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<!DOCTYPE html>
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<title>Chapter 8 VQSR | A practical introduction to GATK 4 on Biowulf (NIH HPC)</title>
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<meta name="twitter:title" content="Chapter 8 VQSR | A practical introduction to GATK 4 on Biowulf (NIH HPC)" />
<meta name="author" content="Qi Yu and Wolfgang Resch" />
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<li><a href="./">GATK on biowulf</a></li>
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<li class="chapter" data-level="" data-path="index.html"><a href="index.html#software"><i class="fa fa-check"></i>Software</a></li>
<li class="chapter" data-level="" data-path="index.html"><a href="index.html#sequencing-data"><i class="fa fa-check"></i>Sequencing data</a></li>
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<li class="chapter" data-level="1" data-path="preproc.html"><a href="preproc.html"><i class="fa fa-check"></i><b>1</b> Data preprocessing</a>
<ul>
<li class="chapter" data-level="1.1" data-path="preproc.html"><a href="preproc.html#brief-introduction"><i class="fa fa-check"></i><b>1.1</b> Brief introduction</a></li>
<li class="chapter" data-level="1.2" data-path="preproc.html"><a href="preproc.html#preproc-tools"><i class="fa fa-check"></i><b>1.2</b> Tools</a></li>
<li class="chapter" data-level="1.3" data-path="preproc.html"><a href="preproc.html#preproc-single-tools"><i class="fa fa-check"></i><b>1.3</b> Individual tool benchmarks</a>
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<li class="chapter" data-level="1.3.1" data-path="preproc.html"><a href="preproc.html#preproc-trim"><i class="fa fa-check"></i><b>1.3.1</b> Adapter trimming</a></li>
<li class="chapter" data-level="1.3.2" data-path="preproc.html"><a href="preproc.html#preproc-align"><i class="fa fa-check"></i><b>1.3.2</b> Alignment</a></li>
<li class="chapter" data-level="1.3.3" data-path="preproc.html"><a href="preproc.html#preproc-sort"><i class="fa fa-check"></i><b>1.3.3</b> Alternative A: Sorting by coordinate</a></li>
<li class="chapter" data-level="1.3.4" data-path="preproc.html"><a href="preproc.html#preproc-view"><i class="fa fa-check"></i><b>1.3.4</b> Alternative B: Queryname-grouped (unsorted) BAM</a></li>
</ul></li>
<li class="chapter" data-level="1.4" data-path="preproc.html"><a href="preproc.html#optimized-script"><i class="fa fa-check"></i><b>1.4</b> Optimized script</a>
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<li class="chapter" data-level="1.4.1" data-path="preproc.html"><a href="preproc.html#alternative-a-producing-sorted-bam-output"><i class="fa fa-check"></i><b>1.4.1</b> Alternative A: producing sorted BAM output</a></li>
<li class="chapter" data-level="1.4.2" data-path="preproc.html"><a href="preproc.html#preproc-nosort-pipeline"><i class="fa fa-check"></i><b>1.4.2</b> Alternative B: BAM output without coordinate sorting</a></li>
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<li class="chapter" data-level="1.5" data-path="preproc.html"><a href="preproc.html#read-groups"><i class="fa fa-check"></i><b>1.5</b> Read groups</a></li>
<li class="chapter" data-level="1.6" data-path="preproc.html"><a href="preproc.html#processing-the-example-trio"><i class="fa fa-check"></i><b>1.6</b> Processing the example trio</a></li>
</ul></li>
<li class="chapter" data-level="2" data-path="workflow-overview.html"><a href="workflow-overview.html"><i class="fa fa-check"></i><b>2</b> GATK practice workflow</a>
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<li class="chapter" data-level="2.1" data-path="workflow-overview.html"><a href="workflow-overview.html#cleaning-up-raw-alignments"><i class="fa fa-check"></i><b>2.1</b> Cleaning up raw alignments</a></li>
