ngs-course.github.io/Course_Materials/variant_calling/tutorial/030_example.html at master · imedina/ngs-course.github.io · GitHub

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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 08-06-2014)</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://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="exercise-3-somatic-calling">Exercise 3: Somatic calling</h1>
<h2 id="prepare-bam-file">1. Prepare BAM file</h2>
<p>Go to the example3 folder:</p>
<pre><code>cd /home/participant/cambridge_mda14/calling/example3</code></pre>
<p>Sort:</p>
<pre><code>samtools sort 000-normal.bam 001-normal_sorted
samtools sort 000-tumor.bam 001-tumor_sorted</code></pre>
<p>Index the BAM file:</p>
<pre><code>samtools index 001-normal_sorted.bam
samtools index 001-tumor_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>java -jar ../mutect/muTect-1.1.4.jar --analysis_type MuTect --reference_sequence ../genome/f000_chr21_ref_genome_sequence.fa --dbsnp 000-dbsnp_132_b37.leftAligned.vcf.gz --cosmic 000-b37_cosmic_v54_120711.vcf.gz --input_file:normal 001-normal_sorted.bam --input_file:tumor 001-tumor_sorted.bam --out 002-call_stats.out --coverage_file 002-coverage.wig --vcf 003-somatic_variants.vcf</code></pre>
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