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TrinotateWeb.html
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<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.1//EN"
"http://www.w3.org/TR/xhtml11/DTD/xhtml11.dtd">
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<head>
<meta http-equiv="Content-Type" content="application/xhtml+xml; charset=UTF-8" />
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<title>TrinotateWeb: Graphical Interface for Navigating Trinotate Annotations and Expression Analyses</title>
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<body class="article">
<div id="header">
<h1>TrinotateWeb: Graphical Interface for Navigating Trinotate Annotations and Expression Analyses</h1>
</div>
<div id="content">
<div class="sect1">
<h2 id="_background">Background</h2>
<div class="sectionbody">
<div class="paragraph"><p>TrinotateWeb provides a web-based graphical interface to support local user-based navigation of Trinotate annotations and differential expression data. Simply run the included light-weight webserver, open your web browser and point it to your Trinotate SQLite database, and begin your data exploration. Note, Trinotate is not yet a full-featured application, but is instead in a very early state of development. As an open source community-driven software development project, contributions are always welcome.</p></div>
<div class="imageblock">
<div class="content">
<img src="images/canvasXpressLogoBig.png" alt="CanvasXpress logo" width="200" />
</div>
</div>
<div class="paragraph"><p>Trinotate makes extensive use of the <a href="http://canvasxpress.org">CanvasXpress</a> infrastructure, as developed and made publicly available by <strong>Isaac Neuhaus</strong>. "CanvasXpress was developed as the core visualization component for bioinformatics and systems biology analysis at Bristol-Myers Squibb" (<a href="http://canvasxpress.org/about.html">More about CanvasXpress</a>).</p></div>
</div>
</div>
<div class="sect1">
<h2 id="_trinotateweb_showcase">TrinotateWeb Showcase</h2>
<div class="sectionbody">
<div class="paragraph"><p>The current capabilities of TrinotateWeb are showcased below (note, the interface is continually improved across software releases):</p></div>
<div class="imageblock">
<div class="content">
<img src="images/TrinotateWebCollage.png" alt="TrinotateWebCollage image" width="800" />
</div>
</div>
</div>
</div>
<div class="sect1">
<h2 id="_populating_trinotate_sqlite_with_expression_data">Populating Trinotate.sqlite with Expression Data</h2>
<div class="sectionbody">
<div class="paragraph"><p>TrinotateWeb currently revolves around using the differential expression analysis pipeline supported by the Trinity suite, as described <a href="http://trinityrnaseq.sourceforge.net/#Downstream_analyses">here</a>, specifically first generating abundance estimates using the <a href="http://trinityrnaseq.sourceforge.net/analysis/abundance_estimation.html">RSEM pipeline</a> and then performing the <a href="http://trinityrnaseq.sourceforge.net/analysis/diff_expression_analysis.html">DE analysis using Bioconductor</a>.</p></div>
<div class="paragraph"><p>After having run the above abundance estimation and DE analysis steps, you can begin loading the expression data like so:</p></div>
<div class="literalblock">
<div class="content">
<pre><code># import the fpkm and DE analysis stuff
$TRINOTATE_HOME/util/transcript_expression/import_expression_and_DE_results.pl \
--sqlite Trinotate.sqlite \
--samples_file samples_n_reads_described.txt \
--count_matrix Trinity_trans.counts.matrix \
--fpkm_matrix Trinity_trans.counts.matrix.TMM_normalized.FPKM \
--DE_dir edgeR_trans/ \
--transcript_mode</code></pre>
</div></div>
<div class="admonitionblock">
<table><tr>
<td class="icon">
<div class="title">Note</div>
</td>
<td class="content">The above is based on using the transcript-level abundance estimates and corresponding DE analyses. Abundance estimation and DE analysis can be performed at the <em>gene</em> level as well. Optionally, run the above using <em>--gene_mode</em> with the corresponding gene-based files. Both transcript-level and gene-level data can be loaded into the single Trinotate.sqlite instance for analysis.</td>