<li class="chapter" data-level="2.2" data-path="workflow-overview.html"><a href="workflow-overview.html#joint-calling"><i class="fa fa-check"></i><b>2.2</b> Joint Calling</a></li>
<li class="chapter" data-level="2.3" data-path="workflow-overview.html"><a href="workflow-overview.html#variant-filtering"><i class="fa fa-check"></i><b>2.3</b> Variant filtering</a></li>
</ul></li>
<li class="chapter" data-level="3" data-path="markdup.html"><a href="markdup.html"><i class="fa fa-check"></i><b>3</b> MarkDuplicates</a>
<ul>
<li class="chapter" data-level="3.1" data-path="markdup.html"><a href="markdup.html#brief-introduction-1"><i class="fa fa-check"></i><b>3.1</b> Brief introduction</a></li>
<li class="chapter" data-level="3.2" data-path="markdup.html"><a href="markdup.html#benchmarks-of-markduplicatesspark"><i class="fa fa-check"></i><b>3.2</b> Benchmarks of <code>MarkDuplicatesSpark</code></a>
<ul>
<li class="chapter" data-level="3.2.1" data-path="markdup.html"><a href="markdup.html#queryname-grouped-input-data-as-generated-by-the-aligner"><i class="fa fa-check"></i><b>3.2.1</b> Queryname-grouped input data (as generated by the aligner)</a></li>
<li class="chapter" data-level="3.2.2" data-path="markdup.html"><a href="markdup.html#coordinate-sorted-input-data"><i class="fa fa-check"></i><b>3.2.2</b> Coordinate-sorted input data</a></li>
<li class="chapter" data-level="3.2.3" data-path="markdup.html"><a href="markdup.html#performance-comparing-between-queryname-grouped-and-coordinate-sorted-inputs"><i class="fa fa-check"></i><b>3.2.3</b> Performance comparing between queryname-grouped and coordinate-sorted inputs</a></li>
</ul></li>
<li class="chapter" data-level="3.3" data-path="markdup.html"><a href="markdup.html#optimized-script-1"><i class="fa fa-check"></i><b>3.3</b> Optimized script</a></li>
</ul></li>
<li class="chapter" data-level="4" data-path="bqsr.html"><a href="bqsr.html"><i class="fa fa-check"></i><b>4</b> BQSR</a>
<ul>
<li class="chapter" data-level="4.1" data-path="bqsr.html"><a href="bqsr.html#brief-introduction-2"><i class="fa fa-check"></i><b>4.1</b> Brief introduction</a></li>
<li class="chapter" data-level="4.2" data-path="bqsr.html"><a href="bqsr.html#benchmarks-of-baserecalibrator"><i class="fa fa-check"></i><b>4.2</b> Benchmarks of <code>BaseRecalibrator</code></a></li>
<li class="chapter" data-level="4.3" data-path="bqsr.html"><a href="bqsr.html#optimized-script-for-baserecalibrator"><i class="fa fa-check"></i><b>4.3</b> Optimized script for <code>BaseRecalibrator</code></a></li>
<li class="chapter" data-level="4.4" data-path="bqsr.html"><a href="bqsr.html#benchmarks-of-applybqsr"><i class="fa fa-check"></i><b>4.4</b> Benchmarks of <code>ApplyBQSR</code></a></li>
<li class="chapter" data-level="4.5" data-path="bqsr.html"><a href="bqsr.html#optimized-script-for-applybqsr"><i class="fa fa-check"></i><b>4.5</b> Optimized script for <code>ApplyBQSR</code></a></li>
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<li class="chapter" data-level="5" data-path="haplotype-caller.html"><a href="haplotype-caller.html"><i class="fa fa-check"></i><b>5</b> HaplotypeCaller</a>
<ul>
<li class="chapter" data-level="5.1" data-path="haplotype-caller.html"><a href="haplotype-caller.html#brief-introduction-3"><i class="fa fa-check"></i><b>5.1</b> Brief introduction</a></li>
<li class="chapter" data-level="5.2" data-path="haplotype-caller.html"><a href="haplotype-caller.html#benchmarks"><i class="fa fa-check"></i><b>5.2</b> Benchmarks</a></li>
