Commit 326dc474 authored by tekath's avatar tekath
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Test with first vignette snippet.

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      <h1 data-toc-skip>Preprocessing of the Hoffman et al. human single-cell RNA-seq data</h1>

                        <h4 class="author">Tobias Tekath</h4>

            

            <h4 class="date">2020-06-30</h4>

      

      

      <div class="hidden name"><code>Hoffman_human_single-cell_preprocess.Rmd</code></div>



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<p>This vignette examplifies the preprocessing of <strong>single-cell RNA-seq data for analysis with DTUrtle</strong>. The data used in this vignette is publicly available as <em>Bioproject PRJNA594939</em> and the used <em>FASTQ</em>-files can be downloaded from <a href="https://www.ebi.ac.uk/ena/browser/view/PRJNA594939">here</a>. The corresponding publication from Hoffman et al. to this dataset can be found <a href="https://doi.org/10.1038/s42003-020-0837-0">here</a>.</p>

<p>For this vignette we focus on just two of the available single-cell RNA-seq samples:</p>

<ul>

<li>

<a href="https://www.ebi.ac.uk/ena/browser/view/SAMN13541133">SAMN13541133</a> which represents a control sample (18 hour treatment with ethanol (EtOH))</li>

<li>

<a href="https://www.ebi.ac.uk/ena/browser/view/SAMN13541131">SAMN13541131</a> which represants a sample after 2 hourse of Dexamethasone treatment (Dex2hr)</li>

</ul>

<p>The <em>FASTQ</em>-files can be directly obtained from ENA, alternatively they are also available as <em>SRA</em>-files from <a href="https://www.ncbi.nlm.nih.gov/bioproject/PRJNA594939">GEO</a>, which can be converted to <em>FASTQ</em>-format.</p>

<p>After downloading a <strong>MD5-check</strong> is strongly encouraged.</p>

<div id="preparing-fastq-files" class="section level3">

<h3 class="hasAnchor">

<a href="#preparing-fastq-files" class="anchor"></a>Preparing FASTQ-files</h3>

<p>For this vignette, it is assumed that the <em>FASTQ</em>-files mentioned above have been downloaded to a directory called <em>samples</em>.</p>

<p>After downloading the <em>FASTQ</em>-files, the first step is to rename the files and split them into two files each. This dataset was prodcued using a Illumina SureCell 3’ kit, which produces paired end reads (as all current single-cell protocols). For unknown reasons the original two <em>FASTQ</em>-files per sample have been appended for the upload. More information about the SureCell format can be found <a href="https://teichlab.github.io/scg_lib_structs/methods_html/SureCell.html">here</a>.</p>

<p>The read files can be split with the following <em>bash</em> commands:</p>

<div class="sourceCode" id="cb1"><pre class="sourceCode bash"><code class="sourceCode bash"><a class="sourceLine" id="cb1-1" data-line-number="1"><span class="bu">cd</span> <span class="st">'YOUR_PATH'</span>/samples</a>

<a class="sourceLine" id="cb1-2" data-line-number="2"></a>

<a class="sourceLine" id="cb1-3" data-line-number="3"><span class="co">#rename</span></a>

<a class="sourceLine" id="cb1-4" data-line-number="4"><span class="fu">mv</span> SRR10669459.fastq.gz sc_EtOH.fastq.gz</a>

<a class="sourceLine" id="cb1-5" data-line-number="5"><span class="fu">mv</span> SRR10669461.fastq.gz sc_Dex2hr.fastq.gz</a>

<a class="sourceLine" id="cb1-6" data-line-number="6"></a>

<a class="sourceLine" id="cb1-7" data-line-number="7"><span class="co">#unpack the files</span></a>

<a class="sourceLine" id="cb1-8" data-line-number="8"><span class="fu">gunzip</span> sc_*.fastq.gz </a>

<a class="sourceLine" id="cb1-9" data-line-number="9"></a>

<a class="sourceLine" id="cb1-10" data-line-number="10"><span class="co">#split</span></a>

<a class="sourceLine" id="cb1-11" data-line-number="11"><span class="co"># the line numbers can be computed grepping the first linker sequence </span></a>

<a class="sourceLine" id="cb1-12" data-line-number="12"><span class="co"># or counting the number of '1'  at the end of the read name.</span></a>

<a class="sourceLine" id="cb1-13" data-line-number="13"><span class="fu">split</span> -l 1215291200 --numeric-suffixes=1 --additional-suffix=.fastq.gz sc_EtOH.fastq sc_EtOH_</a>

<a class="sourceLine" id="cb1-14" data-line-number="14"><span class="fu">split</span> -l 1394785580 --numeric-suffixes=1 --additional-suffix=.fastq.gz sc_Dex2hr.fastq sc_Dex2hr_</a></code></pre></div>

<p>Another drawback of the concatenation of the files for the upload is, that the read names do not match between the samples. We also can overcome this with the following <em>bash</em> commands:</p>

<div class="sourceCode" id="cb2"><pre class="sourceCode bash"><code class="sourceCode bash"><a class="sourceLine" id="cb2-1" data-line-number="1"><span class="co">#create files with matching names</span></a>

