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<h2 id="toc-title">Table of contents</h2>
<ul>
<li><a href="#rnaseq-report" id="toc-rnaseq-report" class="nav-link active" data-scroll-target="#rnaseq-report">RNASeq Report</a></li>
<li><a href="#load-data" id="toc-load-data" class="nav-link" data-scroll-target="#load-data">Load Data</a></li>
<li><a href="#transcript-level" id="toc-transcript-level" class="nav-link" data-scroll-target="#transcript-level">Transcript Level</a>
<ul class="collapse">
<li><a href="#quality-control" id="toc-quality-control" class="nav-link" data-scroll-target="#quality-control">Quality Control</a></li>
<li><a href="#differential-expression-test" id="toc-differential-expression-test" class="nav-link" data-scroll-target="#differential-expression-test">Differential Expression Test</a></li>
<li><a href="#funtional-annotation" id="toc-funtional-annotation" class="nav-link" data-scroll-target="#funtional-annotation">Funtional Annotation</a></li>
</ul></li>
<li><a href="#gene-level" id="toc-gene-level" class="nav-link" data-scroll-target="#gene-level">Gene Level</a>
<ul class="collapse">
<li><a href="#quality-control-1" id="toc-quality-control-1" class="nav-link" data-scroll-target="#quality-control-1">Quality Control</a></li>
<li><a href="#differential-expression-test-1" id="toc-differential-expression-test-1" class="nav-link" data-scroll-target="#differential-expression-test-1">Differential Expression Test</a></li>
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<h1 class="title">RNASeq Navarra</h1>
</div>
<div class="quarto-title-meta">
</div>
</header>
<p><a href="https://irycisbioinfo.github.io/ReportNavarraRNASeq/" class="uri">https://irycisbioinfo.github.io/ReportNavarraRNASeq/</a></p>
<section id="rnaseq-report" class="level1">
<h1>RNASeq Report</h1>
<p>All the data of this analysis can be found in <a href="https://github.com/irycisBioinfo/ReportNavarraRNASeq" class="uri">https://github.com/irycisBioinfo/ReportNavarraRNASeq</a></p>
<div class="cell">
<div class="sourceCode cell-code" id="cb1"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb1-1"><a href="#cb1-1" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(DESeq2)</span>
<span id="cb1-2"><a href="#cb1-2" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(tidyverse)</span>
<span id="cb1-3"><a href="#cb1-3" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(ggrepel)</span>
<span id="cb1-4"><a href="#cb1-4" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(ggsci)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</div>
</section>
<section id="load-data" class="level1">
<h1>Load Data</h1>
<p>Data from primary analisis workflow <a href="https://nf-co.re/rnaseq/3.14.0/" class="uri">https://nf-co.re/rnaseq/3.14.0/</a> are loaded among metadata.</p>
<div class="cell">
<div class="sourceCode cell-code" id="cb2"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb2-1"><a href="#cb2-1" aria-hidden="true" tabindex="-1"></a>reads_genes <span class="ot"><-</span> <span class="fu">read_tsv</span>(<span class="st">"/storage2/Hincode/S-60-47-61-2024-RNASeq_0240725_LH00206_0043_A22MLN2LT3/navarra/results_strand/star_salmon/salmon.merged.gene_counts_length_scaled.tsv"</span>)</span>
<span id="cb2-2"><a href="#cb2-2" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-3"><a href="#cb2-3" aria-hidden="true" tabindex="-1"></a>reads_transcripts <span class="ot"><-</span> <span class="fu">read_tsv</span>(<span class="st">"/storage2/Hincode/S-60-47-61-2024-RNASeq_0240725_LH00206_0043_A22MLN2LT3/navarra/results_strand/star_salmon/salmon.merged.transcript_counts.tsv"</span>)</span>
<span id="cb2-4"><a href="#cb2-4" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-5"><a href="#cb2-5" aria-hidden="true" tabindex="-1"></a>ids <span class="ot"><-</span> <span class="fu">read_tsv</span>(<span class="st">"/storage2/Hincode/S-60-47-61-2024-RNASeq_0240725_LH00206_0043_A22MLN2LT3/navarra/results_strand/star_salmon/salmon_tx2gene.tsv"</span>, <span class="at">col_names =</span> <span class="fu">c</span>(<span class="st">"Transcrip_ID"</span>,<span class="st">"Gene_ID"</span>,<span class="st">"Gene_Name"</span>))</span>
<span id="cb2-6"><a href="#cb2-6" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb2-7"><a href="#cb2-7" aria-hidden="true" tabindex="-1"></a>metadata <span class="ot"><-</span> <span class="fu">read_csv</span>(<span class="st">"/storage2/Hincode/S-60-47-61-2024-RNASeq_0240725_LH00206_0043_A22MLN2LT3/navarra/metadata.csv"</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
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<div class="cell">
<div class="sourceCode cell-code" id="cb3"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb3-1"><a href="#cb3-1" aria-hidden="true" tabindex="-1"></a>mart <span class="ot"><-</span> biomaRt<span class="sc">::</span><span class="fu">useMart</span>(<span class="at">biomart =</span> <span class="st">"ENSEMBL_MART_ENSEMBL"</span>,</span>
<span id="cb3-2"><a href="#cb3-2" aria-hidden="true" tabindex="-1"></a> <span class="at">dataset =</span> <span class="st">"hsapiens_gene_ensembl"</span>,</span>
<span id="cb3-3"><a href="#cb3-3" aria-hidden="true" tabindex="-1"></a> <span class="at">host =</span> <span class="st">'https://www.ensembl.org'</span>)</span>
<span id="cb3-4"><a href="#cb3-4" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb3-5"><a href="#cb3-5" aria-hidden="true" tabindex="-1"></a>ttg <span class="ot"><-</span> biomaRt<span class="sc">::</span><span class="fu">getBM</span>(</span>
<span id="cb3-6"><a href="#cb3-6" aria-hidden="true" tabindex="-1"></a> <span class="at">attributes =</span> <span class="fu">c</span>(<span class="st">"ensembl_transcript_id"</span>,<span class="st">"ensembl_gene_id"</span>,<span class="st">"external_gene_name"</span>, <span class="st">"description"</span>,</span>
<span id="cb3-7"><a href="#cb3-7" aria-hidden="true" tabindex="-1"></a> <span class="st">"entrezgene_id"</span>,<span class="st">"gene_biotype"</span>,<span class="st">"transcript_biotype"</span>),</span>
<span id="cb3-8"><a href="#cb3-8" aria-hidden="true" tabindex="-1"></a> <span class="at">mart =</span> mart)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</div>
<p>Quality Control from the primary analysis could be found <a href="https://irycisbioinfo.github.io/ReportNavarraRNASeq/multiqc/star_salmon/multiqc_report.html" class="uri">https://irycisbioinfo.github.io/ReportNavarraRNASeq/multiqc/star_salmon/multiqc_report.html</a></p>
<p>The analysis workflow aligns the reads to the reference genome’s transcripts. This allows us to perform analysis at two levels: the transcript level and the gene level, which provides a summary of all transcripts associated with each gene.</p>
</section>
<section id="transcript-level" class="level1">
<h1>Transcript Level</h1>
<p>Preparing data format</p>
<div class="cell">
<div class="sourceCode cell-code" id="cb4"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb4-1"><a href="#cb4-1" aria-hidden="true" tabindex="-1"></a>table_sum <span class="ot"><-</span> reads_transcripts <span class="sc">%>%</span> </span>
<span id="cb4-2"><a href="#cb4-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">pivot_longer</span>(<span class="at">names_to =</span> <span class="st">"Sample"</span>, <span class="at">values_to =</span> <span class="st">"counts"</span>, <span class="sc">-</span>(tx<span class="sc">:</span>gene_id)) <span class="sc">%>%</span> </span>
<span id="cb4-3"><a href="#cb4-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">group_by</span>(tx,gene_id) <span class="sc">%>%</span> </span>
<span id="cb4-4"><a href="#cb4-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">mutate</span>(<span class="at">TotalGeneCounts =</span> <span class="fu">sum</span>(counts), </span>
