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<title>Mapping traits in biparental populations with genotyping-by-sequencing data</title>
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</head>
<body class="article toc2 toc-left">
<div id="header">
<h1>Mapping traits in biparental populations with genotyping-by-sequencing data</h1>
<div id="toc" class="toc2">
<div id="toctitle">Table of Contents</div>
<ul class="sectlevel1">
<li><a href="#_open_a_unix_shell">1. Open a UNIX shell</a></li>
<li><a href="#_set_up_your_analysis_directory">2. Set up your analysis directory</a></li>
<li><a href="#_read_mapping">3. Read mapping</a></li>
<li><a href="#_variant_calling_and_filtration">4. Variant calling and filtration</a></li>
<li><a href="#_import_the_genetic_variant_matrix_into_r">5. Import the genetic variant matrix into R</a></li>
<li><a href="#_quality_control_and_linkage_map_construction">6. Quality control and linkage map construction</a></li>
<li><a href="#_trait_mapping">7. Trait mapping</a></li>
<li><a href="#_mapping_the_kasp_markers">8. Mapping the KASP markers</a></li>
</ul>
</div>
</div>
<div id="content">
<div id="preamble">
<div class="sectionbody">
<link rel="stylesheet" href="http://cdnjs.cloudflare.com/ajax/libs/font-awesome/3.1.0/css/font-awesome.min.css">
<div class="imageblock text-center">
<div class="content">
<img src="GBStutorial_flowchart.jpeg" alt="GBStutorial flowchart">
</div>
</div>
</div>
</div>
<div class="sect1">
<h2 id="_open_a_unix_shell">1. Open a UNIX shell</h2>
<div class="sectionbody">
<div class="olist arabic">
<ol class="arabic">
<li>
<p>Connect with <a href="https://en.wikipedia.org/wiki/PuTTY">PuTTY</a> to the server slurm-01.ipk-gatersleben.de.</p>
</li>
<li>
<p>Obtain a Kerberos ticket to access our filer resources.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh">kinit
klist <i class="conum" data-value="1"></i><b>(1)</b></code></pre>
</div>
</div>
<div class="colist arabic">
<table>
<tr>
<td><i class="conum" data-value="1"></i><b>1</b></td>
<td>A ticket is valid for seven days.</td>
</tr>
</table>
</div>
</li>
<li>
<p>You will see the prompt of the UNIX command-line interpreter, the <a href="https://en.wikipedia.org/wiki/Unix_shell">shell</a>.
It is called "shell" because it executes commands that do not belong to the operating system, the "kernel".</p>
</li>
<li>
<p>Check out your home directory.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh"><span class="nb">pwd
ls</span></code></pre>
</div>
</div>
</li>
</ol>
</div>
</div>
</div>
<div class="sect1">
<h2 id="_set_up_your_analysis_directory">2. Set up your analysis directory</h2>
<div class="sectionbody">
<div class="olist arabic">
<ol class="arabic">
<li>
<p>Let’s have a look at the GBS data.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh"><span class="nb">cd</span> /filer/projekte/gbs_course
<span class="nb">cd </span>data
<span class="nb">ls
ls</span> <span class="nt">-lhS</span> gbs_reads | less <i class="conum" data-value="1"></i><b>(1)</b>
man <span class="nb">ls
cd </span>gbs_reads
<span class="nb">ls</span> | <span class="nb">wc</span> <span class="nt">-l</span>
zcat Morex.fastq.gz | less <span class="nt">-S</span> <i class="conum" data-value="2"></i><b>(2)</b>
<span class="nb">cd</span> ..