</tr></table>
</div>
</div>
</div>
<div class="sect1">
<h2 id="_populate_trinotate_sqlite_with_transcript_expression_clusters">Populate Trinotate.sqlite with Transcript Expression Clusters</h2>
<div class="sectionbody">
<div class="paragraph"><p>The Trinity analysis framework describes methods for clustering transcripts based on related expression patterns <a href="http://trinityrnaseq.sourceforge.net/analysis/diff_expression_analysis.html">here</a>. After defining clusters, load these into the Trinotate.sqlite database like so:</p></div>
<div class="literalblock">
<div class="content">
<pre><code># import transcript clusters
$TRINOTATE_HOME/util/transcript_expression/import_transcript_clusters.pl \
--group_name edgeR_DE_analysis \
--analysis_name edgeR_trans/diffExpr.P0.001_C2.matrix.R.all.RData.clusters_fixed_P_20 \
--sqlite Trinotate.sqlite \
edgeR_trans/diffExpr.P0.001_C2.matrix.R.all.RData.clusters_fixed_P_20/*matrix</code></pre>
</div></div>
<div class="paragraph"><p>The step of defining expression clusters can be run multiple times using different parameters according to the <a href="https://github.com/trinityrnaseq/trinityrnaseq/wiki/Trinity-Differential-Expression">Trinity documentation</a>. Each set of clusters can be loaded in separately as above and then separately studied within TrinotateWeb.</p></div>
</div>
</div>
<div class="sect1">
<h2 id="_populate_trinotate_sqlite_with_annotation_text">Populate Trinotate.sqlite with Annotation Text</h2>
<div class="sectionbody">
<div class="paragraph"><p>Any free-text annotation can be applied to your input transcripts, and these data can then be queried by text searches within TrinotateWeb. This was the simplest way to get text-searching up and running in a flexible way, and more sophisticated methods for querying the data will be included in future releases. If you’ve used Trinotate to generate annotations (ex. generated the Trinotate_report.xls tab-delimited summary data file), you can simply import that data table back into Trinotate as the textual annotation for the transcripts (Highly Recommended). Alternatively, if you have a file containing a tab delimited format of:</p></div>
<div class="literalblock">
<div class="content">
<pre><code>gene_id (tab) transcript_id (tab) annotation text</code></pre>
</div></div>
<div class="paragraph"><p>you can use that file instead. Just realize that the TrinotateWeb text-querying is currently soley based on this <em>annotation text</em>. Load the annotations into the Trinotate.sqlite database like so:</p></div>
<div class="literalblock">
<div class="content">
<pre><code># Load annotations
$TRINOTATE_HOME/util/annotation_importer/import_transcript_names.pl Trinotate.sqlite Trinotate_report.xls</code></pre>
</div></div>
</div>
</div>
<div class="sect1">
<h2 id="_sample_data">Sample data</h2>
<div class="sectionbody">
<div class="paragraph"><p>A full set of sample data for loading a Trinotate.sqlite database and populating it with expression and annotation data according to the above steps is provided at</p></div>
<div class="literalblock">
<div class="content">
<pre><code>$TRINOTATE_HOME/sample_data/</code></pre>
</div></div>
<div class="paragraph"><p>Simply <em>./runMe.sh</em> in that directory to generate the fully populated <em>Trinotate.sqlite</em> database that’s ready for exploration using TrinotateWeb.</p></div>
</div>
</div>
<div class="sect1">
<h2 id="_running_trinotateweb">Running TrinotateWeb</h2>
<div class="sectionbody">
<div class="paragraph"><p>To run TrinotateWeb, you’ll need a light-weight webserver installed. We recommend using <a href="https://www.lighttpd.net/">lighttpd</a>, which is free and easy to obtain for Linux and Mac. Once installed, you can launch it via:</p></div>
<div class="literalblock">
<div class="content">
<pre><code>cd $TRINOTATE_HOME</code></pre>
</div></div>
<div class="literalblock">
<div class="content">
<pre><code>./run_TrinotateWebserver.pl 8080</code></pre>
</div></div>
<div class="paragraph"><p>and leave it running within your terminal window. To stop it, type cntrl-C or exit the terminal.</p></div>
<div class="admonitionblock">
<table><tr>
<td class="icon">