<span id="cb4-5"><a href="#cb4-5" aria-hidden="true" tabindex="-1"></a> <span class="at">TotalZerosCounts =</span> <span class="fu">sum</span>(counts<span class="sc">==</span><span class="dv">0</span>))</span>
<span id="cb4-6"><a href="#cb4-6" aria-hidden="true" tabindex="-1"></a> </span>
<span id="cb4-7"><a href="#cb4-7" aria-hidden="true" tabindex="-1"></a>table_sum <span class="sc">%>%</span> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x =</span> TotalGeneCounts)) <span class="sc">+</span> <span class="fu">geom_histogram</span>() <span class="sc">+</span> <span class="fu">geom_vline</span>(<span class="at">xintercept =</span> <span class="dv">10</span>)<span class="sc">+</span><span class="fu">scale_x_log10</span>()</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
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<p><img src="Analisis_files/figure-html/unnamed-chunk-4-1.png" class="img-fluid" width="672"></p>
</div>
</div>
<p>Seen the distribution of the read counts we decide to filter those transcript with less than 10 reads or that are 0 in more than 3 samples.</p>
<div class="cell">
<div class="sourceCode cell-code" id="cb5"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb5-1"><a href="#cb5-1" aria-hidden="true" tabindex="-1"></a>table_counts <span class="ot"><-</span> table_sum <span class="sc">%>%</span></span>
<span id="cb5-2"><a href="#cb5-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">ungroup</span>() <span class="sc">%>%</span> </span>
<span id="cb5-3"><a href="#cb5-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">filter</span>(TotalGeneCounts <span class="sc">></span> <span class="dv">10</span>) <span class="sc">%>%</span> </span>
<span id="cb5-4"><a href="#cb5-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">filter</span>(TotalZerosCounts <span class="sc"><</span> <span class="dv">3</span>) <span class="sc">%>%</span> </span>
<span id="cb5-5"><a href="#cb5-5" aria-hidden="true" tabindex="-1"></a> dplyr<span class="sc">::</span><span class="fu">select</span>(tx,Sample,counts) <span class="sc">%>%</span> </span>
<span id="cb5-6"><a href="#cb5-6" aria-hidden="true" tabindex="-1"></a> <span class="fu">pivot_wider</span>(<span class="at">names_from =</span> Sample, <span class="at">values_from =</span> counts, <span class="at">values_fill =</span> <span class="dv">0</span>) <span class="sc">%>%</span> </span>
<span id="cb5-7"><a href="#cb5-7" aria-hidden="true" tabindex="-1"></a> <span class="fu">column_to_rownames</span>(<span class="st">"tx"</span>) <span class="sc">%>%</span> </span>
<span id="cb5-8"><a href="#cb5-8" aria-hidden="true" tabindex="-1"></a> <span class="fu">round</span>() <span class="sc">%>%</span> </span>
<span id="cb5-9"><a href="#cb5-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">as.matrix</span>()</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</div>
<p>Creating a regression model and performing Differential Expression Analysis</p>
<div class="cell">
<div class="sourceCode cell-code" id="cb6"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb6-1"><a href="#cb6-1" aria-hidden="true" tabindex="-1"></a>dds_transcript <span class="ot"><-</span><span class="fu">DESeqDataSetFromMatrix</span>(<span class="at">countData =</span> table_counts, </span>
<span id="cb6-2"><a href="#cb6-2" aria-hidden="true" tabindex="-1"></a> <span class="at">colData =</span> metadata <span class="sc">%>%</span> <span class="fu">column_to_rownames</span>(<span class="st">"Sample"</span>), </span>
<span id="cb6-3"><a href="#cb6-3" aria-hidden="true" tabindex="-1"></a> <span class="at">design =</span> <span class="sc">~</span>Grupo) </span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>converting counts to integer mode</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
design formula are characters, converting to factors</code></pre>
</div>
<div class="sourceCode cell-code" id="cb9"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb9-1"><a href="#cb9-1" aria-hidden="true" tabindex="-1"></a>dds_transcript <span class="ot"><-</span> <span class="fu">DESeq</span>(dds_transcript,<span class="at">sfType =</span> <span class="st">"poscounts"</span>, <span class="at">fitType =</span> <span class="st">"mean"</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>estimating size factors</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>estimating dispersions</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>gene-wise dispersion estimates</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>mean-dispersion relationship</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>final dispersion estimates</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>fitting model and testing</code></pre>
</div>
</div>
<section id="quality-control" class="level2">
<h2 class="anchored" data-anchor-id="quality-control">Quality Control</h2>
<p>Check the normalization and DE results</p>
<div class="cell">
<div class="sourceCode cell-code" id="cb16"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb16-1"><a href="#cb16-1" aria-hidden="true" tabindex="-1"></a>reads_transcripts <span class="sc">%>%</span> <span class="fu">pivot_longer</span>(<span class="at">names_to =</span> <span class="st">"Sample"</span>, <span class="at">values_to =</span> <span class="st">"counts"</span>, <span class="sc">-</span>(tx<span class="sc">:</span>gene_id)) <span class="sc">%>%</span> </span>
<span id="cb16-2"><a href="#cb16-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">group_by</span>(Sample) <span class="sc">%>%</span> </span>
<span id="cb16-3"><a href="#cb16-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">summarise</span>(<span class="at">TotalCounts =</span> <span class="fu">sum</span>(counts)) <span class="sc">%>%</span> </span>
<span id="cb16-4"><a href="#cb16-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">full_join</span>(metadata) <span class="sc">%>%</span> </span>
<span id="cb16-5"><a href="#cb16-5" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x=</span> Sample, <span class="at">y =</span> TotalCounts, <span class="at">fill =</span> Grupo)) <span class="sc">+</span> <span class="fu">geom_col</span>() <span class="sc">+</span> <span class="fu">coord_flip</span>() <span class="sc">+</span> <span class="fu">theme_light</span>()<span class="sc">+</span> <span class="fu">labs</span>(<span class="at">title =</span> <span class="st">"Raw Data"</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Joining with `by = join_by(Sample)`</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-7-1.png" class="img-fluid" width="672"></p>
</div>
</div>
<div class="cell">
<div class="sourceCode cell-code" id="cb18"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb18-1"><a href="#cb18-1" aria-hidden="true" tabindex="-1"></a><span class="fu">counts</span>(dds_transcript, <span class="at">normalized =</span> T) <span class="sc">%>%</span> <span class="fu">as_tibble</span>(<span class="at">rownames =</span> <span class="st">"tx"</span>) <span class="sc">%>%</span> <span class="fu">pivot_longer</span>(<span class="at">names_to =</span> <span class="st">"Sample"</span>, <span class="at">values_to =</span> <span class="st">"counts"</span>, <span class="sc">-</span>tx) <span class="sc">%>%</span> </span>
<span id="cb18-2"><a href="#cb18-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">group_by</span>(Sample) <span class="sc">%>%</span> </span>
<span id="cb18-3"><a href="#cb18-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">summarise</span>(<span class="at">TotalCounts =</span> <span class="fu">sum</span>(counts)) <span class="sc">%>%</span> </span>
<span id="cb18-4"><a href="#cb18-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">full_join</span>(metadata) <span class="sc">%>%</span> </span>