<li class="chapter" data-level="5.3" data-path="haplotype-caller.html"><a href="haplotype-caller.html#optimized-script-2"><i class="fa fa-check"></i><b>5.3</b> Optimized script</a></li>
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<li class="chapter" data-level="6" data-path="genomics-db-import.html"><a href="genomics-db-import.html"><i class="fa fa-check"></i><b>6</b> GenomicsDBImport (replaces CombineGVCFs)</a>
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<li class="chapter" data-level="6.1" data-path="genomics-db-import.html"><a href="genomics-db-import.html#brief-introduction-4"><i class="fa fa-check"></i><b>6.1</b> Brief introduction</a></li>
<li class="chapter" data-level="6.2" data-path="genomics-db-import.html"><a href="genomics-db-import.html#benchmarks-1"><i class="fa fa-check"></i><b>6.2</b> Benchmarks</a></li>
<li class="chapter" data-level="6.3" data-path="genomics-db-import.html"><a href="genomics-db-import.html#optimized-script-3"><i class="fa fa-check"></i><b>6.3</b> Optimized script</a></li>
</ul></li>
<li class="chapter" data-level="7" data-path="genotype-gvcfs.html"><a href="genotype-gvcfs.html"><i class="fa fa-check"></i><b>7</b> GenotypeGVCFs</a>
<ul>
<li class="chapter" data-level="7.1" data-path="genotype-gvcfs.html"><a href="genotype-gvcfs.html#brief-introduction-5"><i class="fa fa-check"></i><b>7.1</b> Brief introduction</a></li>
<li class="chapter" data-level="7.2" data-path="genotype-gvcfs.html"><a href="genotype-gvcfs.html#benchmarks-2"><i class="fa fa-check"></i><b>7.2</b> Benchmarks</a></li>
<li class="chapter" data-level="7.3" data-path="genotype-gvcfs.html"><a href="genotype-gvcfs.html#optimized-script-4"><i class="fa fa-check"></i><b>7.3</b> Optimized script</a></li>
</ul></li>
<li class="chapter" data-level="8" data-path="vqsr.html"><a href="vqsr.html"><i class="fa fa-check"></i><b>8</b> VQSR</a>
<ul>
<li class="chapter" data-level="8.1" data-path="vqsr.html"><a href="vqsr.html#brief-introduction-6"><i class="fa fa-check"></i><b>8.1</b> Brief introduction</a></li>
<li class="chapter" data-level="8.2" data-path="vqsr.html"><a href="vqsr.html#benchmarks-of-variantrecalibrator"><i class="fa fa-check"></i><b>8.2</b> Benchmarks of <code>VariantRecalibrator</code></a></li>
<li class="chapter" data-level="8.3" data-path="vqsr.html"><a href="vqsr.html#optimized-script-for-variantrecalibrator"><i class="fa fa-check"></i><b>8.3</b> Optimized script for <code>VariantRecalibrator</code></a></li>
<li class="chapter" data-level="8.4" data-path="vqsr.html"><a href="vqsr.html#benchmarks-of-applyvqsr"><i class="fa fa-check"></i><b>8.4</b> Benchmarks of <code>ApplyVQSR</code></a></li>
<li class="chapter" data-level="8.5" data-path="vqsr.html"><a href="vqsr.html#optimized-script-for-applyvqsr"><i class="fa fa-check"></i><b>8.5</b> Optimized script for <code>ApplyVQSR</code></a></li>
</ul></li>
<li class="chapter" data-level="9" data-path="gt-posteriors.html"><a href="gt-posteriors.html"><i class="fa fa-check"></i><b>9</b> Calculate genotype posteriors</a>
<ul>
<li class="chapter" data-level="9.1" data-path="gt-posteriors.html"><a href="gt-posteriors.html#brief-introduction-7"><i class="fa fa-check"></i><b>9.1</b> Brief introduction</a></li>
<li class="chapter" data-level="9.2" data-path="gt-posteriors.html"><a href="gt-posteriors.html#benchmarks-3"><i class="fa fa-check"></i><b>9.2</b> Benchmarks</a></li>
<li class="chapter" data-level="9.3" data-path="gt-posteriors.html"><a href="gt-posteriors.html#optimized-script-5"><i class="fa fa-check"></i><b>9.3</b> Optimized script</a></li>
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<li class="chapter" data-level="" data-path="references.html"><a href="references.html"><i class="fa fa-check"></i>References</a></li>