<span class="nb">du</span> <span class="nt">-ch</span> gbs_reads</code></pre>
</div>
</div>
<div class="colist arabic">
<table>
<tr>
<td><i class="conum" data-value="1"></i><b>1</b></td>
<td>To close <code>less</code>, press <code>q</code>.</td>
</tr>
<tr>
<td><i class="conum" data-value="2"></i><b>2</b></td>
<td>The reads are in <a href="https://en.wikipedia.org/wiki/FASTQ_format">FASTQ format</a>.</td>
</tr>
</table>
</div>
</li>
<li>
<p>Take a look at the phenotypes. There are two. One is binary: lateral spikelet fertility (commonly referred to in barley as row type). The other is quantitative (plant height).</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh">less MxB_92_RILs_phenotypes.csv</code></pre>
</div>
</div>
</li>
<li>
<p>Create your working directory and change into it.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh"><span class="nb">cd</span> /filer/projekte/gbs_course
<span class="nb">mkdir</span> <span class="nt">-p</span> mascher/gbs <i class="conum" data-value="1"></i><b>(1)</b>
<span class="nb">cd </span>mascher/gbs</code></pre>
</div>
</div>
<div class="colist arabic">
<table>
<tr>
<td><i class="conum" data-value="1"></i><b>1</b></td>
<td>Change <code>mascher</code> to your name.</td>
</tr>
</table>
</div>
</li>
<li>
<p>Create symbolic links to the GBS reads and the phenotype data.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh"><span class="nb">pwd <i class="conum" data-value="1"></i><b>(1)</b>
mkdir </span>gbs_reads
<span class="nv">datadir</span><span class="o">=</span><span class="s2">"/filer/projekte/gbs_course/data/"</span>
<span class="nb">ln</span> <span class="nt">-s</span> <span class="nv">$datadir</span>/gbs_reads/<span class="k">*</span>fastq.gz gbs_reads
<span class="nb">ls</span> <span class="nt">-l</span> gbs_reads | less <span class="nt">-S</span>
<span class="nb">ls </span>gbs_reads | <span class="nb">wc</span> <span class="nt">-l</span>
<span class="nb">ln</span> <span class="nt">-s</span> <span class="nv">$datadir</span>/MxB_92_RILs_phenotypes.csv</code></pre>
</div>
</div>
<div class="colist arabic">
<table>
<tr>
<td><i class="conum" data-value="1"></i><b>1</b></td>
<td>Check that you are in your working directory (<code>/filer/projekte/gbs_course/USER/gbs</code>).</td>
</tr>
</table>
</div>
</li>
<li>
<p>Count the reads. We use a for-loop to process all samples at once.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="r"><span class="n">zcat</span><span class="w"> </span><span class="n">gbs_reads</span><span class="o">/</span><span class="n">Morex.fastq.gz</span><span class="w"> </span><span class="o">|</span><span class="w"> </span><span class="n">wc</span><span class="w"> </span><span class="o">-</span><span class="n">l</span><span class="w"> </span><span class="o">></span><span class="w"> </span><span class="n">Morex.len</span><span class="w">
</span><span class="k">for</span><span class="w"> </span><span class="n">i</span><span class="w"> </span><span class="k">in</span><span class="w"> </span><span class="n">gbs_reads</span><span class="o">/*</span><span class="n">fastq.gz</span><span class="p">;</span><span class="w"> </span><span class="n">do</span><span class="w">
</span><span class="n">name</span><span class="o">=</span><span class="n">`basename $i | cut -d . -f 1`</span><span class="w">
</span><span class="n">count</span><span class="o">=</span><span class="n">`zcat $i | wc -l`</span><span class="w">
</span><span class="n">echo</span><span class="w"> </span><span class="o">$</span><span class="n">name</span><span class="w"> </span><span class="o">$</span><span class="n">count</span><span class="w">
</span><span class="n">done</span><span class="w"> </span><span class="o">></span><span class="w"> </span><span class="n">fastq_line_counts.txt</span><span class="w">
