATK to call variants from short-read sequencing ==
    33
    4 This information comes from the [[http://www.broadinstitute.org/gatk/guide/events?id=3093#materials|slides for "Best Practices for Variant Calling with the GATK"]] from the Broad Institute.  This page summarizes and formats their detailed documentation.
     4This information comes from the [[http://www.broadinstitute.org/gatk/guide/events?id=3093#materials|slides for "Best Practices for Variant Calling with the GATK"]] from the Broad Institute.  This page summarizes and formats their detailed documentation.  '''GATK3 (v3 or higher) is recommended.'''
    55\\ \\
    66Note that if you're calling variants from RNA-seq reads, follow the somewhat different commands optimized for this, as described in GATK's [[https://www.broadinstitute.org/gatk/guide/article?id=3891|Calling variants in RNAseq]].
    … …  
    2424\\
    25253 - '''Validate VCF file or known variants''' (with GATK's [[http://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_sting_gatk_walkers_variantutils_ValidateVariants.html|ValidateVariants]])
    26   * java -jar /usr/local/gatk/GenomeAnalysisTK.jar -T ValidateVariants -R /path/to/genome/genome.fa --variant:VCF SNPs_from_NCBI.sorted.vcf \\
     26  * java -jar /usr/local/gatk3/GenomeAnalysisTK.jar -T ValidateVariants -R /path/to/genome/genome.fa --variant:VCF SNPs_from_NCBI.sorted.vcf \\
    2727Respond to errors (by correcting or removing problematic variants), run command again, etc., until validation is successful. \\
    2828Otherwise GATK will not run on any subsequent commands that require this file.
    … …  
    5151\\
    52529 - '''Run Indel Realignment''' (with [[http://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_sting_gatk_walkers_indels_RealignerTargetCreator.html|RealignerTargetCreator]] and [[http://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_sting_gatk_walkers_indels_IndelRealigner|IndelRealigner]]) \\
    53   * Example 1: java -jar /usr/local/gatk/GenomeAnalysisTK.jar -T RealignerTargetCreator -R human.fasta -I original.bam -known indels.vcf -o realigner.intervals \\
    54   * Example 2: java -jar /usr/local/gatk/GenomeAnalysisTK.jar -T IndelRealigner -R human.fasta -I original.bam -known indels.vcf -targetIntervals realigner.intervals -o realigned.bam \\
    55   * java -jar /usr/local/gatk/GenomeAnalysisTK.jar -T RealignerTargetCreator -R /path/to/genome/genome.fa -I Reads_1.bwa.dedup.good.bam -o Reads_1.realigner.intervals  --fix_misencoded_quality_scores
    56   * java -jar /usr/local/gatk/GenomeAnalysisTK.jar -T IndelRealigner -R /path/to/genome/genome.fa -I Reads_1.bwa.dedup.good.bam -targetIntervals Reads_1.realigner.intervals -o Reads_1.bwa.dedup.realigned.bam --fix_misencoded_quality_scores
     53  * Example 1: java -jar /usr/local/gatk3/GenomeAnalysisTK.jar -T RealignerTargetCreator -R human.fasta -I original.bam -known indels.vcf -o realigner.intervals \\
     54  * Example 2: java -jar /usr/local/gatk3/GenomeAnalysisTK.jar -T IndelRealigner -R human.fasta -I original.bam -known indels.vcf -targetIntervals realigner.intervals -o realigned.bam \\
     55  * java -jar /usr/local/gatk3/GenomeAnalysisTK.jar -T RealignerTargetCreator -R /path/to/genome/genome.fa -I Reads_1.bwa.dedup.good.bam -o Reads_1.realigner.intervals  --fix_misencoded_quality_scores