<span id="cb18-5"><a href="#cb18-5" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x=</span> Sample, <span class="at">y =</span> TotalCounts, <span class="at">fill =</span> Grupo)) <span class="sc">+</span> <span class="fu">geom_col</span>() <span class="sc">+</span> <span class="fu">coord_flip</span>() <span class="sc">+</span> <span class="fu">theme_light</span>() <span class="sc">+</span> <span class="fu">labs</span>(<span class="at">title =</span> <span class="st">"Normalize Data"</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Joining with `by = join_by(Sample)`</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-8-1.png" class="img-fluid" width="672"></p>
</div>
</div>
<div class="cell">
<div class="sourceCode cell-code" id="cb20"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb20-1"><a href="#cb20-1" aria-hidden="true" tabindex="-1"></a>mds <span class="ot"><-</span> vegan<span class="sc">::</span><span class="fu">metaMDS</span>(<span class="fu">counts</span>(dds_transcript, <span class="at">normalized =</span> T) <span class="sc">%>%</span> <span class="fu">t</span>())</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stdout">
<pre><code>Square root transformation
Wisconsin double standardization
Run 0 stress 0
Run 1 stress 0
... Procrustes: rmse 0.0291793 max resid 0.04557835
Run 2 stress 9.548865e-05
... Procrustes: rmse 0.2081779 max resid 0.290838
Run 3 stress 0.0003889057
... Procrustes: rmse 0.1453818 max resid 0.2098872
Run 4 stress 5.617026e-05
... Procrustes: rmse 0.2247752 max resid 0.4649492
Run 5 stress 8.098997e-05
... Procrustes: rmse 0.2104377 max resid 0.3823652
Run 6 stress 9.942537e-05
... Procrustes: rmse 0.08345882 max resid 0.1133512
Run 7 stress 0
... Procrustes: rmse 0.06501365 max resid 0.1200745
Run 8 stress 0
... Procrustes: rmse 0.07343435 max resid 0.1365268
Run 9 stress 0
... Procrustes: rmse 0.1403157 max resid 0.2655851
Run 10 stress 9.327804e-05
... Procrustes: rmse 0.2119679 max resid 0.4193939
Run 11 stress 9.734295e-05
... Procrustes: rmse 0.07975466 max resid 0.1116435
Run 12 stress 2.928156e-05
... Procrustes: rmse 0.1756845 max resid 0.3418646
Run 13 stress 4.334659e-05
... Procrustes: rmse 0.1720234 max resid 0.3282976
Run 14 stress 0
... Procrustes: rmse 0.1460413 max resid 0.2309672
Run 15 stress 9.944218e-06
... Procrustes: rmse 0.1606056 max resid 0.2815027
Run 16 stress 0
... Procrustes: rmse 0.09459989 max resid 0.185337
Run 17 stress 0
... Procrustes: rmse 0.0997192 max resid 0.1768916
Run 18 stress 8.52166e-05
... Procrustes: rmse 0.2131113 max resid 0.3332029
Run 19 stress 9.190148e-05
... Procrustes: rmse 0.2257334 max resid 0.461816
Run 20 stress 9.751302e-05
... Procrustes: rmse 0.08435188 max resid 0.1137656
*** Best solution was not repeated -- monoMDS stopping criteria:
1: no. of iterations >= maxit
19: stress < smin</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning in vegan::metaMDS(counts(dds_transcript, normalized = T) %>% t()):
stress is (nearly) zero: you may have insufficient data</code></pre>
</div>
<div class="sourceCode cell-code" id="cb23"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb23-1"><a href="#cb23-1" aria-hidden="true" tabindex="-1"></a>mds<span class="sc">$</span>points <span class="sc">%>%</span> </span>
<span id="cb23-2"><a href="#cb23-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">as.data.frame</span>() <span class="sc">%>%</span> </span>
<span id="cb23-3"><a href="#cb23-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">rownames_to_column</span>(<span class="st">"Sample"</span>) <span class="sc">%>%</span> </span>
<span id="cb23-4"><a href="#cb23-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">inner_join</span>(metadata) <span class="sc">%>%</span> </span>
<span id="cb23-5"><a href="#cb23-5" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x =</span> MDS1, <span class="at">y =</span> MDS2, <span class="at">fill =</span> Grupo, <span class="at">label =</span>Sample)) <span class="sc">+</span> <span class="fu">geom_label</span>() <span class="sc">+</span></span>
<span id="cb23-6"><a href="#cb23-6" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_light</span>()</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Joining with `by = join_by(Sample)`</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-9-1.png" class="img-fluid" width="672"></p>
</div>
</div>
<p>The X Group seens more heteregenous than the group P. This get worse the quality of the Differential Expression Test.</p>
</section>
<section id="differential-expression-test" class="level2">
<h2 class="anchored" data-anchor-id="differential-expression-test">Differential Expression Test</h2>
<div class="cell">
<div class="sourceCode cell-code" id="cb25"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb25-1"><a href="#cb25-1" aria-hidden="true" tabindex="-1"></a><span class="fu">resultsNames</span>(dds_transcript)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stdout">
<pre><code>[1] "Intercept" "Grupo_X_vs_P"</code></pre>
</div>
<div class="sourceCode cell-code" id="cb27"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb27-1"><a href="#cb27-1" aria-hidden="true" tabindex="-1"></a>de_test <span class="ot"><-</span> <span class="fu">results</span>(dds_transcript) <span class="sc">%>%</span> </span>
<span id="cb27-2"><a href="#cb27-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">as_tibble</span>(<span class="at">rownames =</span> <span class="st">"Transcrip_ID"</span>) <span class="sc">%>%</span> <span class="fu">inner_join</span>(ids)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Joining with `by = join_by(Transcrip_ID)`</code></pre>
</div>
<div class="sourceCode cell-code" id="cb29"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb29-1"><a href="#cb29-1" aria-hidden="true" tabindex="-1"></a>de_test<span class="sc">%>%</span> </span>
<span id="cb29-2"><a href="#cb29-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">mutate</span>(<span class="at">sig =</span> <span class="fu">ifelse</span>(padj <span class="sc"><</span> <span class="fl">0.005</span>,<span class="st">"Significative"</span>,<span class="st">"No-Significative"</span>)) <span class="sc">%>%</span></span>
<span id="cb29-3"><a href="#cb29-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">mutate</span>(<span class="at">label =</span> <span class="fu">ifelse</span>(sig <span class="sc">==</span> <span class="st">"Significative"</span>,Gene_Name,<span class="cn">NA</span>)) <span class="sc">%>%</span> </span>
<span id="cb29-4"><a href="#cb29-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x=</span> log2FoldChange, </span>
<span id="cb29-5"><a href="#cb29-5" aria-hidden="true" tabindex="-1"></a> <span class="at">y =</span> <span class="sc">-</span><span class="fu">log10</span>(padj), </span>
<span id="cb29-6"><a href="#cb29-6" aria-hidden="true" tabindex="-1"></a> <span class="at">color =</span> sig, </span>
<span id="cb29-7"><a href="#cb29-7" aria-hidden="true" tabindex="-1"></a> <span class="at">size =</span> <span class="fu">sqrt</span>(baseMean), </span>
<span id="cb29-8"><a href="#cb29-8" aria-hidden="true" tabindex="-1"></a> <span class="at">label =</span> label)) <span class="sc">+</span></span>
<span id="cb29-9"><a href="#cb29-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_point</span>(<span class="at">alpha =</span> <span class="fl">0.5</span>) <span class="sc">+</span></span>
<span id="cb29-10"><a href="#cb29-10" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_text_repel</span>(<span class="at">size =</span> <span class="dv">2</span>)<span class="sc">+</span></span>
<span id="cb29-11"><a href="#cb29-11" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_color_d3</span>() <span class="sc">+</span></span>
