java -Xmx4g -Djava.io.tmpdir=/tmp -jar /work/01866/phr254/gshare/Tools_And_Programs/bin/CalculateHsMetrics.jar BI=/work/01866/phr254/gshare/data/hg19/target_intervals.reduced.withhead.intervallist TI=/work/01866/phr254/gshare/data/hg19/target_intervals.reduced.withhead.intervallist I=DEG03_01.merged.realigned.marked2.bam O=DEG03_01.picard.stats R=/work/01866/phr254/gshare/data/hg19/bwa0.5.4_index_cs/Homo_sapiens.GRCh37.60.dna.fasta PER_TARGET_COVERAGE=DEG03_01.picardEvaluating capture metrics
There are many ways to measure sequence capture. You might care more about minimizing off-target capture, to make your sequencing dollars go as far as possible. Or you might care more about maximizing on-target capture, to make sure you get data from every region of interest. These two are usually negatively correlated.
Using Picard's "CalculateHSMetrics" function to evaluate capture
Here is a link to the full documentation.
To run the program on Lonestar, there are three prerequisites: 1) A bam file and 2) a list of the genomic intervals that were to be captured and 3) the reference (.fa). As you would guess, the BAM and interval list both have to be based on exactly the same genomic reference file.
For our tutorial, the bam files are one of these:
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| title | BAM files for exome capture evaluation tutorial |
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/corral-repl/utexas/BioITeam/ngs_course/human_variation/NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam
/corral-repl/utexas/BioITeam/ngs_course/human_variation/NA12892.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam
/corral-repl/utexas/BioITeam/ngs_course/human_variation/NA12891.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam |
I've started with one of Illumina's target capture definitions (the vendor of your capture kit will provide this) but since the bam files only represent chr21 data I've created a target definitions file from chr21 only as well. Here they are:
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| title | Two relevant target list definitions |
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/corral-repl/utexas/BioITeam/ngs_course/human_variation/target_intervals.chr21.reduced.withhead.intervallist
/corral-repl/utexas/BioITeam/ngs_course/human_variation/target_intervals.reduced.withhead.intervallist |
And the relevant reference is:
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| title | Reference for exome metrics |
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/corral-repl/utexas/BioITeam/ngs_course/human_variation/ref/hs37d5.fa |
If you'd like to try this, copy the intervals, bam files, and reference (.fa and .fai) to a temporary directory on your $SCRATCH, and don't forget to "module load picard" first!
| Expand |
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| title | If you're in a hurry... |
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cds
mkdir tmpE
cd tmpE
cp /corral-repl/utexas/BioITeam/ngs_course/human_variation/NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam .
cp /corral-repl/utexas/BioITeam/ngs_course/human_variation/target_intervals.chr21.reduced.withhead.intervallist .
cp /corral-repl/utexas/BioITeam/ngs_course/human_variation/ref/hs37d5.fa .
cp /corral-repl/utexas/BioITeam/ngs_course/human_variation/ref/hs37d5.fa.fai . |
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The run command looks long but isn't that complicated (like most java programs):
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| title | How to run exactly these files on Lonestar |
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java -Xmx4g -Djava.io.tmpdir=/tmp -jar $TACC_PICARD_DIR/CalculateHsMetrics.jar BI=target_intervals.chr21.reduced.withhead.intervallist TI=target_intervals.chr21.reduced.withhead.intervallist I=NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam R=hs37d5.fa O=exome.picard.stats PER_TARGET_COVERAGE=exome.pertarget.stats |