GATK #

• GenomeAnalysisToolKit
• BroadInstitute에서 개발된 NGS 데이터를 이용한 유전체 분석 tool package

Requirements #

• Mapping file은 반듯이 .bam 파일
• bam 파일은 indexing 되어 있어야 함
• Mapping reads는 refernece ordering을 이용한 sorting이 되어 있어야함
• Reads는 하나 이상의 reads group 정보를 가지고 있어야함

Install programs #

• BWA
• SAMtools
• HTSlib (optional)
• Picard
• Genome Analysis Toolkit (GATK)
• IGV
• R
• FASTQToolkit

Manual #

• Pre-Processing ( FASTQToolkit )

  $ fastx_clipper –a [adapter_seq] -n –o [output.fastq] –i [input.fastq] > [report.txt]
  $ fastx_quality_filter [-i INFILE] [-o OUTFILE] > [report.txt]
  

• Mapping ( BWA )

  $ bwa index [-p prefix] [-a bwtsw|is] <in.fasta>
  $ bwa mem [reference_genome] [read1] [read2] > [output.sam]
  

• Mark Duplicates ( Picard )

  #ordering 
  $java -jar picard.jar SortSam I=[input.sam] O=[output.bam] SO=coordinate > [log]
  
  #duplicate marking
  $java -jar picard.jar MarkDuplicates I=[input.bam] O=[output.dup.bam] M=[matrix] > [log]
  
  #RG tag insertion
  $java -jar picard.jar \
  AddOrReplaceReadGroups \
  I=[input.dup.bam] \
  O=[output.dup.RG.bam]\
  RGID=[id] RGLB=[library] RGPL=[illumina] \
  RGPU=[barcord] RGSM=[group] RGCN=[center]> [log]
  

• Indel Realignment ( GATK )

  # index / dictionary file
  $ java -jar picard.jar\
  CreateSequenceDictionary \
  R=[input_ref_seq.fasta] \
  O=[ouput_ref_seq.dict]
  
  $ samtools faidx [genome.fasta]
  $ samtools index [input.bam]
  
  # RealignerTargetCreator
  $ java -jar GenomeAnalysisTK.jar \
  -T RealignerTargetCreator \
  -R [reference] \
  -I [original bam] \
  -known [vcf_file] \
  -o [output_candiate_region]
  
  # Realignment
  $ java -jar GenomeAnalysisTK.jar\
  -T IndelRealigner\
  -R [reference] \
  -I [original bam] \
  -known [vcf_file] \
  –targetIntervals [file with target region] \
  -o [output_candiate_region] \
  –filterNoBases
  

• Base Recalibration ( GATK )

  #searching
  $java -jar GenomeAnalysisTK.jar\
  -T BaseRecalibrator \
  -R [reference]\
  -I [realigned.bam] \
  -knownSites [.vcf] \
  –knowSites [.vcf] \
  -o [output_file]
  
  #ReCalibration
  $ java -jar GenomeAnalysisTK.jar \
  -T PrintReads \
  -R [genome.fasta] \
  -I [original.bam] \
  -BQSR [recal_searching.table] \
  -o [output.bam]
  

• Variation Calling

• UnifiedGenotyper ( GATK )

  $ java -jar GenomeAnalysisTK.jar \
  -T UnifiedGenotyper \
  -R [reference] \
  -I [input.bam] \
  -o [output.vcf] \
  -stand_call_conf 30 \
  -stand_emit_conf 10
  

• HaplotypeCaller ( GATK )

  $ java -jar GenomeAnalysisTK.jar \
  -T HaplotypeCaller \
  -R [reference] \
  -I [input.bam] \
  -o [output.vcf] \
  -stand_call_conf 30 \
  -stand_emit_conf 10 \
  -minPruning 3
  

• Variant Recalibration ( GATK )

  # Recalibration training
  $java -jar GenomeAnalysisTK.jar \
  -T VariantRecalibrator \
  –R [human_refernece] \
  –input CEUTrio.HiSeq.WGS.b37.bestPractices.b37.chr20.vcf \
   -resource:hapmap,known=false,training=true,truth=true,prior=15.0   hapmap_3.3.b37.vcf \
  -resource:dbsnp,known=true,training=false,truth=false,prior=2.0  dbsnp_138.b37.vcf \
  -an DP -an QD -an FS \ 
  -mode [SNP|inde] \
  -recalFile [recalibration_file] \
  -tranchesFile [reacal.tranches] \ 
  -rscriptFile [R_recal.plots.R]
  
  # Recalibration apply for VQSR
  $java -jar /DATA/1.src/bin/GenomeAnalysisTK.jar \
  -T ApplyRecalibration \ 
  -R  [human_refernece] \
  -input [input.vcf] \
  -mode SNP \
  -recalFile [recalibration_file]  \
  -tranchesFile [reacal.tranches] \
  -ts_filter_level [99.0] \
  -o [output.vcf]
  

• Genotype annotation ( snpEff )

  # databases search
  $java -jar snpEff.jar databases
  
  # databases download
  $java -jar snpEff.jar download -v [GRCh37.71|rice5]
  
  # datbases building
  $java -jar /DATA/1.src/snpEff/snpEff.jar \
  -v -onlyProtein \
  -i vcf \
  -o gatk [database]\
  [input.vcf] > [snpEff.output.vcf]
  
  # Annotation with GATK
  $java -jar /DATA/1.src/bin/GenomeAnalysisTK.jar \ 
  -T VariantAnnotator \
  -R [human_reference.fasta]\
  -A SnpEff \
  --variant [input.SNP.vcf] \
  --snpEffFile [snpEff.vcf] \
  -o [output.SNP.anno.vcf]
  

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