</span><span class="n">cat</span><span class="w"> </span><span class="n">fastq_line_counts.txt</span><span class="w"> </span><span class="o">|</span><span class="w"> </span><span class="n">awk</span><span class="w"> </span><span class="s1">'{print $1,$2/4}'</span><span class="w"> </span><span class="o">|</span><span class="w"> </span><span class="n">sort</span><span class="w"> </span><span class="o">-</span><span class="n">nk</span><span class="w"> </span><span class="m">2</span><span class="w"> </span><span class="o">></span><span class="w"> </span><span class="n">fastq_read_counts.txt</span><span class="w"> <i class="conum" data-value="1"></i><b>(1)</b>
</span></code></pre>
</div>
</div>
<div class="colist arabic">
<table>
<tr>
<td><i class="conum" data-value="1"></i><b>1</b></td>
<td>We use <a href="https://www.tutorialspoint.com/awk/index.htm">AWK</a> to divide the second column by 4.</td>
</tr>
</table>
</div>
</li>
</ol>
</div>
</div>
</div>
<div class="sect1">
<h2 id="_read_mapping">3. Read mapping</h2>
<div class="sectionbody">
<div class="olist arabic">
<ol class="arabic">
<li>
<p>We will use the tools <a href="https://github.com/lh3/bwa">BWA</a> and <a href="http://www.htslib.org/doc/samtools.html">samtools</a> for read mapping and processing of alignment records, respectively.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh">module load bwa samtools
module list</code></pre>
</div>
</div>
</li>
<li>
<p>Load the BWA index into shared memory. This step can be skipped, but pre-loadeding the index makes mapping a lot faster.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh"><span class="nv">ref</span><span class="o">=</span><span class="s1">'/filer/projekte/gbs_course/data/reference/Hordeum_vulgare_MorexV3_assembly.fasta'</span> <i class="conum" data-value="1"></i><b>(1)</b>
bwa shm <span class="nv">$ref</span>
bwa shm <span class="nt">-l</span></code></pre>
</div>
</div>
<div class="colist arabic">
<table>
<tr>
<td><i class="conum" data-value="1"></i><b>1</b></td>
<td>We use the <a href="https://academic.oup.com/plcell/article/33/6/1888/6169005">MorexV3</a> reference genome sequence.</td>
</tr>
</table>
</div>
</li>
<li>
<p>We will first run a toy example with 10,000 reads of the Morex sample.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh">zcat gbs_reads/Morex.fastq.gz | <span class="nb">head</span> <span class="nt">-n</span> 40000 <span class="o">></span> Morex_10k.fastq
bwa mem <span class="nv">$ref</span> Morex_10k.fastq <span class="o">></span> Morex_10k.sam <i class="conum" data-value="1"></i><b>(1)</b>
samtools <span class="nb">sort </span>Morex_10k.sam <span class="o">></span> Morex_10k.bam <i class="conum" data-value="1"></i><b>(1)</b>
samtools view Morex_10k.bam | less <span class="nt">-S</span></code></pre>
</div>
</div>
<div class="colist arabic">
<table>
<tr>
<td><i class="conum" data-value="1"></i><b>1</b></td>
<td>Alignments are stored in the <a href="https://en.wikipedia.org/wiki/SAM_(file_format)">Sequence Alignment Map format</a> (SAM) or its binary equivalent BAM.</td>
</tr>
</table>
</div>
</li>
<li>
<p>Combine mapping and sorting into one command.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh">bwa mem <span class="nv">$ref</span> Morex_10k.fastq | samtools <span class="nb">sort</span> <span class="o">></span> Morex_10k.bam</code></pre>
</div>
</div>
</li>
<li>
<p>Open a new PuTTY session. Start <a href="https://www.hamvocke.com/blog/a-quick-and-easy-guide-to-tmux/">tmux</a> to keep your commands running in the background.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh">k5reauth tmux <i class="conum" data-value="1"></i><b>(1)</b></code></pre>
</div>
</div>
<div class="colist arabic">
<table>
<tr>
<td><i class="conum" data-value="1"></i><b>1</b></td>
<td>k5reauth is used to prolong the Kerberos ticket for as long as the tmux session is running.</td>
</tr>
</table>
</div>