<a class="sourceLine" id="cb2-2" data-line-number="2"><span class="fu">cat</span> sc_EtOH_2.fastq <span class="kw">|</span> <span class="fu">sed</span> -e <span class="st">'s/^\(@SRR10669459.\)\(.*\) /echo \1$((\2-303822800)) /e'</span> <span class="op">&gt;</span> sc_EtOH_2_matched.fastq</a>

<a class="sourceLine" id="cb2-3" data-line-number="3"><span class="fu">cat</span> sc_Dex2hr_2.fastq <span class="kw">|</span> <span class="fu">sed</span> -e <span class="st">'s/^\(@SRR10669461.\)\(.*\) /echo \1$((\2-348696395)) /e'</span> <span class="op">&gt;</span> sc_Dex2hr_2_matched.fastq</a>

<a class="sourceLine" id="cb2-4" data-line-number="4"></a>

<a class="sourceLine" id="cb2-5" data-line-number="5"><span class="co">#pack again</span></a>

<a class="sourceLine" id="cb2-6" data-line-number="6"><span class="fu">gzip</span> sc_*_1.fastq</a>

<a class="sourceLine" id="cb2-7" data-line-number="7"><span class="fu">gzip</span> sc_*_matched.fastq</a>

<a class="sourceLine" id="cb2-8" data-line-number="8"></a>

<a class="sourceLine" id="cb2-9" data-line-number="9"><span class="co">#optionally: remove temporary files</span></a>

<a class="sourceLine" id="cb2-10" data-line-number="10"><span class="fu">rm</span> *.fastq</a>

<a class="sourceLine" id="cb2-11" data-line-number="11"></a>

<a class="sourceLine" id="cb2-12" data-line-number="12"><span class="co">#optionally: remove concatenated files</span></a>

<a class="sourceLine" id="cb2-13" data-line-number="13"><span class="fu">rm</span> sc_EtOH.fastq.gz </a>

<a class="sourceLine" id="cb2-14" data-line-number="14"><span class="fu">rm</span> sc_Dex2hr.fastq.gz</a></code></pre></div>

<p>There should be four packed <em>FASTQ</em>-files present in the folder, two per sample.</p>

<p>Vignettes are long form documentation commonly included in packages. Because they are part of the distribution of the package, they need to be as compact as possible. The <code>html_vignette</code> output type provides a custom style sheet (and tweaks some options) to ensure that the resulting html is as small as possible. The <code>html_vignette</code> format:</p>

<ul>

<li>Never uses retina figures</li>

<li>Has a smaller default figure size</li>

<li>Uses a custom CSS stylesheet instead of the default Twitter Bootstrap style</li>

</ul>

</div>

<div id="vignette-info" class="section level2">

<h2 class="hasAnchor">

<a href="#vignette-info" class="anchor"></a>Vignette Info</h2>

<p>Note the various macros within the <code>vignette</code> section of the metadata block above. These are required in order to instruct R how to build the vignette. Note that you should change the <code>title</code> field and the <code>\VignetteIndexEntry</code> to match the title of your vignette.</p>

</div>

<div id="styles" class="section level2">

<h2 class="hasAnchor">

<a href="#styles" class="anchor"></a>Styles</h2>

<p>The <code>html_vignette</code> template includes a basic CSS theme. To override this theme you can specify your own CSS in the document metadata as follows:</p>

<pre><code>output: 

  rmarkdown::html_vignette:

    css: mystyles.css</code></pre>

</div>

<div id="figures" class="section level2">

<h2 class="hasAnchor">

<a href="#figures" class="anchor"></a>Figures</h2>

<p>The figure sizes have been customised so that you can easily put two images side-by-side.</p>

<div class="sourceCode" id="cb4"><html><body><pre class="r"><span class="fu"><a href="https://rdrr.io/r/graphics/plot.html">plot</a></span>(<span class="fl">1</span>:<span class="fl">10</span>)

<span class="fu"><a href="https://rdrr.io/r/graphics/plot.html">plot</a></span>(<span class="fl">10</span>:<span class="fl">1</span>)</pre></body></html></div>

<p><img src="Hoffman_human_single-cell_preprocess_files/figure-html/unnamed-chunk-3-1.png" width="700"><img src="Hoffman_human_single-cell_preprocess_files/figure-html/unnamed-chunk-3-2.png" width="700"></p>

<p>You can enable figure captions by <code>fig_caption: yes</code> in YAML:</p>

<pre><code>output:

  rmarkdown::html_vignette:

    fig_caption: yes</code></pre>

<p>Then you can use the chunk option <code>fig.cap = "Your figure caption."</code> in <strong>knitr</strong>.</p>

</div>

<div id="more-examples" class="section level2">

<h2 class="hasAnchor">

<a href="#more-examples" class="anchor"></a>More Examples</h2>

<p>You can write math expressions, e.g. <span class="math inline">\(Y = X\beta + \epsilon\)</span>, footnotes<a href="#fn1" class="footnote-ref" id="fnref1"><sup>1</sup></a>, and tables, e.g. using <code><a href="https://rdrr.io/pkg/knitr/man/kable.html">knitr::kable()</a></code>.</p>