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<section class="normal" id="section-">
<div id="vqsr" class="section level1" number="8">
<h1><span class="header-section-number">Chapter 8</span> VQSR</h1>
<div id="brief-introduction-6" class="section level2" number="8.1">
<h2><span class="header-section-number">8.1</span> Brief introduction</h2>
<p>Raw variant calls include many artifacts.</p>
<p>The core algorithm in VQSR is a Gaussian mixture model that aims to classify
variants based on how their annotation values cluster given a training set of
high-confidence variants.</p>
<p>Then the VQSR tools use this model to assign a new confidence score to each
variant, called VQSLOD. This is a log-ratio of the variant’s probabilities
belonging to the positive and negative model.</p>
</div>
<div id="benchmarks-of-variantrecalibrator" class="section level2" number="8.2">
<h2><span class="header-section-number">8.2</span> Benchmarks of <code>VariantRecalibrator</code></h2>
<p>We did a benchmark on the performance of <code>VariantRecalibrator</code> with different
numbers of CPUs and memory. As show in figure <a href="vqsr.html#fig:vqsr-cpu">8.1</a>, the running
time didn’t reduce much after increasing threads.</p>
<div class="figure" style="text-align: center"><span id="fig:vqsr-cpu"></span>
<img src="images/08-VariantRecalibrator_benchmark_cpu_constrain.png" alt="Runtime of VariantRecalibrator as a function of the number of threads" width="70%" />
<p class="caption">
Figure 8.1: Runtime of <code>VariantRecalibrator</code> as a function of the number of threads
</p>
</div>
<p>We normally recommend running jobs with 70%-80% efficiency. Based on the efficiency calculated from the runtime (figure <a href="vqsr.html#fig:vqsr-eff">8.2</a>),
<code>variantRecalibrator</code> should not be run with more than 2 threads.</p>
<div class="figure" style="text-align: center"><span id="fig:vqsr-eff"></span>
<img src="images/08-VariantRecalibrator_efficiency_cpu_constrain.png" alt="Efficiency of VariantRecalibrator as a function of the number of threads" width="70%" />
<p class="caption">
Figure 8.2: Efficiency of <code>VariantRecalibrator</code> as a function of the number of threads
</p>
</div>
<p>As for memory, increasing memory didn’t improve performance (figure
<a href="vqsr.html#fig:vqsr-mem">8.3</a>), although a minimal of 4GB memory is required for the job
to finish with 2 threads.</p>
<div class="figure" style="text-align: center"><span id="fig:vqsr-mem"></span>
<img src="images/08-VariantRecalibrator_benchmark_mem_constrain.png" alt="Runtime of VariantRecalibrator as a function of memory" width="70%" />
<p class="caption">
Figure 8.3: Runtime of <code>VariantRecalibrator</code> as a function of memory
</p>
</div>
</div>
<div id="optimized-script-for-variantrecalibrator" class="section level2" number="8.3">
<h2><span class="header-section-number">8.3</span> Optimized script for <code>VariantRecalibrator</code></h2>
<p>Step 1: We use <code>VariantRecalibrator</code> to build the model and calculate VQSLOD
for SNP and INDEL separately:</p>
<div class="sourceCode" id="cb22"><pre class="sourceCode sh"><code class="sourceCode bash"><span id="cb22-1"><a href="vqsr.html#cb22-1" aria-hidden="true" tabindex="-1"></a><span class="bu">cd</span> data/<span class="kw">;</span> <span class="dt">\</span></span>
<span id="cb22-2"><a href="vqsr.html#cb22-2" aria-hidden="true" tabindex="-1"></a><span class="ex">gatk</span> <span class="at">--java-options</span> <span class="st">"-Djava.io.tmpdir=/lscratch/</span><span class="va">$SLURM_JOBID</span><span class="st"> -Xms4G -Xmx4G -XX:ParallelGCThreads=2"</span> VariantRecalibrator <span class="dt">\</span></span>