<span id="cb29-12"><a href="#cb29-12" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_light</span>() <span class="sc">+</span> </span>
<span id="cb29-13"><a href="#cb29-13" aria-hidden="true" tabindex="-1"></a> <span class="fu">labs</span>(<span class="at">title =</span> <span class="st">"Volcano Plot Grupo_X_vs_P"</span>) </span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: Removed 7693 rows containing missing values or values outside the scale range
(`geom_point()`).</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: Removed 94886 rows containing missing values or values outside the scale range
(`geom_text_repel()`).</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: ggrepel: 49 unlabeled data points (too many overlaps). Consider
increasing max.overlaps</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-10-1.png" class="img-fluid" width="672"></p>
</div>
<div class="sourceCode cell-code" id="cb33"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb33-1"><a href="#cb33-1" aria-hidden="true" tabindex="-1"></a>de_test<span class="sc">%>%</span> </span>
<span id="cb33-2"><a href="#cb33-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">mutate</span>(<span class="at">sig =</span> <span class="fu">ifelse</span>(padj <span class="sc"><</span> <span class="fl">0.005</span>,<span class="st">"Significative"</span>,<span class="st">"No-Significative"</span>)) <span class="sc">%>%</span></span>
<span id="cb33-3"><a href="#cb33-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">mutate</span>(<span class="at">label =</span> <span class="fu">ifelse</span>(sig <span class="sc">==</span> <span class="st">"Significative"</span>,Gene_Name,<span class="cn">NA</span>)) <span class="sc">%>%</span> </span>
<span id="cb33-4"><a href="#cb33-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x=</span> baseMean, </span>
<span id="cb33-5"><a href="#cb33-5" aria-hidden="true" tabindex="-1"></a> <span class="at">y =</span> log2FoldChange, </span>
<span id="cb33-6"><a href="#cb33-6" aria-hidden="true" tabindex="-1"></a> <span class="at">color =</span> sig, </span>
<span id="cb33-7"><a href="#cb33-7" aria-hidden="true" tabindex="-1"></a> <span class="at">size =</span> <span class="fu">sqrt</span>(baseMean), </span>
<span id="cb33-8"><a href="#cb33-8" aria-hidden="true" tabindex="-1"></a> <span class="at">label =</span> label)) <span class="sc">+</span></span>
<span id="cb33-9"><a href="#cb33-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_point</span>(<span class="at">alpha =</span> <span class="fl">0.5</span>) <span class="sc">+</span></span>
<span id="cb33-10"><a href="#cb33-10" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_text_repel</span>(<span class="at">size =</span> <span class="dv">1</span>)<span class="sc">+</span></span>
<span id="cb33-11"><a href="#cb33-11" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_color_d3</span>() <span class="sc">+</span></span>
<span id="cb33-12"><a href="#cb33-12" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_x_log10</span>()<span class="sc">+</span></span>
<span id="cb33-13"><a href="#cb33-13" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_light</span>() <span class="sc">+</span> </span>
<span id="cb33-14"><a href="#cb33-14" aria-hidden="true" tabindex="-1"></a> <span class="fu">labs</span>(<span class="at">title =</span> <span class="st">"MA Plot Grupo_X_vs_P"</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: Removed 7693 rows containing missing values or values outside the scale range
(`geom_point()`).</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: Removed 94886 rows containing missing values or values outside the scale range
(`geom_text_repel()`).</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: ggrepel: 13 unlabeled data points (too many overlaps). Consider
increasing max.overlaps</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-10-2.png" class="img-fluid" width="672"></p>
</div>
</div>
<p>Volcano and MA plot shown several transcript differential expressed. In order to see better this differences we plot all the comparison individually.</p>
<div class="cell">
<div class="sourceCode cell-code" id="cb37"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb37-1"><a href="#cb37-1" aria-hidden="true" tabindex="-1"></a>significativos <span class="ot"><-</span> de_test<span class="sc">%>%</span> </span>
<span id="cb37-2"><a href="#cb37-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">filter</span>(padj <span class="sc"><</span> <span class="fl">0.005</span>) <span class="sc">%>%</span> <span class="fu">pull</span>(Transcrip_ID)</span>
<span id="cb37-3"><a href="#cb37-3" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb37-4"><a href="#cb37-4" aria-hidden="true" tabindex="-1"></a><span class="fu">counts</span>(dds_transcript, <span class="at">normalized =</span> T) <span class="sc">%>%</span> </span>
<span id="cb37-5"><a href="#cb37-5" aria-hidden="true" tabindex="-1"></a> <span class="fu">as.data.frame</span>() <span class="sc">%>%</span> </span>
<span id="cb37-6"><a href="#cb37-6" aria-hidden="true" tabindex="-1"></a> <span class="fu">rownames_to_column</span>(<span class="st">"tx"</span>) <span class="sc">%>%</span> </span>
<span id="cb37-7"><a href="#cb37-7" aria-hidden="true" tabindex="-1"></a> <span class="fu">filter</span>(tx <span class="sc">%in%</span> significativos) <span class="sc">%>%</span> </span>
<span id="cb37-8"><a href="#cb37-8" aria-hidden="true" tabindex="-1"></a> <span class="fu">pivot_longer</span>(<span class="at">names_to =</span> <span class="st">"Sample"</span>,<span class="at">values_to =</span> <span class="st">"counts"</span>, <span class="sc">-</span>tx) <span class="sc">%>%</span> </span>
<span id="cb37-9"><a href="#cb37-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">inner_join</span>(metadata) <span class="sc">%>%</span> </span>
<span id="cb37-10"><a href="#cb37-10" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x =</span> Grupo, <span class="at">y =</span> counts, <span class="at">fill =</span> Grupo)) <span class="sc">+</span> <span class="fu">geom_boxplot</span>() <span class="sc">+</span> <span class="fu">facet_wrap</span>(<span class="sc">~</span>tx, <span class="at">scales =</span> <span class="st">"free_y"</span>) <span class="sc">+</span> <span class="fu">scale_fill_d3</span>() <span class="sc">+</span> <span class="fu">theme_light</span>()</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Joining with `by = join_by(Sample)`</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-11-1.png" class="img-fluid" width="1920"></p>
</div>
</div>
</section>
<section id="funtional-annotation" class="level2">
<h2 class="anchored" data-anchor-id="funtional-annotation">Funtional Annotation</h2>
<p>To create a biological context for the differential expressed transcript we are traying to infer if there are some funtional annotation (GO Terms) or pathway (KEGG) over-represented in the transcript-set.</p>
<div class="cell">
<div class="sourceCode cell-code" id="cb39"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb39-1"><a href="#cb39-1" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(clusterProfiler)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code></code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>clusterProfiler v4.12.6 Learn more at https://yulab-smu.top/contribution-knowledge-mining/
Please cite:
T Wu, E Hu, S Xu, M Chen, P Guo, Z Dai, T Feng, L Zhou, W Tang, L Zhan,
X Fu, S Liu, X Bo, and G Yu. clusterProfiler 4.0: A universal
enrichment tool for interpreting omics data. The Innovation. 2021,
2(3):100141</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>
Adjuntando el paquete: 'clusterProfiler'</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>The following object is masked from 'package:purrr':
