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mkatari-bioinformatics-august-2013-gatknotes [2014/07/02 15:19] – mkatarimkatari-bioinformatics-august-2013-gatknotes [2016/08/10 20:45] – mkatari
Line 36: Line 36:
  
 <code> <code>
-bowtie2 -x PTC_Human -U Cohen.fastq -S Cohen.sam +bowtie2 -x PTC_Human -U Sample1.fastq -S Sample1.sam 
-samtools view -bS Cohen.sam > Cohen.bam+samtools view -bS Sample1.sam > Sample1.bam 
 + 
 +bowtie2 -x PTC_Human -U Sample2.fastq -S Sample2.sam 
 +samtools view -bS Sample2.sam > Sample2.bam 
 + 
 +bowtie2 -x PTC_Human -U Sample3.fastq -S Sample3.sam 
 +samtools view -bS Sample3.sam > Sample3.bam 
 + 
 +bowtie2 -x PTC_Human -U Sample4.fastq -S Sample4.sam 
 +samtools view -bS Sample4.sam > Sample4.bam
 </code> </code>
  
-The picard method to sort is preferred by GATK+The picard method to sort is preferred by GATK. In some cases PICARD uses the temp directory to do its sorting. You may run into an error that complains about running out of space. To avoid this problem simply create your own tmp directory and tell java that it should use it. See details [[https://www.biostars.org/p/42613/|here]]. 
 <code> <code>
-java -jar /export/apps/picard-tools/1.112/SortSam.jar \ +module load picard/1.133 
-   INPUT=Cohen.bam \ + 
-   OUTPUT=Cohen.sorted.bam \ +picard SortSam INPUT=Sample1.bam OUTPUT=Sample1.sorted.bam \ 
-   SORT_ORDER=coordinate +    SORT_ORDER=coordinate 
-   + 
 +picard SortSam INPUT=Sample2.bam OUTPUT=Sample2.sorted.bam \ 
 +    SORT_ORDER=coordinate 
 + 
 +picard SortSam INPUT=Sample3.bam OUTPUT=Sample3.sorted.bam \ 
 +    SORT_ORDER=coordinate 
 + 
 +picard SortSam INPUT=Sample4.bam OUTPUT=Sample4.sorted.bam \ 
 +    SORT_ORDER=coordinate 
  
 </code> </code>
Line 53: Line 72:
  
 <code> <code>
 +picard AddOrReplaceReadGroups \
 +   INPUT=Sample1.sorted.bam \
 +   OUTPUT=Sample1RG.bam \
 +   RGLB=Sample1 \
 +   RGPL=IonTorrent \
 +   RGPU=None \
 +   RGSM=Sample1
  
-java -jar /export/apps/picard-tools/1.112/AddOrReplaceReadGroups.jar \ +picard AddOrReplaceReadGroups \ 
-   INPUT=Cohen.sorted.bam \ +   INPUT=Sample2.sorted.bam \ 
-   OUTPUT=CohenRG.bam \ +   OUTPUT=Sample2RG.bam \ 
-   RGLB=Cohen \+   RGLB=Sample2 \
    RGPL=IonTorrent \    RGPL=IonTorrent \
    RGPU=None \    RGPU=None \
-   RGSM=Cohen+   RGSM=Sample2 
 + 
 +picard AddOrReplaceReadGroups \ 
 +   INPUT=Sample3.sorted.bam \ 
 +   OUTPUT=Sample3RG.bam \ 
 +   RGLB=Sample3 \ 
 +   RGPL=IonTorrent \ 
 +   RGPU=None \ 
 +   RGSM=Sample3 
 + 
 +picard AddOrReplaceReadGroups \ 
 +   INPUT=Sample4.sorted.bam \ 
 +   OUTPUT=Sample4RG.bam \ 
 +   RGLB=Sample4 \ 
 +   RGPL=IonTorrent \ 
 +   RGPU=None \ 
 +   RGSM=Sample4 
 + 
 </code> </code>
  