<span id="cb22-3"><a href="vqsr.html#cb22-3" aria-hidden="true" tabindex="-1"></a> <span class="at">-tranche</span> 100.0 <span class="at">-tranche</span> 99.95 <span class="at">-tranche</span> 99.9 <span class="dt">\</span></span>
<span id="cb22-4"><a href="vqsr.html#cb22-4" aria-hidden="true" tabindex="-1"></a> <span class="at">-tranche</span> 99.5 <span class="at">-tranche</span> 99.0 <span class="at">-tranche</span> 97.0 <span class="at">-tranche</span> 96.0 <span class="dt">\</span></span>
<span id="cb22-5"><a href="vqsr.html#cb22-5" aria-hidden="true" tabindex="-1"></a> <span class="at">-tranche</span> 95.0 <span class="at">-tranche</span> 94.0 <span class="dt">\</span></span>
<span id="cb22-6"><a href="vqsr.html#cb22-6" aria-hidden="true" tabindex="-1"></a> <span class="at">-tranche</span> 93.5 <span class="at">-tranche</span> 93.0 <span class="at">-tranche</span> 92.0 <span class="at">-tranche</span> 91.0 <span class="at">-tranche</span> 90.0 <span class="dt">\</span></span>
<span id="cb22-7"><a href="vqsr.html#cb22-7" aria-hidden="true" tabindex="-1"></a> <span class="at">-R</span> /fdb/igenomes/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/genome.fa <span class="dt">\</span></span>
<span id="cb22-8"><a href="vqsr.html#cb22-8" aria-hidden="true" tabindex="-1"></a> <span class="at">-V</span> merged.vcf.gz <span class="dt">\</span></span>
<span id="cb22-9"><a href="vqsr.html#cb22-9" aria-hidden="true" tabindex="-1"></a> <span class="at">--resource:hapmap,known</span><span class="op">=</span>false,training=true,truth=true,prior=15.0 <span class="dt">\</span></span>
<span id="cb22-10"><a href="vqsr.html#cb22-10" aria-hidden="true" tabindex="-1"></a> /fdb/GATK_resource_bundle/hg38/hapmap_3.3.hg38.vcf.gz <span class="dt">\</span></span>
<span id="cb22-11"><a href="vqsr.html#cb22-11" aria-hidden="true" tabindex="-1"></a> <span class="at">--resource:omni,known</span><span class="op">=</span>false,training=true,truth=false,prior=12.0 <span class="dt">\</span></span>
<span id="cb22-12"><a href="vqsr.html#cb22-12" aria-hidden="true" tabindex="-1"></a> /fdb/GATK_resource_bundle/hg38/1000G_omni2.5.hg38.vcf.gz <span class="dt">\</span></span>
<span id="cb22-13"><a href="vqsr.html#cb22-13" aria-hidden="true" tabindex="-1"></a> <span class="at">--resource:1000G,known</span><span class="op">=</span>false,training=true,truth=false,prior=10.0 <span class="dt">\</span></span>
<span id="cb22-14"><a href="vqsr.html#cb22-14" aria-hidden="true" tabindex="-1"></a> /fdb/GATK_resource_bundle/hg38/1000G_phase1.snps.high_confidence.hg38.vcf.gz <span class="dt">\</span></span>
<span id="cb22-15"><a href="vqsr.html#cb22-15" aria-hidden="true" tabindex="-1"></a> <span class="at">-an</span> QD <span class="at">-an</span> MQ <span class="at">-an</span> MQRankSum <span class="at">-an</span> ReadPosRankSum <span class="at">-an</span> FS <span class="at">-an</span> SOR <span class="dt">\</span></span>
<span id="cb22-16"><a href="vqsr.html#cb22-16" aria-hidden="true" tabindex="-1"></a> <span class="at">-mode</span> SNP <span class="at">-O</span> merged_SNP1.recal <span class="at">--tranches-file</span> output_SNP1.tranches <span class="dt">\</span></span>
<span id="cb22-17"><a href="vqsr.html#cb22-17" aria-hidden="true" tabindex="-1"></a> <span class="at">--rscript-file</span> output_SNP1.plots.R</span>
<span id="cb22-18"><a href="vqsr.html#cb22-18" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb22-19"><a href="vqsr.html#cb22-19" aria-hidden="true" tabindex="-1"></a><span class="bu">cd</span> data/<span class="kw">;</span> <span class="dt">\</span></span>