simplify</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>The following object is masked from 'package:IRanges':
slice</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>The following object is masked from 'package:S4Vectors':
rename</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>The following object is masked from 'package:stats':
filter</code></pre>
</div>
<div class="sourceCode cell-code" id="cb47"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb47-1"><a href="#cb47-1" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(org.Hs.eg.db)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Cargando paquete requerido: AnnotationDbi</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>
Adjuntando el paquete: 'AnnotationDbi'</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>The following object is masked from 'package:clusterProfiler':
select</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>The following object is masked from 'package:dplyr':
select</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code></code></pre>
</div>
</div>
<div class="cell">
<div class="sourceCode cell-code" id="cb53"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb53-1"><a href="#cb53-1" aria-hidden="true" tabindex="-1"></a>sig_transcripts <span class="ot"><-</span> de_test <span class="sc">%>%</span> </span>
<span id="cb53-2"><a href="#cb53-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">inner_join</span>(ttg, <span class="at">by =</span> <span class="fu">c</span>(<span class="st">"Transcrip_ID"</span> <span class="ot">=</span> <span class="st">"ensembl_transcript_id"</span>)) <span class="sc">%>%</span> <span class="fu">filter</span>(padj <span class="sc"><</span> <span class="fl">0.005</span>) <span class="sc">%>%</span> </span>
<span id="cb53-3"><a href="#cb53-3" aria-hidden="true" tabindex="-1"></a> dplyr<span class="sc">::</span><span class="fu">select</span>(entrezgene_id) <span class="sc">%>%</span> </span>
<span id="cb53-4"><a href="#cb53-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">distinct</span>() <span class="sc">%>%</span> </span>
<span id="cb53-5"><a href="#cb53-5" aria-hidden="true" tabindex="-1"></a> <span class="fu">drop_na</span>() <span class="sc">%>%</span> </span>
<span id="cb53-6"><a href="#cb53-6" aria-hidden="true" tabindex="-1"></a> <span class="fu">pull</span>(entrezgene_id)</span>
<span id="cb53-7"><a href="#cb53-7" aria-hidden="true" tabindex="-1"></a> </span>
<span id="cb53-8"><a href="#cb53-8" aria-hidden="true" tabindex="-1"></a>GO_enrichment <span class="ot"><-</span> <span class="fu">enrichGO</span>(<span class="at">gene =</span>sig_transcripts,</span>
<span id="cb53-9"><a href="#cb53-9" aria-hidden="true" tabindex="-1"></a> <span class="at">OrgDb =</span> org.Hs.eg.db,</span>
<span id="cb53-10"><a href="#cb53-10" aria-hidden="true" tabindex="-1"></a> <span class="at">ont =</span> <span class="st">"ALL"</span>,</span>
<span id="cb53-11"><a href="#cb53-11" aria-hidden="true" tabindex="-1"></a> <span class="at">pAdjustMethod =</span> <span class="st">"BH"</span>,</span>
<span id="cb53-12"><a href="#cb53-12" aria-hidden="true" tabindex="-1"></a> <span class="at">pvalueCutoff =</span> <span class="fl">0.01</span>,</span>
<span id="cb53-13"><a href="#cb53-13" aria-hidden="true" tabindex="-1"></a> <span class="at">qvalueCutoff =</span> <span class="fl">0.05</span>,</span>
<span id="cb53-14"><a href="#cb53-14" aria-hidden="true" tabindex="-1"></a> <span class="at">readable =</span> <span class="cn">TRUE</span>)</span>
<span id="cb53-15"><a href="#cb53-15" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb53-16"><a href="#cb53-16" aria-hidden="true" tabindex="-1"></a><span class="cf">if</span> ((GO_enrichment <span class="sc">%>%</span> <span class="fu">as.data.frame</span>() <span class="sc">%>%</span> <span class="fu">nrow</span>()) <span class="sc">></span> <span class="dv">0</span> )</span>
<span id="cb53-17"><a href="#cb53-17" aria-hidden="true" tabindex="-1"></a>{</span>
<span id="cb53-18"><a href="#cb53-18" aria-hidden="true" tabindex="-1"></a> <span class="fu">barplot</span>(GO_enrichment, <span class="at">showCategory =</span> <span class="dv">20</span>)</span>
<span id="cb53-19"><a href="#cb53-19" aria-hidden="true" tabindex="-1"></a>} <span class="cf">else</span>{</span>
<span id="cb53-20"><a href="#cb53-20" aria-hidden="true" tabindex="-1"></a> <span class="fu">print</span>(<span class="st">"No enriched Pathways"</span>)</span>
<span id="cb53-21"><a href="#cb53-21" aria-hidden="true" tabindex="-1"></a>}</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stdout">
<pre><code>[1] "No enriched Pathways"</code></pre>
</div>
<div class="sourceCode cell-code" id="cb55"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb55-1"><a href="#cb55-1" aria-hidden="true" tabindex="-1"></a>KEGG_enrichment <span class="ot"><-</span> <span class="fu">enrichKEGG</span>(<span class="at">gene =</span> sig_transcripts,</span>
<span id="cb55-2"><a href="#cb55-2" aria-hidden="true" tabindex="-1"></a> <span class="at">organism =</span> <span class="st">'hsa'</span>,</span>
<span id="cb55-3"><a href="#cb55-3" aria-hidden="true" tabindex="-1"></a> <span class="at">pvalueCutoff =</span> <span class="fl">0.05</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Reading KEGG annotation online: "https://rest.kegg.jp/link/hsa/pathway"...</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Reading KEGG annotation online: "https://rest.kegg.jp/list/pathway/hsa"...</code></pre>
</div>
<div class="sourceCode cell-code" id="cb58"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb58-1"><a href="#cb58-1" aria-hidden="true" tabindex="-1"></a><span class="cf">if</span> ((KEGG_enrichment <span class="sc">%>%</span> <span class="fu">as.data.frame</span>() <span class="sc">%>%</span> <span class="fu">nrow</span>()) <span class="sc">></span> <span class="dv">0</span> )</span>
<span id="cb58-2"><a href="#cb58-2" aria-hidden="true" tabindex="-1"></a>{</span>
<span id="cb58-3"><a href="#cb58-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">barplot</span>(KEGG_enrichment, <span class="at">showCategory =</span> <span class="dv">20</span>)</span>
<span id="cb58-4"><a href="#cb58-4" aria-hidden="true" tabindex="-1"></a>} <span class="cf">else</span>{</span>
<span id="cb58-5"><a href="#cb58-5" aria-hidden="true" tabindex="-1"></a> <span class="fu">print</span>(<span class="st">"No enriched Pathways"</span>)</span>
<span id="cb58-6"><a href="#cb58-6" aria-hidden="true" tabindex="-1"></a>}</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stdout">
<pre><code>[1] "No enriched Pathways"</code></pre>
</div>
</div>
<p>There are not over-represented funtional annotation or pathway.</p>
</section>
</section>
<section id="gene-level" class="level1">
<h1>Gene Level</h1>
<p>At this point we repeat de analysis but at Gene Level.</p>
<div class="cell">
<div class="sourceCode cell-code" id="cb60"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb60-1"><a href="#cb60-1" aria-hidden="true" tabindex="-1"></a>table_sum <span class="ot"><-</span> reads_genes <span class="sc">%>%</span> </span>
<span id="cb60-2"><a href="#cb60-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">pivot_longer</span>(<span class="at">names_to =</span> <span class="st">"Sample"</span>, <span class="at">values_to =</span> <span class="st">"counts"</span>, <span class="sc">-</span>(gene_id<span class="sc">:</span>gene_name)) <span class="sc">%>%</span> </span>
<span id="cb60-3"><a href="#cb60-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">group_by</span>(gene_id) <span class="sc">%>%</span> </span>
<span id="cb60-4"><a href="#cb60-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">mutate</span>(<span class="at">TotalGeneCounts =</span> <span class="fu">sum</span>(counts), </span>
