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<h1 class="title"><a href="http://ngscourse.github.io/">NGS data analysis course</a></h1>
<h2 class="author"><strong>Visualization of mapped reads</strong></h2>
<h3 class="date"><em>(updated 21-10-2015)</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://www.broadinstitute.org/igv/home" title="IGV">IGV</a> : The Integrative Genomics Viewer is a program for reading several types of indexed database information, including mapped reads and variant calls, and displaying them on a reference genome. It is invaluable as a tool for viewing and interpreting the “raw data” of many NGS data analysis pipelines.</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>
</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>
</ul>
<h1 id="guided-exercise-igv-introduction">Guided exercise: IGV introduction</h1>
<p>Directory used for the tutorials:</p>
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mkdir -p /home/participant/cambridge_mda15/
cp -r /home/participant/Desktop/Open_Share/visualization /home/participant/cambridge_mda15/
cd /home/participant/cambridge_mda15/visualization
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<!--    cd /home/participant/Desktop/Course_Materials/visualization -->

<pre><code>cd /home/training/ngs_course/visualization</code></pre>
<p>Enter in the example folder:</p>
<pre><code>cd example_0</code></pre>
<p>This BAM file contains reads only for <strong>chromosome 11</strong>.</p>
<h2 id="creating-indexed-files">Creating indexed files</h2>
<p>Use <code>samtools</code> to index the bam files:</p>
<pre><code>samtools index igv1.bam</code></pre>
<h2 id="run-igv">Run IGV</h2>
<p>You can run IGV using this command from the terminal:</p>
<pre><code>igv.sh &amp;</code></pre>
<!--or you can also use the link in your Desktop.-->


<h2 id="download-a-reference-genome">Download a reference genome</h2>
<p>By default, IGV loads Human hg18. Bear in mind that we must work with the <strong>same assembly used to mapped our reads</strong>, in this case Human hg19.<br />Genome assemblies for several species can be downloaded using IGV. To explore the list of available genomes:</p>
<ul>
<li>Go to <code>Genomes</code> –&gt; <code>Load Genome From Server...</code></li>
</ul>
<p>In this practical we are using <strong>Human hg19</strong></p>
<h2 id="loading-and-browsing-files">Loading and browsing files</h2>
<ul>
<li>Go to <code>File</code> –&gt; <code>Load from file...</code> Select <code>igv1.bam</code></li>
</ul>
<p><strong>Steps:</strong></p>
<ol style="list-style-type: decimal">
<li>Go to the location box and insert ‘’chr11:996096-1036047’’ in the search box and hit <code>Go</code>.</li>
<li>Move across the alignment</li>
<li>Zoom in</li>
<li>Grey boxes are reads:
<ul>
<li>point to the left –&gt; map reverse strand</li>
<li>point to the right –&gt; map forward strand</li>
</ul></li>
<li>At the top –&gt; histogram of coverage</li>
<li>Zoom further
<ul>
<li>Colour representation of the sequence</li>
<li>Zoom in –&gt; Bases are shown</li>
<li>Amminoacids corresponding to each base</li>
<li>See all possible translation reading frames</li>
</ul></li>
<li>Change read information panels</li>
</ol>
<h2 id="what-we-want-to-do-with-igv">What we want to do with IGV?</h2>
<ol style="list-style-type: decimal">
<li>Examine coverage
<ul>
<li>High/low coverage</li>
<li>No coverage</li>
</ul></li>
<li>Visualise alternative splicing
<ul>
<li>RNA alignment against DNA, not cDNA</li>
</ul></li>
<li>Look for base changes – &gt; SNPs
<ul>
<li>Complete grey reads –&gt; no mismatches</li>
<li>Coloured bases –&gt; Change
<ul>
<li>Coverage track os only coloured if &gt;20% of the reads support the variant</li>
</ul></li>
<li>Reads are shadowed according to quality
<ul>
<li>Darker –&gt; good quality</li>
<li>Lighter –&gt; bad quality</li>
</ul></li>
<li>Bases are also shadowed according to quality
<ul>
<li>Darker –&gt; good quality</li>
<li>Lighter –&gt; bad quality</li>
</ul></li>
<li>Inspecting a SNP (<code>chr11:1,018,335-1,018,412</code>)
<ul>
<li>Look at the coverage bar –&gt; percentage and counts</li>
<li>Sort alignment by base</li>
</ul></li>
<li>Inspecting a homozygous variant (<code>chr11:1,019,336-1,019,413</code>) - All reads support the variant but 1 - The reference read quality is 0</li>
</ul></li>
<li>Load SNP data
<ul>
<li>File &gt; Load from server &gt; Annotations &gt; Variation and repeats &gt; dbSNP 1.3.7</li>
</ul></li>
</ol>
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