<span id="cb22-20"><a href="vqsr.html#cb22-20" aria-hidden="true" tabindex="-1"></a><span class="ex">gatk</span> <span class="at">--java-options</span> <span class="st">"-Djava.io.tmpdir=/lscratch/</span><span class="va">$SLURM_JOBID</span><span class="st"> -Xms4G -Xmx4G -XX:ParallelGCThreads=2"</span> VariantRecalibrator <span class="dt">\</span></span>
<span id="cb22-21"><a href="vqsr.html#cb22-21" aria-hidden="true" tabindex="-1"></a> <span class="at">-tranche</span> 100.0 <span class="at">-tranche</span> 99.95 <span class="at">-tranche</span> 99.9 <span class="dt">\</span></span>
<span id="cb22-22"><a href="vqsr.html#cb22-22" aria-hidden="true" tabindex="-1"></a> <span class="at">-tranche</span> 99.5 <span class="at">-tranche</span> 99.0 <span class="at">-tranche</span> 97.0 <span class="at">-tranche</span> 96.0 <span class="dt">\</span></span>
<span id="cb22-23"><a href="vqsr.html#cb22-23" aria-hidden="true" tabindex="-1"></a> <span class="at">-tranche</span> 95.0 <span class="at">-tranche</span> 94.0 <span class="at">-tranche</span> 93.5 <span class="at">-tranche</span> 93.0 <span class="dt">\</span></span>
<span id="cb22-24"><a href="vqsr.html#cb22-24" aria-hidden="true" tabindex="-1"></a> <span class="at">-tranche</span> 92.0 <span class="at">-tranche</span> 91.0 <span class="at">-tranche</span> 90.0 <span class="dt">\</span></span>
<span id="cb22-25"><a href="vqsr.html#cb22-25" aria-hidden="true" tabindex="-1"></a> <span class="at">-R</span> /fdb/igenomes/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/genome.fa <span class="dt">\</span></span>
<span id="cb22-26"><a href="vqsr.html#cb22-26" aria-hidden="true" tabindex="-1"></a> <span class="at">-V</span> merged.vcf.gz <span class="dt">\</span></span>
<span id="cb22-27"><a href="vqsr.html#cb22-27" aria-hidden="true" tabindex="-1"></a> <span class="at">--resource:mills,known</span><span class="op">=</span>false,training=true,truth=true,prior=12.0 <span class="dt">\</span></span>
<span id="cb22-28"><a href="vqsr.html#cb22-28" aria-hidden="true" tabindex="-1"></a> /fdb/GATK_resource_bundle/hg38/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz <span class="dt">\</span></span>
<span id="cb22-29"><a href="vqsr.html#cb22-29" aria-hidden="true" tabindex="-1"></a> <span class="at">--resource:dbsnp,known</span><span class="op">=</span>true,training=false,truth=false,prior=2.0 <span class="dt">\</span></span>
<span id="cb22-30"><a href="vqsr.html#cb22-30" aria-hidden="true" tabindex="-1"></a> /fdb/GATK_resource_bundle/hg38/dbsnp_146.hg38.vcf.gz <span class="dt">\</span></span>
<span id="cb22-31"><a href="vqsr.html#cb22-31" aria-hidden="true" tabindex="-1"></a> <span class="at">-an</span> QD <span class="at">-an</span> MQRankSum <span class="at">-an</span> ReadPosRankSum <span class="at">-an</span> FS <span class="at">-an</span> SOR <span class="at">-an</span> DP <span class="dt">\</span></span>
<span id="cb22-32"><a href="vqsr.html#cb22-32" aria-hidden="true" tabindex="-1"></a> <span class="at">-mode</span> INDEL <span class="at">-O</span> merged_indel1.recal <span class="at">--tranches-file</span> output_indel1.tranches <span class="dt">\</span></span>
<span id="cb22-33"><a href="vqsr.html#cb22-33" aria-hidden="true" tabindex="-1"></a> <span class="at">--rscript-file</span> output_indel1.plots.R</span></code></pre></div>
<p>Jobs can be submitted with <a href="https://hpc.nih.gov/apps/swarm.html">swarm</a>:</p>
<div class="sourceCode" id="cb23"><pre class="sourceCode sh"><code class="sourceCode bash"><span id="cb23-1"><a href="vqsr.html#cb23-1" aria-hidden="true" tabindex="-1"></a><span class="ex">swarm</span> <span class="at">-t</span> 2 <span class="at">-g</span> 4 <span class="at">--gres</span><span class="op">=</span>lscratch:400 <span class="at">--time</span><span class="op">=</span>2:00:00 <span class="at">-m</span> GATK/4.1.8.1 <span class="at">-f</span> 08-GATK_VariantRecalibrator.sh</span></code></pre></div>