<span id="cb60-5"><a href="#cb60-5" aria-hidden="true" tabindex="-1"></a> <span class="at">TotalZerosCounts =</span> <span class="fu">sum</span>(counts<span class="sc">==</span><span class="dv">0</span>))</span>
<span id="cb60-6"><a href="#cb60-6" aria-hidden="true" tabindex="-1"></a> </span>
<span id="cb60-7"><a href="#cb60-7" aria-hidden="true" tabindex="-1"></a>table_sum <span class="sc">%>%</span> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x =</span> TotalGeneCounts)) <span class="sc">+</span> <span class="fu">geom_histogram</span>() <span class="sc">+</span> <span class="fu">geom_vline</span>(<span class="at">xintercept =</span> <span class="dv">10</span>)<span class="sc">+</span><span class="fu">scale_x_log10</span>()</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning in scale_x_log10(): log-10 transformation introduced infinite values.</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>`stat_bin()` using `bins = 30`. Pick better value with `binwidth`.</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: Removed 144900 rows containing non-finite outside the scale range
(`stat_bin()`).</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-14-1.png" class="img-fluid" width="672"></p>
</div>
<div class="sourceCode cell-code" id="cb64"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb64-1"><a href="#cb64-1" aria-hidden="true" tabindex="-1"></a>table_counts <span class="ot"><-</span> table_sum <span class="sc">%>%</span> </span>
<span id="cb64-2"><a href="#cb64-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">ungroup</span>() <span class="sc">%>%</span> </span>
<span id="cb64-3"><a href="#cb64-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">filter</span>(TotalGeneCounts <span class="sc">></span> <span class="dv">10</span>) <span class="sc">%>%</span> </span>
<span id="cb64-4"><a href="#cb64-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">filter</span>(TotalZerosCounts <span class="sc"><</span> <span class="dv">3</span>) <span class="sc">%>%</span> </span>
<span id="cb64-5"><a href="#cb64-5" aria-hidden="true" tabindex="-1"></a> dplyr<span class="sc">::</span><span class="fu">select</span>(gene_id,Sample,counts) <span class="sc">%>%</span> </span>
<span id="cb64-6"><a href="#cb64-6" aria-hidden="true" tabindex="-1"></a> <span class="fu">pivot_wider</span>(<span class="at">names_from =</span> Sample, <span class="at">values_from =</span> counts, <span class="at">values_fill =</span> <span class="dv">0</span>) <span class="sc">%>%</span> </span>
<span id="cb64-7"><a href="#cb64-7" aria-hidden="true" tabindex="-1"></a> <span class="fu">column_to_rownames</span>(<span class="st">"gene_id"</span>) <span class="sc">%>%</span> </span>
<span id="cb64-8"><a href="#cb64-8" aria-hidden="true" tabindex="-1"></a> <span class="fu">round</span>() <span class="sc">%>%</span> </span>
<span id="cb64-9"><a href="#cb64-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">as.matrix</span>()</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
</div>
<p>We use the same criteria to filter low quality genes.</p>
<div class="cell">
<div class="sourceCode cell-code" id="cb65"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb65-1"><a href="#cb65-1" aria-hidden="true" tabindex="-1"></a>dds_genes <span class="ot"><-</span><span class="fu">DESeqDataSetFromMatrix</span>(<span class="at">countData =</span> table_counts, </span>
<span id="cb65-2"><a href="#cb65-2" aria-hidden="true" tabindex="-1"></a> <span class="at">colData =</span> metadata <span class="sc">%>%</span> <span class="fu">column_to_rownames</span>(<span class="st">"Sample"</span>), </span>
<span id="cb65-3"><a href="#cb65-3" aria-hidden="true" tabindex="-1"></a> <span class="at">design =</span> <span class="sc">~</span>Grupo) </span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>converting counts to integer mode</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
design formula are characters, converting to factors</code></pre>
</div>
<div class="sourceCode cell-code" id="cb68"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb68-1"><a href="#cb68-1" aria-hidden="true" tabindex="-1"></a>dds_genes <span class="ot"><-</span> <span class="fu">DESeq</span>(dds_genes,<span class="at">sfType =</span> <span class="st">"poscounts"</span>, <span class="at">fitType =</span> <span class="st">"mean"</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>estimating size factors</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>estimating dispersions</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>gene-wise dispersion estimates</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>mean-dispersion relationship</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>final dispersion estimates</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>fitting model and testing</code></pre>
</div>
</div>
<section id="quality-control-1" class="level2">
<h2 class="anchored" data-anchor-id="quality-control-1">Quality Control</h2>
<div class="cell">
<div class="sourceCode cell-code" id="cb75"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb75-1"><a href="#cb75-1" aria-hidden="true" tabindex="-1"></a>reads_genes <span class="sc">%>%</span> <span class="fu">pivot_longer</span>(<span class="at">names_to =</span> <span class="st">"Sample"</span>, <span class="at">values_to =</span> <span class="st">"counts"</span>, <span class="sc">-</span>(gene_id<span class="sc">:</span>gene_name)) <span class="sc">%>%</span> </span>
<span id="cb75-2"><a href="#cb75-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">group_by</span>(Sample) <span class="sc">%>%</span> </span>
<span id="cb75-3"><a href="#cb75-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">summarise</span>(<span class="at">TotalCounts =</span> <span class="fu">sum</span>(counts)) <span class="sc">%>%</span> </span>
<span id="cb75-4"><a href="#cb75-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">full_join</span>(metadata) <span class="sc">%>%</span> </span>
<span id="cb75-5"><a href="#cb75-5" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x=</span> Sample, <span class="at">y =</span> TotalCounts, <span class="at">fill =</span> Grupo)) <span class="sc">+</span> <span class="fu">geom_col</span>() <span class="sc">+</span> <span class="fu">coord_flip</span>() <span class="sc">+</span> <span class="fu">theme_light</span>()<span class="sc">+</span> <span class="fu">labs</span>(<span class="at">title =</span> <span class="st">"Raw Data"</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Joining with `by = join_by(Sample)`</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-16-1.png" class="img-fluid" width="672"></p>
</div>
</div>
<div class="cell">
<div class="sourceCode cell-code" id="cb77"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb77-1"><a href="#cb77-1" aria-hidden="true" tabindex="-1"></a><span class="fu">counts</span>(dds_genes, <span class="at">normalized =</span> T) <span class="sc">%>%</span> <span class="fu">as_tibble</span>(<span class="at">rownames =</span> <span class="st">"genes_id"</span>) <span class="sc">%>%</span> <span class="fu">pivot_longer</span>(<span class="at">names_to =</span> <span class="st">"Sample"</span>, <span class="at">values_to =</span> <span class="st">"counts"</span>, <span class="sc">-</span>genes_id) <span class="sc">%>%</span> </span>
<span id="cb77-2"><a href="#cb77-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">group_by</span>(Sample) <span class="sc">%>%</span> </span>
<span id="cb77-3"><a href="#cb77-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">summarise</span>(<span class="at">TotalCounts =</span> <span class="fu">sum</span>(counts)) <span class="sc">%>%</span> </span>