<div class="title">Note</div>
</td>
<td class="content">The number 8080 is the port at which it will be listening to connections from your web browser. You can use whatever open port is available.</td>
</tr></table>
</div>
<div class="paragraph"><p>Then, go to your web browser and visit the URL: <em>http://localhost:8080/cgi-bin/index.cgi</em></p></div>
<div class="paragraph"><p>You should be prompted to enter in the path to your Trinotate sqlite database.</p></div>
<div class="imageblock">
<div class="content">
<img src="images/TrinotateEntry.png" alt="Trinotate Web Entry Page unfilled" width="500" />
</div>
</div>
</div>
</div>
<div class="sect1">
<h2 id="_navigating_trinotateweb">Navigating TrinotateWeb</h2>
<div class="sectionbody">
<div class="sect2">
<h3 id="_overview_tab">Overview Tab</h3>
<div class="paragraph"><p>The overview tab will show basic summary statistics.</p></div>
<div class="imageblock">
<div class="content">
<img src="images/TrinotateOverview.png" alt="Trinotate overview tab" width="500" />
</div>
</div>
</div>
<div class="sect2">
<h3 id="_differential_expression_analysis">Differential Expression Analysis</h3>
<div class="paragraph"><p>Differential expression can be explored from interactive volcano plots, MA plots, and heatmaps, starting from the <em>Differential Expression</em> tab.</p></div>
<div class="imageblock">
<div class="content">
<img src="images/TrinotateDEselection.png" alt="DE selection page" width="500" />
</div>
</div>
<div class="paragraph"><p>An example volcano plot for a pairwise comparison between two samples is shown below:</p></div>
<div class="imageblock">
<div class="content">
<img src="images/TrinotateVolcano.png" alt="Trinotate Volcano Plot page" width="500" />
</div>
</div>
<div class="paragraph"><p>Just double-click on a point to visit that gene or transcript’s expression and annotation report. Drag and select a range to zoom in.</p></div>
</div>
<div class="sect2">
<h3 id="_gene_transcript_id_or_keyword_searches">Gene/Transcript ID or Keyword Searches</h3>
<div class="sect3">
<h4 id="_keyword_search">Keyword Search</h4>
<div class="paragraph"><p>You can search for genes or transcripts via keyword searches. For example, from the keyword search tab, I type in <em>transporter</em> like so:</p></div>
<div class="imageblock">
<div class="content">
<img src="images/TrinotateKeywordSelectionInput.png" alt="Trinotate keyword search entry" width="500" />
</div>
</div>
<div class="paragraph"><p>Submitting the search results in a list of all entries where <em>transporter</em> was found among the annotation text.</p></div>
<div class="imageblock">
<div class="content">
<img src="images/TrinotateKeywordSelectionResult.png" alt="Trinotate keyword search results" width="500" />
</div>
</div>
</div>
<div class="sect3">
<h4 id="_gene_transcript_id_search">Gene/Transcript ID Search</h4>
<div class="paragraph"><p>Given a gene or transcript identifier, the feature can be searched for directly via the <em>Gene or Transcript ID Search</em> tab:</p></div>
<div class="imageblock">
<div class="content">
<img src="images/TrinotateGeneIDselection.png" alt="Trinotate gene ID selection" width="500" />
</div>
</div>
</div>
</div>
<div class="sect2">
<h3 id="_gene_transcript_expression_and_annotation_reports">Gene/Transcript Expression and Annotation Reports</h3>
<div class="paragraph"><p>A given gene or transcript search will lead to a gene/transcript expression and annotation report page:</p></div>
<div class="imageblock">
<div class="content">
<img src="images/TrinotateGeneExpressionReport.png" alt="Trinotate gene expression report" width="500" />
</div>
</div>
<div class="paragraph"><p>Annotation information will be displayed below, including a view of the position of the ORF on the transcript and any homology match information.</p></div>
<div class="imageblock">
<div class="content">
<img src="images/TrinotateTranscriptReport.png" alt="Trinotate transcript annotation report" width="500" />
</div>
</div>
</div>
</div>
</div>
</div>
<div id="footnotes"><hr /></div>
<div id="footer">
<div id="footer-text">
Last updated 2016-03-08 16:11:51 EST
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