mkatari-bioinformatics-august-2013-gatknotes
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| mkatari-bioinformatics-august-2013-gatknotes [2014/06/11 12:49] – created mkatari | mkatari-bioinformatics-august-2013-gatknotes [2014/07/09 07:18] – mkatari | ||
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| bowtie2-build PTC_Human.fasta PTC_Human | bowtie2-build PTC_Human.fasta PTC_Human | ||
| samtools faidx PTC_Human.fasta | samtools faidx PTC_Human.fasta | ||
| - | java -jar / | + | java -jar / |
| + | R=PTC_Human.fasta | ||
| + | O=PTC_Human.dict | ||
| </ | </ | ||
| Line 27: | Line 29: | ||
| * create a bam index for dedup bam files | * create a bam index for dedup bam files | ||
| * realign samples | * realign samples | ||
| + | |||
| + | Once they are all processed | ||
| * merge all samples to one bam file | * merge all samples to one bam file | ||
| * sort and index this merged bam file | * sort and index this merged bam file | ||
| Line 34: | Line 38: | ||
| bowtie2 -x PTC_Human -U Cohen.fastq -S Cohen.sam | bowtie2 -x PTC_Human -U Cohen.fastq -S Cohen.sam | ||
| samtools view -bS Cohen.sam > Cohen.bam | samtools view -bS Cohen.sam > Cohen.bam | ||
| - | bowtie2 -x PTC_Human -U Sherman.fastq -S Sherman.sam | ||
| - | samtools view -bS Sherman.sam > Sherman.bam | ||
| </ | </ | ||
| - | 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:// |
| < | < | ||
| - | java -jar /export/apps/picard-tools/1.112/SortSam.jar INPUT=Cohen.bam OUTPUT=Cohen.sorted.bam SORT_ORDER=coordinate | + | mkdir /var/scratch/mkatari |
| - | java -jar / | + | mkdir /var/ |
| + | |||
| + | java -Djava.io.tmpdir=/ | ||
| + | INPUT=Cohen.bam \ | ||
| + | OUTPUT=Cohen.sorted.bam | ||
| + | SORT_ORDER=coordinate | ||
| + | |||
| </ | </ | ||
| Line 47: | Line 57: | ||
| < | < | ||
| - | java -jar / | ||
| - | java -jar / | + | java -Djava.io.tmpdir=/ |
| + | INPUT=Cohen.sorted.bam | ||
| + | OUTPUT=CohenRG.bam | ||
| + | RGLB=Cohen | ||
| + | RGPL=IonTorrent | ||
| + | RGPU=None | ||
| + | RGSM=Cohen | ||
| </ | </ | ||
| 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. | ||
| < | < | ||
| - | java -jar / | ||
| - | java -jar / | + | java -Djava.io.tmpdir=/ |
| + | INPUT=CohenRG.bam \ | ||
| + | OUTPUT=Cohen.dedup.bam | ||
| + | METRICS_FILE=Cohen.dedup.metrics | ||
| + | REMOVE_DUPLICATES=TRUE | ||
| + | ASSUME_SORTED=TRUE | ||
| + | | ||
| </ | </ | ||
| Line 62: | Line 83: | ||
| < | < | ||
| samtools index Cohen.dedup.bam | samtools index Cohen.dedup.bam | ||
| - | samtools index Sherman.dedup.bam | ||
| # | # | ||
| - | java -Xmx2g -jar / | + | java -Xmx2g |
| -T RealignerTargetCreator \ | -T RealignerTargetCreator \ | ||
| -R PTC_Human.fasta \ | -R PTC_Human.fasta \ | ||
| Line 71: | Line 91: | ||
| -o CohenforIndelRealigner.intervals | -o CohenforIndelRealigner.intervals | ||
| - | # | ||
| - | java -Xmx2g -jar / | ||
| - | -T RealignerTargetCreator \ | ||
| - | -R PTC_Human.fasta \ | ||
| - | -I Sherman.dedup.bam \ | ||
| - | -o ShermanforIndelRealigner.intervals | ||
| - | java -Xmx4g -jar / | + | java -Xmx4g |
| -T IndelRealigner \ | -T IndelRealigner \ | ||
| -R PTC_Human.fasta \ | -R PTC_Human.fasta \ | ||
| Line 86: | Line 100: | ||
| -o Cohen.dedup.realign.bam | -o Cohen.dedup.realign.bam | ||
| - | java -Xmx4g -jar /export/ | + | </code> |
| - | -T IndelRealigner \ | + | |
| - | -R PTC_Human.fasta \ | + | |
| - | -I Sherman.dedup.bam \ | + | |
| - | -targetIntervals ShermanforIndelRealigner.intervals \ | + | |
| - | -o Sherman.dedup.realign.bam | + | |
| + | In some cases there may be a need to clean the sam/bam file(s) (soft-trimming the coordinates). To do this use CleanSam in Picard tools. You may want to just do it to all to avoid the error in a workflow, but it may not be necessary. | ||
| + | |||
| + | < | ||
| + | java -Djava.io.tmpdir=/ | ||
| + | | ||
| + | | ||
| </ | </ | ||
| - | Now we merge the bam files and then sort and index them | + | Now we merge the bam files and then sort and index them. If you cleaned the bam file, remember to use the cleaned ones. |
| < | < | ||
| - | java -jar / | + | java -Djava.io.tmpdir=/ |
| + | INPUT=Sherman.clean.dedup.realign.bam | ||
| + | INPUT=Cohen.dedup.realign.bam | ||
| + | OUTPUT=ShermanCohenMerged.bam | ||
| samtools sort ShermanCohenMerged.bam ShermanCohenMerged.sorted | samtools sort ShermanCohenMerged.bam ShermanCohenMerged.sorted | ||
| + | |||
| samtools index ShermanCohenMerged.sorted.bam | samtools index ShermanCohenMerged.sorted.bam | ||
| </ | </ | ||
| - | Finall | + | Finally |
| < | < | ||
| - | java -jar / | + | java -Djava.io.tmpdir=/ |
| -T UnifiedGenotyper \ | -T UnifiedGenotyper \ | ||
| -I ShermanCohenMerged.sorted.bam \ | -I ShermanCohenMerged.sorted.bam \ | ||
| Line 115: | Line 135: | ||
| -glm SNP \ | -glm SNP \ | ||
| -o PTC_human.gatk.vcf | -o PTC_human.gatk.vcf | ||
| + | |||
| + | </ | ||
| + | |||
| + | If you want to load the vcf file into IGV, remember to index it first. | ||
| + | < | ||
| + | module load igvtools | ||
| + | igvtools index PTC_human.gatk.vcf | ||
| + | </ | ||
| + | |||
| + | If you would like to generate a table of from the vcf file use the following command | ||
| + | < | ||
| + | java --Djava.io.tmpdir=/ | ||
| + | -R PTC_Human.fasta | ||
| + | -T VariantsToTable \ | ||
| + | -V PTC_human.gatk.vcf \ | ||
| + | -F CHROM -F POS -F ID -F QUAL -F AC \ | ||
| + | -GF GT -GF GQ \ | ||
| + | -o PTC_human.gatk.vcf.table | ||
| </ | </ | ||
mkatari-bioinformatics-august-2013-gatknotes.txt · Last modified: 2016/08/17 08:37 by mkatari