<span id="cb77-4"><a href="#cb77-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">full_join</span>(metadata) <span class="sc">%>%</span> </span>
<span id="cb77-5"><a href="#cb77-5" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x=</span> Sample, <span class="at">y =</span> TotalCounts, <span class="at">fill =</span> Grupo)) <span class="sc">+</span> <span class="fu">geom_col</span>() <span class="sc">+</span> <span class="fu">coord_flip</span>() <span class="sc">+</span> <span class="fu">theme_light</span>() <span class="sc">+</span> <span class="fu">labs</span>(<span class="at">title =</span> <span class="st">"Normalize Data"</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Joining with `by = join_by(Sample)`</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-17-1.png" class="img-fluid" width="672"></p>
</div>
</div>
<div class="cell">
<div class="sourceCode cell-code" id="cb79"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb79-1"><a href="#cb79-1" aria-hidden="true" tabindex="-1"></a>pca <span class="ot"><-</span> vegan<span class="sc">::</span><span class="fu">metaMDS</span>(<span class="fu">counts</span>(dds_genes, <span class="at">normalized =</span> T) <span class="sc">%>%</span> <span class="fu">t</span>())</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stdout">
<pre><code>Square root transformation
Wisconsin double standardization
Run 0 stress 3.011127e-06
Run 1 stress 6.479562e-06
... Procrustes: rmse 0.1722707 max resid 0.2337154
Run 2 stress 9.960007e-05
... Procrustes: rmse 0.1717837 max resid 0.3605271
Run 3 stress 9.864728e-05
... Procrustes: rmse 0.09142828 max resid 0.1268886
Run 4 stress 9.846717e-05
... Procrustes: rmse 0.06694584 max resid 0.1107112
Run 5 stress 0.0002527055
... Procrustes: rmse 0.2839842 max resid 0.4818229
Run 6 stress 0
... New best solution
... Procrustes: rmse 0.1247765 max resid 0.2513806
Run 7 stress 9.870621e-05
... Procrustes: rmse 0.1013454 max resid 0.192716
Run 8 stress 8.199781e-05
... Procrustes: rmse 0.138157 max resid 0.1901614
Run 9 stress 9.471227e-05
... Procrustes: rmse 0.1359267 max resid 0.207019
Run 10 stress 9.618336e-05
... Procrustes: rmse 0.217148 max resid 0.3902685
Run 11 stress 9.473049e-05
... Procrustes: rmse 0.1148755 max resid 0.2127429
Run 12 stress 0.0007052933
Run 13 stress 9.536384e-05
... Procrustes: rmse 0.1207609 max resid 0.2144415
Run 14 stress 8.61864e-05
... Procrustes: rmse 0.1503647 max resid 0.2618885
Run 15 stress 9.920266e-05
... Procrustes: rmse 0.2216172 max resid 0.2976934
Run 16 stress 0.0007465923
Run 17 stress 0.276134
Run 18 stress 9.766822e-05
... Procrustes: rmse 0.1251696 max resid 0.1791651
Run 19 stress 0.0001227039
... Procrustes: rmse 0.2613389 max resid 0.4705428
Run 20 stress 6.484234e-05
... Procrustes: rmse 0.1479421 max resid 0.2603061
*** Best solution was not repeated -- monoMDS stopping criteria:
4: no. of iterations >= maxit
15: stress < smin
1: stress ratio > sratmax</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning in vegan::metaMDS(counts(dds_genes, normalized = T) %>% t()): stress is
(nearly) zero: you may have insufficient data</code></pre>
</div>
<div class="sourceCode cell-code" id="cb82"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb82-1"><a href="#cb82-1" aria-hidden="true" tabindex="-1"></a>pca<span class="sc">$</span>points <span class="sc">%>%</span> </span>
<span id="cb82-2"><a href="#cb82-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">as.data.frame</span>() <span class="sc">%>%</span> </span>
<span id="cb82-3"><a href="#cb82-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">rownames_to_column</span>(<span class="st">"Sample"</span>) <span class="sc">%>%</span> </span>
<span id="cb82-4"><a href="#cb82-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">inner_join</span>(metadata) <span class="sc">%>%</span> </span>
<span id="cb82-5"><a href="#cb82-5" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x =</span> MDS1, <span class="at">y =</span> MDS2, <span class="at">fill =</span> Grupo, <span class="at">label =</span>Sample)) <span class="sc">+</span> <span class="fu">geom_label</span>() <span class="sc">+</span></span>
<span id="cb82-6"><a href="#cb82-6" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_light</span>()</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Joining with `by = join_by(Sample)`</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-18-1.png" class="img-fluid" width="672"></p>
</div>
</div>
<p>We obtain the same results at heterogeneity level of the samples.</p>
</section>
<section id="differential-expression-test-1" class="level2">
<h2 class="anchored" data-anchor-id="differential-expression-test-1">Differential Expression Test</h2>
<div class="cell">
<div class="sourceCode cell-code" id="cb84"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb84-1"><a href="#cb84-1" aria-hidden="true" tabindex="-1"></a><span class="fu">resultsNames</span>(dds_genes)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stdout">
<pre><code>[1] "Intercept" "Grupo_X_vs_P"</code></pre>
</div>
<div class="sourceCode cell-code" id="cb86"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb86-1"><a href="#cb86-1" aria-hidden="true" tabindex="-1"></a>de_test <span class="ot"><-</span> <span class="fu">results</span>(dds_genes) <span class="sc">%>%</span> </span>
<span id="cb86-2"><a href="#cb86-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">as_tibble</span>(<span class="at">rownames =</span> <span class="st">"Gene_ID"</span>) <span class="sc">%>%</span> <span class="fu">inner_join</span>(ids)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Joining with `by = join_by(Gene_ID)`</code></pre>
</div>
<div class="sourceCode cell-code" id="cb88"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb88-1"><a href="#cb88-1" aria-hidden="true" tabindex="-1"></a>de_test<span class="sc">%>%</span> </span>
<span id="cb88-2"><a href="#cb88-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">mutate</span>(<span class="at">sig =</span> <span class="fu">ifelse</span>(padj <span class="sc"><</span> <span class="fl">0.01</span>,<span class="st">"Significative"</span>,<span class="st">"No-Significative"</span>)) <span class="sc">%>%</span></span>
<span id="cb88-3"><a href="#cb88-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">mutate</span>(<span class="at">label =</span> <span class="fu">ifelse</span>(sig <span class="sc">==</span> <span class="st">"Significative"</span>,Gene_Name,<span class="cn">NA</span>)) <span class="sc">%>%</span> </span>
<span id="cb88-4"><a href="#cb88-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x=</span> log2FoldChange, </span>
<span id="cb88-5"><a href="#cb88-5" aria-hidden="true" tabindex="-1"></a> <span class="at">y =</span> <span class="sc">-</span><span class="fu">log10</span>(padj), </span>
<span id="cb88-6"><a href="#cb88-6" aria-hidden="true" tabindex="-1"></a> <span class="at">color =</span> sig, </span>
<span id="cb88-7"><a href="#cb88-7" aria-hidden="true" tabindex="-1"></a> <span class="at">size =</span> <span class="fu">sqrt</span>(baseMean), </span>
<span id="cb88-8"><a href="#cb88-8" aria-hidden="true" tabindex="-1"></a> <span class="at">label =</span> label)) <span class="sc">+</span></span>
<span id="cb88-9"><a href="#cb88-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_point</span>(<span class="at">alpha =</span> <span class="fl">0.5</span>) <span class="sc">+</span></span>
<span id="cb88-10"><a href="#cb88-10" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_text_repel</span>(<span class="at">size =</span> <span class="dv">1</span>)<span class="sc">+</span></span>
<span id="cb88-11"><a href="#cb88-11" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_color_d3</span>() <span class="sc">+</span></span>