</div>
<div id="benchmarks-of-applyvqsr" class="section level2" number="8.4">
<h2><span class="header-section-number">8.4</span> Benchmarks of <code>ApplyVQSR</code></h2>
<p>We did benchmarks on the performance of <code>ApplyVQSR</code> with different numbers of
CPUs and memory. As show in figure <a href="vqsr.html#fig:avqsr-cpu">8.4</a>, the running time is
not reduced much when using multiple threads as with the other essentially single
threaded tools.</p>
<div class="figure" style="text-align: center"><span id="fig:avqsr-cpu"></span>
<img src="images/08-ApplyVQSR_benchmark_cpu_constrain.png" alt="Runtime of ApplyVQSR as a function of the number of threads" width="70%" />
<p class="caption">
Figure 8.4: Runtime of <code>ApplyVQSR</code> as a function of the number of threads
</p>
</div>
<p>We normally recommend running jobs with 70%-80% efficiency. Based on figure <a href="vqsr.html#fig:avqsr-eff">8.5</a> we recommend running <code>ApplyVQSR</code> with
no more than two threads.</p>
<div class="figure" style="text-align: center"><span id="fig:avqsr-eff"></span>
<img src="images/08-ApplyVQSR_efficiency_cpu_constrain.png" alt="Efficiency of ApplyVQSR as a function of the number of threads" width="70%" />
<p class="caption">
Figure 8.5: Efficiency of <code>ApplyVQSR</code> as a function of the number of threads
</p>
</div>
<p>Increasing memory beyond the minimum needed did not improve performance
(figure <a href="vqsr.html#fig:avqsr-mem">8.6</a>). Using the <code>G1GC</code> garbage collector did not improve performance with default parameters, however
it requires extra 6GB memory to avoid out of memory kill.</p>
<div class="figure" style="text-align: center"><span id="fig:avqsr-mem"></span>
<img src="images/08-ApplyVQSR_benchmark_mem_constrain.png" alt="Runtime of ApplyVQSR as a function of memory" width="70%" />
<p class="caption">
Figure 8.6: Runtime of <code>ApplyVQSR</code> as a function of memory
</p>
</div>
</div>
<div id="optimized-script-for-applyvqsr" class="section level2" number="8.5">
<h2><span class="header-section-number">8.5</span> Optimized script for <code>ApplyVQSR</code></h2>
<p>Step 2: We choose the VQSLOD cutoff to filter VCF file using <code>ApplyVQSR</code>:</p>
<div class="sourceCode" id="cb24"><pre class="sourceCode sh"><code class="sourceCode bash"><span id="cb24-1"><a href="vqsr.html#cb24-1" aria-hidden="true" tabindex="-1"></a><span class="co">#! /bin/bash</span></span>
<span id="cb24-2"><a href="vqsr.html#cb24-2" aria-hidden="true" tabindex="-1"></a><span class="bu">set</span> <span class="at">-euo</span></span>
<span id="cb24-3"><a href="vqsr.html#cb24-3" aria-hidden="true" tabindex="-1"></a><span class="co"># run ApplyVQSR on SNP then INDEL</span></span>
<span id="cb24-4"><a href="vqsr.html#cb24-4" aria-hidden="true" tabindex="-1"></a><span class="ex">module</span> load GATK/4.1.8.1</span>
<span id="cb24-5"><a href="vqsr.html#cb24-5" aria-hidden="true" tabindex="-1"></a><span class="bu">cd</span> data/<span class="kw">;</span></span>
<span id="cb24-6"><a href="vqsr.html#cb24-6" aria-hidden="true" tabindex="-1"></a><span class="ex">gatk</span> <span class="at">--java-options</span> <span class="st">"-Djava.io.tmpdir=/lscratch/</span><span class="va">$SLURM_JOBID</span><span class="st"> </span><span class="dt">\</span></span>
<span id="cb24-7"><a href="vqsr.html#cb24-7" aria-hidden="true" tabindex="-1"></a><span class="st"> -Xms2G -Xmx2G -XX:ParallelGCThreads=2"</span> ApplyVQSR <span class="dt">\</span></span>