 This will remove any reads that map to the same exact place. It is helpful to get rid of artifacts.  This will remove any reads that map to the same exact place. It is helpful to get rid of artifacts. 
 <code> <code>
-java -jar /export/apps/picard-tools/1.112/MarkDuplicates.jar \ + 
-   INPUT=CohenRG.bam \ +picard MarkDuplicates \ 
-   OUTPUT=Cohen.dedup.bam \ +   INPUT=Sample1RG.bam \ 
-   METRICS_FILE=Cohen.dedup.metrics \+   OUTPUT=Sample1.dedup.bam \ 
 +   METRICS_FILE=Sample1.dedup.metrics \
    REMOVE_DUPLICATES=TRUE \    REMOVE_DUPLICATES=TRUE \
-   ASSUME_SORTED=TRUE+   ASSUME_SORTED=TRUE \ 
 +   MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=1000 
 + 
 + 
 +picard MarkDuplicates \ 
 +   INPUT=Sample2RG.bam \ 
 +   OUTPUT=Sample2.dedup.bam \ 
 +   METRICS_FILE=Sample2.dedup.metrics \ 
 +   REMOVE_DUPLICATES=TRUE \ 
 +   ASSUME_SORTED=TRUE \ 
 +   MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=1000 
 + 
 +picard MarkDuplicates \ 
 +   INPUT=Sample3RG.bam \ 
 +   OUTPUT=Sample3.dedup.bam \ 
 +   METRICS_FILE=Sample3.dedup.metrics \ 
 +   REMOVE_DUPLICATES=TRUE \ 
 +   ASSUME_SORTED=TRUE \ 
 +   MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=1000 
 + 
 +picard MarkDuplicates \ 
 +   INPUT=Sample4RG.bam \ 
 +   OUTPUT=Sample4.dedup.bam \ 
 +   METRICS_FILE=Sample4.dedup.metrics \ 
 +   REMOVE_DUPLICATES=TRUE \ 
 +   ASSUME_SORTED=TRUE \ 
 +   MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=1000
  
 </code> </code>
Line 76: Line 147:
 Index the files and realign them Index the files and realign them
 <code> <code>
-samtools index Cohen.dedup.bam +samtools index Sample1.dedup.bam  
 +samtools index Sample2.dedup.bam  
 +samtools index Sample3.dedup.bam  
 +samtools index Sample4.dedup.bam 
  
 #identifying indels #identifying indels
-java -Xmx2g -jar /export/apps/GenomeAnalysisTK/GenomeAnalysisTK-2.3-9-ge5ebf34/GenomeAnalysisTK.jar \+ 
 +module load gatk/3.3.0 
 + 
 +GenomeAnalysisTK \
    -T RealignerTargetCreator \    -T RealignerTargetCreator \
    -R PTC_Human.fasta \    -R PTC_Human.fasta \
-   -I Cohen.dedup.bam \ +   -I Sample1.dedup.bam \ 
-   -o CohenforIndelRealigner.intervals+   -o Sample1forIndelRealigner.intervals
    
  
-  +GenomeAnalysisTK \
- java -Xmx4g -jar /export/apps/GenomeAnalysisTK/GenomeAnalysisTK-2.3-9-ge5ebf34/GenomeAnalysisTK.jar \+
    -T IndelRealigner \    -T IndelRealigner \
    -R PTC_Human.fasta \    -R PTC_Human.fasta \
-   -I Cohen.dedup.bam \ +   -I Sample1.dedup.bam \ 
-  -targetIntervals CohenforIndelRealigner.intervals \ +   -targetIntervals Sample1forIndelRealigner.intervals \ 
-   -o Cohen.dedup.realign.bam+   -o Sample1.dedup.realign.bam
  
 </code> </code>
Line 99: Line 175:
  
