COURSE_MATERIALS/variant_calling/example/030_example.html

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<h1 class="title"><a href="http://ngscourse.github.io/">NGS data analysis course</a></h1>
<h2 class="author"><strong>Variant calling</strong></h2>
<h3 class="date"><em>(updated 28-02-2014)</em></h3>
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<h1 id="preliminaries">Preliminaries</h1>
<h2 id="software-used-in-this-practical">Software used in this practical:</h2>
<ul>
<li><a href="http://bio-bwa.sourceforge.net/" title="BWA">BWA</a> : BWA is a software package for mapping low-divergent sequences against a large reference genome, such as the human genome.</li>
<li><a href="http://samtools.sourceforge.net/" title="samtools">SAMTools</a> : SAM Tools provide various utilities for manipulating alignments in the SAM format, including sorting, merging, indexing and generating alignments in a per-position format.</li>
<li><a href="http://picard.sourceforge.net/" title="Picard">Picard</a> : Picard comprises Java-based command-line utilities that manipulate SAM files, and a Java API (SAM-JDK) for creating new programs that read and write SAM files.</li>
<li><a href="http://www.broadinstitute.org/cancer/cga/mutect_download" title="MuTect">MuTect</a> : method developed at the Broad Institute for the reliable and accurate identification of somatic point mutations in next generation sequencing data of cancer genomes.</li>
</ul>
<h2 id="file-formats-explored">File formats explored:</h2>
<ul>
<li><a href="http://samtools.sourceforge.net/SAMv1.pdf">SAM</a></li>
<li><a href="http://www.broadinstitute.org/igv/bam">BAM</a></li>
<li>VCF Variant Call Format: see [1000 Genomes][vcf-format-1000ge] and [Wikipedia][vcf-format-wikipedia] specifications.</li>
</ul>
<h1 id="some-previous-configurations">Some previous configurations</h1>
<p>Download MuTect software from http://www.broadinstitute.org/cancer/cga/mutect_download</p>
<p>You need to register before downloading. Extract the file in your desktop.</p>
<p>Just to run MuTect easier:</p>
<pre><code>echo &quot;alias MuTect.jar=&#39;java -jar /home/Desktop/muTect-1.1.4-bin/muTect-1.1.4.jar&#39;&quot; &gt;&gt; ~/.bashrc
source ~/.bashrc</code></pre>
<h1 id="exercise-3-somatic-calling">Exercise 3: Somatic calling</h1>
<h2 id="prepare-bam-file">1. Prepare BAM file</h2>
<p>Go to the example1 folder:</p>
<pre><code>cd example3</code></pre>
<p>Add read groups:</p>
<pre><code>AddOrReplaceReadGroups.jar I=f000-normal.bam O=f010-normal_fixRG.bam RGID=group1 RGLB=lib1 RGPL=illumina RGSM=sample1 RGPU=unit1
AddOrReplaceReadGroups.jar I=f000-tumor.bam O=f010-tumor_fixRG.bam RGID=group2 RGLB=lib1 RGPL=illumina RGSM=sample1 RGPU=unit2</code></pre>
<p>Sort:</p>
<pre><code>samtools sort f010-normal_fixRG.bam f020-normal_fixRG_sorted
samtools sort f010-tumor_fixRG.bam f020-tumor_fixRG_sorted</code></pre>
<p>Index the BAM file:</p>
<pre><code>samtools index f020-normal_fixRG_sorted.bam
samtools index f020-tumor_fixRG_sorted.bam</code></pre>
<h2 id="somatic-calling">2. Somatic calling</h2>
<p>For brevity, we are not including BAM preprocessing steps. However, in real analysis it is recommended to include them.</p>
<pre><code>MuTect.jar --analysis_type MuTect --reference_sequence ../f000-reference.fa --dbsnp ../f000-dbSNP_chr21.vcf --input_file:normal f020-normal_fixRG_sorted.bam \  
--input_file:tumor f020-tumor_fixRG_sorted.bam --out f030-call_stats.out --coverage_file f030-coverage.wig.txt</code></pre>
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