     56  * java -jar /usr/local/gatk3/GenomeAnalysisTK.jar -T IndelRealigner -R /path/to/genome/genome.fa -I Reads_1.bwa.dedup.good.bam -targetIntervals Reads_1.realigner.intervals -o Reads_1.bwa.dedup.realigned.bam --fix_misencoded_quality_scores
    5757\\
    585810 - '''Run Base Recalibration''' ([[http://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_sting_gatk_walkers_bqsr_BaseRecalibrator.html|BaseRecalibrator]] and [[http://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_sting_gatk_walkers_PrintReads.html|PrintReads]]) \\
    … …  
    6565  * Example 4: java -jar GenomeAnalysisTK.jar -T AnalyzeCovariates -R human.fasta -before recal.table -after after_recal.table -plots recal_plots.pdf
    6666All applied to our sample data:
    67   * bsub java -jar /usr/local/gatk/GenomeAnalysisTK.jar -T BaseRecalibrator -I Reads_1.bwa.dedup.realigned.bam -R /path/to/genome/genome.fa -o Reads_1.bwa.recal_data.txt -knownSites SNPs_from_NCBI.sorted.vcf
    68   * bsub java -jar /usr/local/gatk/GenomeAnalysisTK.jar -T PrintReads -I Reads_1.bwa.dedup.realigned.bam -R /path/to/genome/genome.fa -BQSR Reads_1.bwa.recal_data.txt -o Reads_1.bwa.dedup.realigned.recal.bam
    69   * bsub java -jar /usr/local/gatk/GenomeAnalysisTK.jar -T BaseRecalibrator -I Reads_1.bwa.dedup.realigned.bam -R /path/to/genome/genome.fa -knownSites SNPs_from_NCBI.sorted.vcf -BQSR Reads_1.bwa.recal_data.txt -o Reads_1.bwa.after_recal.txt
    70   * bsub java -jar /usr/local/gatk/GenomeAnalysisTK.jar -T AnalyzeCovariates -R /path/to/genome/genome.fa -before Reads_1.bwa.recal_data.txt -after Reads_1.bwa.after_recal.txt -plots Reads_1.bwa.recal_plots.pdf
     67  * bsub java -jar /usr/local/gatk3/GenomeAnalysisTK.jar -T BaseRecalibrator -I Reads_1.bwa.dedup.realigned.bam -R /path/to/genome/genome.fa -o Reads_1.bwa.recal_data.txt -knownSites SNPs_from_NCBI.sorted.vcf
     68  * bsub java -jar /usr/local/gatk3/GenomeAnalysisTK.jar -T PrintReads -I Reads_1.bwa.dedup.realigned.bam -R /path/to/genome/genome.fa -BQSR Reads_1.bwa.recal_data.txt -o Reads_1.bwa.dedup.realigned.recal.bam
     69  * bsub java -jar /usr/local/gatk3/GenomeAnalysisTK.jar -T BaseRecalibrator -I Reads_1.bwa.dedup.realigned.bam -R /path/to/genome/genome.fa -knownSites SNPs_from_NCBI.sorted.vcf -BQSR Reads_1.bwa.recal_data.txt -o Reads_1.bwa.after_recal.txt
     70  * bsub java -jar /usr/local/gatk3/GenomeAnalysisTK.jar -T AnalyzeCovariates -R /path/to/genome/genome.fa -before Reads_1.bwa.recal_data.txt -after Reads_1.bwa.after_recal.txt -plots Reads_1.bwa.recal_plots.pdf
    7171
    7272\\
    737311 - '''Compress BAM with [[http://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_sting_gatk_walkers_compression_reducereads_ReduceReads.html|ReduceReads]]''' [Optional: this step is not available in GATK Version 3.0 and higher] \\
    7474  * Example 1: java -jar GenomeAnalysisTK.jar -T ReduceReads -R human.fasta -I recal.bam -o reduced.bam
    75   * java -jar /usr/local/gatk/GenomeAnalysisTK.jar -T ReduceReads -R /path/to/genome/genome.fa -I Reads_1.bwa.dedup.realigned.recal.bam -o Reads_1.bwa.dedup.realigned.recal.reduced.bam
     75  * java -jar /usr/local/gatk3/GenomeAnalysisTK.jar -T ReduceReads -R /path/to/genome/genome.fa -I Reads_1.bwa.dedup.realigned.recal.bam -o Reads_1.bwa.dedup.realigned.recal.reduced.bam
