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Also this is in bash so I do not need to get the bam files into R.</p> <div id="install" class="section level4"> <h4>Install</h4> <p>I added the deeptools package to my net-seq conda environment.</p> </div> <div id="running" class="section level4"> <h4>Running:</h4> <p>Step 1: Create bigwig coverage files with bamcoverage</p> <ul> <li>bamCoverage -b reads.bam -o coverage.bw</li> </ul> <p>Step 2: computeMatrix</p> <ul> <li><p>I will need my normalized bigwig reads and the bed interval file (in my case PAS clusters)</p></li> <li><p>ex: computeMatrix scale-regions -S <biwig file(s)> -R <bed file> -b 1000 -a 1000 -out <compressed matrix name.gz></p></li> <li><p>–skipZeros (option- not included in first try)</p></li> </ul> <p>Step 3: Plot heatmap</p> <ul> <li><p>required –matrixFile, -m (from the compute matrix), -out (file name to save image.png)</p></li> <li><p>–sortRegions descending</p></li> <li><p>–plotTitle, -T</p></li> </ul> <p>I will run this first on the hES 3’ seq becasue we expect heavy enrichment.</p> <pre class="bash"><code>#!/bin/bash #SBATCH --job-name=deeptools_hES #SBATCH --time=8:00:00 #SBATCH --partition=gilad #SBATCH --mem=16G #SBATCH --mail-type=END module load Anaconda3 source activate net-seq bamCoverage -b /project2/gilad/briana/Lianoglou.data/hES.hg38.sorted.bam -o hES.cov.bw computeMatrix reference-point -S hES.cov.bw -R /project2/gilad/briana/apa_sites/clusters.hg38.bed -b 1000 -a 1000 -out hES.hg38.gz plotHeatmap --sortRegions descend -m hES.hg38.gz -out hES.hg38.apa.png </code></pre> <p>This plot shows enrichement of the 3’ seq on called PAS sites. The axis are wrong.</p> <div class="figure"> <img src="../data/hES.hg38.apa.png" alt="hES 3’ coverage at APA sites" /> <p class="caption">hES 3’ coverage at APA sites</p> </div> <p>I will make this plot excluding exons. I will use bedtools intersect to get APA sites not in exons.</p> <p>I want A interects B -v. This will give me regions in A not in B. A is the cluster file. /project2/gilad/briana/apa_sites/clusters.hg38.bed and B is /project2/gilad/briana/apa_sites/deeptools/exons_hg38.bed</p> <p>This bash file is /project2/gilad/briana/apa_sites/deeptools/heatmatrix_hES_apa_excludeExons.sh</p> <pre class="bash"><code>#!/bin/bash #SBATCH --job-name=deeptools_hES_noexon #SBATCH --time=8:00:00 #SBATCH --partition=gilad #SBATCH --mem=16G #SBATCH --mail-type=END module load Anaconda3 source activate net-seq bedtools intersect -v -a /project2/gilad/briana/apa_sites/clusters.hg38.bed -b /project2/gilad/yangili/hg38_exons.bed > ../clusters.hg38.noExons.bed #bamCoverage -b /project2/gilad/briana/Lianoglou.data/hES.hg38.sorted.bam -o hES.cov.bw # dont need to redo this. The file exsists computeMatrix reference-point -S hES.cov.bw -R /project2/gilad/briana/apa_sites/clusters.hg38.noExons.bed -b 1000 -a 1000 -out hES.hg38.noExons.gz plotHeatmap --sortRegions descend -T "hES 3` APA enrichment exclude exons" -m hES.hg38.noExons.gz -out hES.hg38.apa.noExons.png </code></pre> <div class="figure"> <img src="../data/hES.hg38.apa.noExons.png" alt="hES 3’ coverage at APA sites not in exons" /> <p class="caption">hES 3’ coverage at APA sites not in exons</p> </div> </div> </div> <div id="session-information" class="section level2"> <h2>Session information</h2> <!-- Insert the session information into the document --> <pre class="r"><code>sessionInfo()</code></pre> <pre><code>R version 3.4.2 (2017-09-28) Platform: x86_64-apple-darwin15.6.0 (64-bit) Running under: macOS Sierra 10.12.6 Matrix products: default BLAS: /Library/Frameworks/R.framework/Versions/3.4/Resources/lib/libRblas.0.dylib LAPACK: /Library/Frameworks/R.framework/Versions/3.4/Resources/lib/libRlapack.dylib locale: [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 attached base packages: [1] stats graphics grDevices utils datasets methods base loaded via a namespace (and not attached): [1] compiler_3.4.2 backports_1.1.2 magrittr_1.5 rprojroot_1.3-2 [5] tools_3.4.2 htmltools_0.3.6 yaml_2.1.16 Rcpp_0.12.15 [9] stringi_1.1.6 rmarkdown_1.8.5 knitr_1.18 git2r_0.21.0 [13] stringr_1.2.0 digest_0.6.14 evaluate_0.10.1</code></pre> </div> <hr> <p> This <a href="http://rmarkdown.rstudio.com">R Markdown</a> site was created with <a href="https://github.com/jdblischak/workflowr">workflowr</a> </p> <hr> <!-- To enable disqus, uncomment the section below and provide your disqus_shortname --> <!-- disqus <div id="disqus_thread"></div> <script type="text/javascript"> /* * * CONFIGURATION VARIABLES: EDIT BEFORE PASTING INTO YOUR WEBPAGE * * */ var disqus_shortname = 'rmarkdown'; // required: replace example with your forum shortname /* * * DON'T EDIT BELOW THIS LINE * * */ (function() { var dsq = document.createElement('script'); dsq.type = 'text/javascript'; dsq.async = true; dsq.src = '//' + disqus_shortname + '.disqus.com/embed.js'; (document.getElementsByTagName('head')[0] || document.getElementsByTagName('body')[0]).appendChild(dsq); })(); </script> <noscript>Please enable JavaScript to view the <a href="http://disqus.com/?ref_noscript">comments powered by Disqus.</a></noscript> <a href="http://disqus.com" class="dsq-brlink">comments powered by <span class="logo-disqus">Disqus</span></a> --> </div> </div> </div> <script> // add bootstrap table styles to pandoc tables function bootstrapStylePandocTables() { $('tr.header').parent('thead').parent('table').addClass('table table-condensed'); } $(document).ready(function () { bootstrapStylePandocTables(); }); </script> <!-- dynamically load mathjax for compatibility with self-contained --> <script> (function () { var script = document.createElement("script"); script.type = "text/javascript"; script.src = "https://mathjax.rstudio.com/latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML"; document.getElementsByTagName("head")[0].appendChild(script); })(); </script> </body> </html>