 <code> <code>
-java -jar /export/apps/picard-tools/1.112/CleanSam.jar \ +picard CleanSam \ 
-   INPUT=Sherman.dedup.realign.bam \ +   INPUT=Sample4.dedup.realign.bam \ 
-   OUTPUT=Sherman.clean.dedup.realign.bam+   OUTPUT=Sample4.clean.dedup.realign.bam
 </code> </code>
  
Line 107: Line 183:
  
 <code> <code>
-java -jar /export/apps/picard-tools/1.112/MergeSamFiles.jar \ +picard MergeSamFiles \ 
-   INPUT=Sherman.clean.dedup.realign.bam \ +   INPUT=Sample1.dedup.realign.bam \ 
-   INPUT=Cohen.dedup.realign.bam \ +   INPUT=Sample2.dedup.realign.bam \ 
-   OUTPUT=ShermanCohenMerged.bam  +   INPUT=Sample3.dedup.realign.bam \ 
 +   INPUT=Sample4.dedup.realign.bam \ 
 +   OUTPUT=AllMerged.bam  
  
-samtools sort ShermanCohenMerged.bam ShermanCohenMerged.sorted+picard SortSam INPUT=AllMerged.bam OUTPUT=AllMerged.sorted.bam SORT_ORDER=coordinate 
 + 
 +samtools index AllMerged.sorted.bam
  
-samtools index ShermanCohenMerged.sorted.bam  
 </code> </code>
  
Line 121: Line 200:
  
 <code> <code>
-java -jar /export/apps/GenomeAnalysisTK/GenomeAnalysisTK-2.3-9-ge5ebf34/GenomeAnalysisTK.jar \ +GenomeAnalysisTK -T UnifiedGenotyper \ 
-   -T UnifiedGenotyper \ +   -I AllMerged.sorted.bam \
-   -I ShermanCohenMerged.sorted.bam \+
    -R PTC_Human.fasta \    -R PTC_Human.fasta \
    --output_mode EMIT_VARIANTS_ONLY \    --output_mode EMIT_VARIANTS_ONLY \
Line 140: Line 218:
 If you would like to generate a table of from the vcf file use the following command If you would like to generate a table of from the vcf file use the following command
 <code> <code>
-java -jar /export/apps/GenomeAnalysisTK/GenomeAnalysisTK-2.3-9-ge5ebf34/GenomeAnalysisTK.jar \+GenomeAnalysisTK \
      -R PTC_Human.fasta      -R PTC_Human.fasta
      -T VariantsToTable \      -T VariantsToTable \
Line 147: Line 225:
      -GF GT -GF GQ \      -GF GT -GF GQ \
      -o PTC_human.gatk.vcf.table      -o PTC_human.gatk.vcf.table
 +</code>
 +
 +In order to filter your vcf file based on quality measures, depth, and also statistical significance, you can use variant filter option in the gatk toolkit. Below is an example of suggested filters for data that has low coverage.
 +
 +<code>
 +GenomeAnalysisTK \
 +    -R PTC_Human.fasta \
 +    -T VariantFiltration \
 +    -o PTC_human.gatk.filter.vcf \
 +    --variant PTC_human.gatk.vcf \
 +    --filterExpression "QD < 2.0 || MQ < 40.0 || FS > 60.0 || HaplotypeScore >13.0" \
 +    --filterName "mannyfilter"
 +
 +</code>
 +
 +Good descriptions of the different information on vcf files [[https://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_gatk_tools_walkers_annotator_HaplotypeScore.php|GATK Docs]]
 +
 +Finally to save the SNPs that passed your filter, you simply use the selectvariant tool.
 +
 +<code>
 +
 +GenomeAnalysisTK \
 +    -T SelectVariants \
 +    --variant PTC_human.gatk.filter.vcf \
 +    -o PTC_human.gatk.filter.only.vcf \
 +    -ef \
 +    -R PTC_Human.fasta
 +
 </code> </code>
mkatari-bioinformatics-august-2013-gatknotes.txt · Last modified: 2016/08/17 08:37 by mkatari