<table class="table">

<thead><tr class="header">

<th></th>

<th align="right">mpg</th>

<th align="right">cyl</th>

<th align="right">disp</th>

<th align="right">hp</th>

<th align="right">drat</th>

<th align="right">wt</th>

<th align="right">qsec</th>

<th align="right">vs</th>

<th align="right">am</th>

<th align="right">gear</th>

<th align="right">carb</th>

</tr></thead>

<tbody>

<tr class="odd">

<td>Mazda RX4</td>

<td align="right">21.0</td>

<td align="right">6</td>

<td align="right">160.0</td>

<td align="right">110</td>

<td align="right">3.90</td>

<td align="right">2.620</td>

<td align="right">16.46</td>

<td align="right">0</td>

<td align="right">1</td>

<td align="right">4</td>

<td align="right">4</td>

</tr>

<tr class="even">

<td>Mazda RX4 Wag</td>

<td align="right">21.0</td>

<td align="right">6</td>

<td align="right">160.0</td>

<td align="right">110</td>

<td align="right">3.90</td>

<td align="right">2.875</td>

<td align="right">17.02</td>

<td align="right">0</td>

<td align="right">1</td>

<td align="right">4</td>

<td align="right">4</td>

</tr>

<tr class="odd">

<td>Datsun 710</td>

<td align="right">22.8</td>

<td align="right">4</td>

<td align="right">108.0</td>

<td align="right">93</td>

<td align="right">3.85</td>

<td align="right">2.320</td>

<td align="right">18.61</td>

<td align="right">1</td>

<td align="right">1</td>

<td align="right">4</td>

<td align="right">1</td>

</tr>

<tr class="even">

<td>Hornet 4 Drive</td>

<td align="right">21.4</td>

<td align="right">6</td>

<td align="right">258.0</td>

<td align="right">110</td>

<td align="right">3.08</td>

<td align="right">3.215</td>

<td align="right">19.44</td>

<td align="right">1</td>

<td align="right">0</td>

<td align="right">3</td>

<td align="right">1</td>

</tr>

<tr class="odd">

<td>Hornet Sportabout</td>

<td align="right">18.7</td>

<td align="right">8</td>

<td align="right">360.0</td>

<td align="right">175</td>

<td align="right">3.15</td>

<td align="right">3.440</td>

<td align="right">17.02</td>

<td align="right">0</td>

<td align="right">0</td>

<td align="right">3</td>

<td align="right">2</td>

</tr>

<tr class="even">

<td>Valiant</td>

<td align="right">18.1</td>

<td align="right">6</td>

<td align="right">225.0</td>

<td align="right">105</td>

<td align="right">2.76</td>

<td align="right">3.460</td>

<td align="right">20.22</td>

<td align="right">1</td>

<td align="right">0</td>

<td align="right">3</td>

<td align="right">1</td>

</tr>

<tr class="odd">

<td>Duster 360</td>

<td align="right">14.3</td>

<td align="right">8</td>

<td align="right">360.0</td>

<td align="right">245</td>

<td align="right">3.21</td>

<td align="right">3.570</td>

<td align="right">15.84</td>

<td align="right">0</td>

<td align="right">0</td>

<td align="right">3</td>

<td align="right">4</td>

</tr>

<tr class="even">

<td>Merc 240D</td>

<td align="right">24.4</td>

<td align="right">4</td>

<td align="right">146.7</td>

<td align="right">62</td>

<td align="right">3.69</td>

<td align="right">3.190</td>

<td align="right">20.00</td>

<td align="right">1</td>

<td align="right">0</td>

<td align="right">4</td>

<td align="right">2</td>

</tr>

<tr class="odd">

<td>Merc 230</td>

<td align="right">22.8</td>

<td align="right">4</td>

<td align="right">140.8</td>

<td align="right">95</td>

<td align="right">3.92</td>

<td align="right">3.150</td>

<td align="right">22.90</td>

<td align="right">1</td>

<td align="right">0</td>

<td align="right">4</td>

<td align="right">2</td>

</tr>

<tr class="even">

<td>Merc 280</td>

<td align="right">19.2</td>

<td align="right">6</td>

<td align="right">167.6</td>

<td align="right">123</td>

<td align="right">3.92</td>

<td align="right">3.440</td>

<td align="right">18.30</td>

<td align="right">1</td>

<td align="right">0</td>

<td align="right">4</td>

<td align="right">4</td>

</tr>

</tbody>

</table>

<p>Also a quote using <code><a href="https://rdrr.io/r/base/Comparison.html">&gt;</a></code>:</p>

<blockquote>

<p>“He who gives up [code] safety for [code] speed deserves neither.” (<a href="https://twitter.com/hadleywickham/status/504368538874703872">via</a>)</p>

</blockquote>

</div>

<div class="footnotes">

<hr>

<ol>

<li id="fn1"><p>A footnote here.<a href="#fnref1" class="footnote-back">↩</a></p></li>

</ol>

</div>

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