</li>
<li>
<p>Try out tmux functionality.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh">tmux <span class="nb">ls
</span>tmux rename mapping
tmux <span class="nb">ls
</span>tmux detach
tmux <span class="nb">ls
</span>tmux attach <span class="nt">-t</span> mapping</code></pre>
</div>
</div>
</li>
<li>
<p>Map all samples.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh"><span class="nv">ref</span><span class="o">=</span><span class="s1">'/filer/projekte/gbs_course/data/reference/Hordeum_vulgare_MorexV3_assembly.fasta'</span>
<span class="k">for </span>i <span class="k">in </span>gbs_reads/<span class="k">*</span>fastq.gz<span class="p">;</span> <span class="k">do
</span><span class="nv">name</span><span class="o">=</span><span class="sb">`</span><span class="nb">echo</span> <span class="nv">$i</span> | <span class="nb">cut</span> <span class="nt">-d</span> <span class="nb">.</span> <span class="nt">-f</span> 1<span class="sb">`</span> <i class="conum" data-value="1"></i><b>(1)</b>
bwa mem <span class="nt">-t</span> 4 <span class="nv">$ref</span> <span class="nv">$i</span> | samtools <span class="nb">sort</span> <span class="o">></span> <span class="nv">$name</span>.bam
<span class="k">done </span>2> bwa.err <i class="conum" data-value="2"></i><b>(2)</b></code></pre>
</div>
</div>
<div class="colist arabic">
<table>
<tr>
<td><i class="conum" data-value="1"></i><b>1</b></td>
<td>Strip the extension: Morex.fastq.gz become Morex.</td>
</tr>
<tr>
<td><i class="conum" data-value="2"></i><b>2</b></td>
<td>To detach the tmux session, press <kbd>Ctrl-B</kbd> followed by <kbd>D</kbd>.</td>
</tr>
</table>
</div>
<div class="admonitionblock important">
<table>
<tr>
<td class="icon">
<i class="fa icon-important" title=":heavy_exclamation_mark:"></i>
</td>
<td class="content">
If you forget to start bwa inside a tmux session, there is no way to prevent your job from aborting when you shutdown your laptop. Also without k5reauth programs cannot access filer resource after a maximum of ten hours.
</td>
</tr>
</table>
</div>
</li>
<li>
<p>Open a new terminal. Look at your jobs in the table of processes (<code>top</code>).</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh">find gbs_reads | <span class="nb">grep</span> <span class="nt">-c</span> <span class="s1">'bam$'</span> <i class="conum" data-value="1"></i><b>(1)</b>
top <span class="nt">-u</span> mascher <i class="conum" data-value="2"></i><b>(2)</b></code></pre>
</div>
</div>
<div class="colist arabic">
<table>
<tr>
<td><i class="conum" data-value="1"></i><b>1</b></td>
<td>Show the number of BAM files created so far.</td>
</tr>
<tr>
<td><i class="conum" data-value="2"></i><b>2</b></td>
<td>Replace mascher with your username.</td>
</tr>
</table>
</div>
</li>
<li>
<p>When the mapping is done, calculate the mapping rates for all samples.</p>
<div class="listingblock">
<div class="content">
<pre class="rouge highlight"><code data-lang="sh"><span class="k">for </span>i <span class="k">in </span>gbs_reads/<span class="k">*</span>bam<span class="p">;</span> <span class="k">do
</span><span class="nv">name</span><span class="o">=</span><span class="sb">`</span><span class="nb">basename</span> <span class="nv">$i</span> | <span class="nb">cut</span> <span class="nt">-d</span> <span class="nb">.</span> <span class="nt">-f</span> 1<span class="sb">`</span>
<span class="nv">count</span><span class="o">=</span><span class="sb">`</span>samtools view <span class="nt">-cq</span> 20 <span class="nv">$i</span><span class="sb">`</span>
<span class="nb">echo</span> <span class="nv">$name</span> <span class="nv">$count</span>
<span class="k">done</span> <span class="o">></span> mapped_reads.txt</code></pre>
</div>
</div>
</li>
<li>
<p>Combine the raw read counts and the mapping rates into one table.</p>
<div class="listingblock">
<div class="content">