Course_Materials/visualization/tutorial/020_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>Visualization of mapped reads</strong></h2>
<h3 class="date"><em>(updated 26-02-2014)</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://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="exercise-2-visualizing-rna-seq-data">Exercise 2: Visualizing RNA-Seq data</h1>
<p>In the following <strong>folder</strong> you wil find mapped RNA-Seq data from Illumina’s Human BodyMap 2.0 project. This two datasets in particular contain reads from brain and heart tissues.</p>
<pre><code>cd /home/participant/cambridge_mda14/visualization/example_1</code></pre>
<h2 id="steps">Steps</h2>
<h5 id="use-samtools-to-index-the-bam-file">1. Use <code>samtools</code> to index the bam file:</h5>
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<pre><code>samtools index heart.bodyMap.bam
samtools index brain.bodyMap.bam</code></pre>
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<h5 id="open-igv-and-load-the-bam-file-heart.bodymap.bam.">2. Open IGV and load the bam file heart.bodyMap.bam.</h5>
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<pre><code>igv</code></pre>
<ul>
<li>Go to <code>File</code> –&gt; <code>Load from file...</code> Select heart.bodyMap.bam and brain.bodyMap.bam</li>
</ul>
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<h5 id="navigate-a-little-and-get-familiar-with-the-software.">3. Navigate a little and get familiar with the software.</h5>
<p>Reads are only on <strong>chromosome 12 from 98Mbp to 100Mbp</strong>. Remember that 1Mbp = 1000000bp.</p>
<p>So, reads will be around <code>12:98000000-100000000</code>.</p>
<h5 id="look-for-ikbip-gene-and-try-to-answer-the-following-questions">4. Look for <strong><em>IKBIP</em></strong> gene and try to answer the following questions:</h5>
<ul>
<li>How many different isoforms does the gene have? Which exon combinations form every isoform? <strong>TIP:</strong> Right-click on the Features panel –&gt; Select <code>Expanded</code> <input class="spoilerbutton" type="button" value="Show answer" onclick="this.value=this.value=='Show answer'?'Hide answer':'Show answer';">
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The gene <strong>IKBIP</strong> contains 4 exons that generate <strong>3 different isoforms</strong>:<br>- Isoform 1: exon 1 + exon 4<br>- Isoform 2: exon 1 + exon 3 + exon 4<br>- Isoform 3: exon 2 + exon 3 + exon 4</li>
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<ul>
<li><p>Is <strong>IKBIP</strong> gene expressed in heart and brain tissues?</p></li>
<li>Create a <strong>Sashimi plot</strong> of the gene. <strong>TIP:</strong> center the gene IKBIP on the Track panel, right-click on the Panel and select <code>Sashimi Plot</code>.<br /> What kind of information can you obtain from this plot? <input class="spoilerbutton" type="button" value="Show answer" onclick="this.value=this.value=='Show answer'?'Hide answer':'Show answer';">
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The Sashimi Plot shows raw RNA-Seq densities along exons and junctions. Lines indicate junctions and the number corresponds to the number of reads that support this junction.</li>
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<ul>
<li>Load SNP data from <strong>dbSNP 1.3.7</strong> and go to position <strong>chr12:99019058</strong>. Is the heart sample homozygous or heterozygous for this position? Would you trust this variant? Is this variant a known SNP? <input class="spoilerbutton" type="button" value="Show answer" onclick="this.value=this.value=='Show answer'?'Hide answer':'Show answer';">
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<code>-</code> Load and look at the SNP track: <code>File</code> –&gt; <code>Load from server</code> –&gt; <code>Annotations</code> –&gt; <code>Variants and Repeats</code> –&gt; <code>dbSNP 1.3.7</code><br>- This individual is heterozygous for this position with alleles A/G.<br> - Yes. There are 17 high quality reads (47%) supporting this variant and the base quality is, in general, high.<br> - Yes, rs7310804.</li>
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