<span id="cb88-12"><a href="#cb88-12" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_light</span>() <span class="sc">+</span> </span>
<span id="cb88-13"><a href="#cb88-13" aria-hidden="true" tabindex="-1"></a> <span class="fu">labs</span>(<span class="at">title =</span> <span class="st">"Volcano Plot Grupo_X_vs_P"</span>) </span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: Removed 5373 rows containing missing values or values outside the scale range
(`geom_point()`).</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: Removed 183875 rows containing missing values or values outside the scale range
(`geom_text_repel()`).</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: ggrepel: 29 unlabeled data points (too many overlaps). Consider
increasing max.overlaps</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-19-1.png" class="img-fluid" width="672"></p>
</div>
<div class="sourceCode cell-code" id="cb92"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb92-1"><a href="#cb92-1" aria-hidden="true" tabindex="-1"></a>de_test<span class="sc">%>%</span> </span>
<span id="cb92-2"><a href="#cb92-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">mutate</span>(<span class="at">sig =</span> <span class="fu">ifelse</span>(padj <span class="sc"><</span> <span class="fl">0.01</span>,<span class="st">"Significative"</span>,<span class="st">"No-Significative"</span>)) <span class="sc">%>%</span></span>
<span id="cb92-3"><a href="#cb92-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">mutate</span>(<span class="at">label =</span> <span class="fu">ifelse</span>(sig <span class="sc">==</span> <span class="st">"Significative"</span>,Gene_Name,<span class="cn">NA</span>)) <span class="sc">%>%</span> </span>
<span id="cb92-4"><a href="#cb92-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x=</span> baseMean, </span>
<span id="cb92-5"><a href="#cb92-5" aria-hidden="true" tabindex="-1"></a> <span class="at">y =</span> log2FoldChange, </span>
<span id="cb92-6"><a href="#cb92-6" aria-hidden="true" tabindex="-1"></a> <span class="at">color =</span> sig, </span>
<span id="cb92-7"><a href="#cb92-7" aria-hidden="true" tabindex="-1"></a> <span class="at">size =</span> <span class="fu">sqrt</span>(baseMean), </span>
<span id="cb92-8"><a href="#cb92-8" aria-hidden="true" tabindex="-1"></a> <span class="at">label =</span> label)) <span class="sc">+</span></span>
<span id="cb92-9"><a href="#cb92-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_point</span>(<span class="at">alpha =</span> <span class="fl">0.5</span>) <span class="sc">+</span></span>
<span id="cb92-10"><a href="#cb92-10" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_text_repel</span>(<span class="at">size =</span> <span class="dv">1</span>)<span class="sc">+</span></span>
<span id="cb92-11"><a href="#cb92-11" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_color_d3</span>() <span class="sc">+</span></span>
<span id="cb92-12"><a href="#cb92-12" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_x_log10</span>()<span class="sc">+</span></span>
<span id="cb92-13"><a href="#cb92-13" aria-hidden="true" tabindex="-1"></a> <span class="fu">theme_light</span>() <span class="sc">+</span> </span>
<span id="cb92-14"><a href="#cb92-14" aria-hidden="true" tabindex="-1"></a> <span class="fu">labs</span>(<span class="at">title =</span> <span class="st">"MA Plot Grupo_X_vs_P"</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: Removed 5373 rows containing missing values or values outside the scale range
(`geom_point()`).</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: Removed 183875 rows containing missing values or values outside the scale range
(`geom_text_repel()`).</code></pre>
</div>
<div class="cell-output cell-output-stderr">
<pre><code>Warning: ggrepel: 29 unlabeled data points (too many overlaps). Consider
increasing max.overlaps</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-19-2.png" class="img-fluid" width="672"></p>
</div>
</div>
<div class="cell">
<div class="sourceCode cell-code" id="cb96"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb96-1"><a href="#cb96-1" aria-hidden="true" tabindex="-1"></a>significativos <span class="ot"><-</span> de_test<span class="sc">%>%</span> </span>
<span id="cb96-2"><a href="#cb96-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">filter</span>(padj <span class="sc"><</span> <span class="fl">0.01</span>) <span class="sc">%>%</span> <span class="fu">pull</span>(Gene_Name)</span>
<span id="cb96-3"><a href="#cb96-3" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb96-4"><a href="#cb96-4" aria-hidden="true" tabindex="-1"></a><span class="fu">counts</span>(dds_genes, <span class="at">normalized =</span> T) <span class="sc">%>%</span> </span>
<span id="cb96-5"><a href="#cb96-5" aria-hidden="true" tabindex="-1"></a> <span class="fu">as.data.frame</span>() <span class="sc">%>%</span> </span>
<span id="cb96-6"><a href="#cb96-6" aria-hidden="true" tabindex="-1"></a> <span class="fu">rownames_to_column</span>(<span class="st">"Gene_Name"</span>) <span class="sc">%>%</span> </span>
<span id="cb96-7"><a href="#cb96-7" aria-hidden="true" tabindex="-1"></a> <span class="fu">filter</span>( Gene_Name <span class="sc">%in%</span> significativos) <span class="sc">%>%</span> </span>
<span id="cb96-8"><a href="#cb96-8" aria-hidden="true" tabindex="-1"></a> <span class="fu">pivot_longer</span>(<span class="at">names_to =</span> <span class="st">"Sample"</span>,<span class="at">values_to =</span> <span class="st">"counts"</span>, <span class="sc">-</span>Gene_Name) <span class="sc">%>%</span> </span>
<span id="cb96-9"><a href="#cb96-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">inner_join</span>(metadata) <span class="sc">%>%</span> </span>
<span id="cb96-10"><a href="#cb96-10" aria-hidden="true" tabindex="-1"></a> <span class="fu">ggplot</span>(<span class="fu">aes</span>(<span class="at">x =</span> Grupo, <span class="at">y =</span> counts, <span class="at">fill =</span> Grupo)) <span class="sc">+</span> <span class="fu">geom_boxplot</span>() <span class="sc">+</span> <span class="fu">facet_wrap</span>(<span class="sc">~</span>Gene_Name, <span class="at">scales =</span> <span class="st">"free_y"</span>) <span class="sc">+</span> <span class="fu">scale_fill_d3</span>() <span class="sc">+</span> <span class="fu">theme_light</span>()</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
<div class="cell-output cell-output-stderr">
<pre><code>Joining with `by = join_by(Sample)`</code></pre>
</div>
<div class="cell-output-display">
<p><img src="Analisis_files/figure-html/unnamed-chunk-20-1.png" class="img-fluid" width="672"></p>
</div>
</div>
<p>In this case we only see two genes deferentially expresed.</p>
</section>
</section>
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// Unselect the line
unselectCodeLines();
clickedEl.classList.remove('code-annotation-active');
}
});
}
const findCites = (el) => {
const parentEl = el.parentElement;
if (parentEl) {
const cites = parentEl.dataset.cites;
if (cites) {
return {
el,
cites: cites.split(' ')
};
} else {
return findCites(el.parentElement)
}
} else {
return undefined;
}
};
var bibliorefs = window.document.querySelectorAll('a[role="doc-biblioref"]');
for (var i=0; i<bibliorefs.length; i++) {
const ref = bibliorefs[i];
const citeInfo = findCites(ref);
if (citeInfo) {
tippyHover(citeInfo.el, function() {
var popup = window.document.createElement('div');
citeInfo.cites.forEach(function(cite) {
var citeDiv = window.document.createElement('div');
citeDiv.classList.add('hanging-indent');
citeDiv.classList.add('csl-entry');
var biblioDiv = window.document.getElementById('ref-' + cite);
if (biblioDiv) {
citeDiv.innerHTML = biblioDiv.innerHTML;
}
popup.appendChild(citeDiv);
});
return popup.innerHTML;
});
}
}
});