<span id="cb24-8"><a href="vqsr.html#cb24-8" aria-hidden="true" tabindex="-1"></a> <span class="at">-V</span> merged.vcf.gz <span class="dt">\</span></span>
<span id="cb24-9"><a href="vqsr.html#cb24-9" aria-hidden="true" tabindex="-1"></a> <span class="at">--recal-file</span> merged_SNP1.recal <span class="dt">\</span></span>
<span id="cb24-10"><a href="vqsr.html#cb24-10" aria-hidden="true" tabindex="-1"></a> <span class="at">-mode</span> SNP <span class="dt">\</span></span>
<span id="cb24-11"><a href="vqsr.html#cb24-11" aria-hidden="true" tabindex="-1"></a> <span class="at">--tranches-file</span> output_SNP1.tranches <span class="dt">\</span></span>
<span id="cb24-12"><a href="vqsr.html#cb24-12" aria-hidden="true" tabindex="-1"></a> <span class="at">--truth-sensitivity-filter-level</span> 99.9 <span class="dt">\</span></span>
<span id="cb24-13"><a href="vqsr.html#cb24-13" aria-hidden="true" tabindex="-1"></a> <span class="at">--create-output-variant-index</span> true <span class="dt">\</span></span>
<span id="cb24-14"><a href="vqsr.html#cb24-14" aria-hidden="true" tabindex="-1"></a> <span class="at">-O</span> SNP.recalibrated_99.9.vcf.gz</span>
<span id="cb24-15"><a href="vqsr.html#cb24-15" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb24-16"><a href="vqsr.html#cb24-16" aria-hidden="true" tabindex="-1"></a><span class="ex">gatk</span> <span class="at">--java-options</span> <span class="st">"-Djava.io.tmpdir=/lscratch/</span><span class="va">$SLURM_JOBID</span><span class="st"> </span><span class="dt">\</span></span>
<span id="cb24-17"><a href="vqsr.html#cb24-17" aria-hidden="true" tabindex="-1"></a><span class="st"> -Xms2G -Xmx2G -XX:ParallelGCThreads=2"</span> ApplyVQSR <span class="dt">\</span></span>
<span id="cb24-18"><a href="vqsr.html#cb24-18" aria-hidden="true" tabindex="-1"></a> <span class="at">-V</span> SNP.recalibrated_99.9.vcf.gz <span class="dt">\</span></span>
<span id="cb24-19"><a href="vqsr.html#cb24-19" aria-hidden="true" tabindex="-1"></a> <span class="at">-mode</span> INDEL <span class="dt">\</span></span>
<span id="cb24-20"><a href="vqsr.html#cb24-20" aria-hidden="true" tabindex="-1"></a> <span class="at">--recal-file</span> merged_indel1.recal <span class="dt">\</span></span>
<span id="cb24-21"><a href="vqsr.html#cb24-21" aria-hidden="true" tabindex="-1"></a> <span class="at">--tranches-file</span> output_indel1.tranches <span class="dt">\</span></span>
<span id="cb24-22"><a href="vqsr.html#cb24-22" aria-hidden="true" tabindex="-1"></a> <span class="at">--truth-sensitivity-filter-level</span> 99.9 <span class="dt">\</span></span>
<span id="cb24-23"><a href="vqsr.html#cb24-23" aria-hidden="true" tabindex="-1"></a> <span class="at">--create-output-variant-index</span> true <span class="dt">\</span></span>
<span id="cb24-24"><a href="vqsr.html#cb24-24" aria-hidden="true" tabindex="-1"></a> <span class="at">-O</span> indel.SNP.recalibrated_99.9.vcf.gz</span></code></pre></div>
<p>99.9% is the recommended default VQSLOD cutoff for SNPs in human genomic analysis.</p>
<p>Job submission:</p>
<div class="sourceCode" id="cb25"><pre class="sourceCode sh"><code class="sourceCode bash"><span id="cb25-1"><a href="vqsr.html#cb25-1" aria-hidden="true" tabindex="-1"></a><span class="ex">sbatch</span> <span class="at">--cpus-per-task</span><span class="op">=</span>2 <span class="at">--mem</span><span class="op">=</span>2g <span class="at">--gres</span><span class="op">=</span>lscratch:100 <span class="at">--time</span><span class="op">=</span>2:00:00 08-GATK_ApplyVQSR.sh</span></code></pre></div>
<p>Note:</p>
<ul>
<li>For INDEL there is no recommended VQSLOD cutoff, here we use 99.9 just as an example.</li>
</ul>
</div>
</div>
</section>
</div>
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