    7676\\
    777712 - '''Finally -- Call variants''' \\
    7878Run [[http://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_sting_gatk_walkers_haplotypecaller_HaplotypeCaller.html|HaplotypeCaller]] ("The HaplotypeCaller is a more recent and sophisticated tool than the UnifiedGenotyper."; HaplotypeCaller is recommended as of GATK Version 3.0)
    7979  * Example: java -jar GenomeAnalysisTK.jar -T HaplotypeCaller -R human.fasta -I input.bam -o output.vcf -stand_call_conf 30 -stand_emit_conf 10 -minPruning 3
    80   * java -jar /usr/local/gatk/GenomeAnalysisTK.jar -T HaplotypeCaller -R /nfs/genomes/a.thaliana_TAIR_10/fasta_whole_genome/TAIR10.fa -I Reads_1.bwa.dedup.realigned.recal.reduced.bam --dbsnp SNPs_from_NCBI.sorted.vcf -o Reads_1.bwa.raw.snps.indels.HaplotypeCaller.vcf -stand_call_conf 30 -stand_emit_conf 10 -minPruning 3
     80  * java -jar /usr/local/gatk3/GenomeAnalysisTK.jar -T HaplotypeCaller -R /nfs/genomes/a.thaliana_TAIR_10/fasta_whole_genome/TAIR10.fa -I Reads_1.bwa.dedup.realigned.recal.reduced.bam --dbsnp SNPs_from_NCBI.sorted.vcf -o Reads_1.bwa.raw.snps.indels.HaplotypeCaller.vcf -stand_call_conf 30 -stand_emit_conf 10 -minPruning 3
    8181
    8282Pay attention to the INFO lines printed when the command is complete to check that too many reads aren't being unexpectedly filtered out.  Using RNA-seq reads mapped by STAR, for example, can result in almost all reads failing the HCMappingQualityFilter or the MappingQualityUnavailableFilter.  A quick fix is to modify the mapping quality of reads with some value (like 255) to some other value (like 60) on the fly by adding the arguments "-rf ReassignOneMappingQuality -RMQF 255 -RMQT 60".  See the [[https://www.broadinstitute.org/gatk/blog?id=4285|GATK blog]] for more information.
    … …  
    8484[If needed] Run [[http://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_sting_gatk_walkers_genotyper_UnifiedGenotyper.html|UnifiedGenotyper]] should be a better choice for nondiploid samples and high sample numbers
    8585  * Example: java -jar GenomeAnalysisTK.jar -T UnifiedGenotyper -R human.fasta -I input.bam -o output.vcf -stand_call_conf 30 -stand_emit_conf 10
    86   * java -jar /usr/local/gatk/GenomeAnalysisTK.jar -T UnifiedGenotyper -R /nfs/genomes/a.thaliana_TAIR_10/fasta_whole_genome/TAIR10.fa -I Reads_1.bwa.dedup.realigned.recal.reduced.bam --dbsnp SNPs_from_NCBI.sorted.vcf -o Reads_1.bwa.raw.snps.indels.UnifiedGenotyper.vcf -stand_call_conf 30 -stand_emit_conf 10
     86  * java -jar /usr/local/gatk3/GenomeAnalysisTK.jar -T UnifiedGenotyper -R /nfs/genomes/a.thaliana_TAIR_10/fasta_whole_genome/TAIR10.fa -I Reads_1.bwa.dedup.realigned.recal.reduced.bam --dbsnp SNPs_from_NCBI.sorted.vcf -o Reads_1.bwa.raw.snps.indels.UnifiedGenotyper.vcf -stand_call_conf 30 -stand_emit_conf 10
    8787\\
    888813 - '''Run Variant Quality Score Recalibration''' ("VQSR", with [[http://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_sting_gatk_walkers_variantrecalibration_VariantRecalibrator.html|VariantRecalibrator] and [[http://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_sting_gatk_walkers_variantrecalibration_ApplyRecalibration.html|ApplyRecalibration]) \\ \\