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| id: rna-seq-viz-with-cummerbund | ||
| name: Visualization of RNA-Seq results with CummeRbund | ||
| description: >- | ||
| In this tutorial we will visualize RNA-Seq results in order to make sense of | ||
| the available RNA-Seq data, and overview the condition-specific gene | ||
| expression levels of the provided samples. | ||
| title_default: rna-seq-viz-with-cummerbund | ||
| steps: | ||
| - title: Introduction | ||
| content: >- | ||
| In this tutorial we will visualize RNA-Seq results in order to make sense | ||
| of the available RNA-Seq data, and overview the condition-specific gene | ||
| expression levels of the provided samples. | ||
| backdrop: true | ||
| - title: Introduction | ||
| content: >- | ||
| RNA-Seq analysis helps researchers annotate new genes and splice variants, | ||
| and provides cell- and context-specific quantification of gene expression. | ||
| RNA-Seq data, however, are complex and require both computer science and | ||
| mathematical knowledge to be managed and interpreted. | ||
| <br><br>Visualization techniques are key to overcome the complexity of | ||
| RNA-Seq data, and represent valuable tools to gather information and | ||
| insights. | ||
| backdrop: true | ||
| - title: Reasons for visualizing RNA-Seq results | ||
| content: >- | ||
| To make sense of the available RNA-Seq data, and overview the | ||
| condition-specific gene expression levels of the provided samples, we need | ||
| to visualize our results. Here we will use <a | ||
| href="http://compbio.mit.edu/cummeRbund/">CummeRbund</a>. | ||
| backdrop: true | ||
| - title: CummeRbund | ||
| content: >- | ||
| CummeRbund is an open-source tool that simplifies the analysis of a | ||
| CuffDiff RNA-Seq output. In particular, it helps researchers:<ul> | ||
| <li>managing, integrating, and visualizing the data produced by CuffDiff</li> | ||
| <li>simplifying data exploration</li> | ||
| <li>providing a bird’s-eye view of the expression analysis by describing relationships betweeen genes, transcripts, transcription start sites, and protein-coding regions</li> | ||
| <li>exploring subfeatures of individual genes or gene-sets</li> | ||
| <li>creating publication-ready plots</li></ul> | ||
| CummeRbund reads your RNA-Seq results from a <a | ||
| href="https://www.sqlite.org/">SQLite</a> database. This database has to | ||
| be created using CuffDiff’s SQLite output option. | ||
| backdrop: true | ||
| - title: History options | ||
| element: '#history-options-button' | ||
| content: >- | ||
| Create a new history for this exercise. Click on this button and then | ||
| "Create New" | ||
| placement: left | ||
| - title: Importing data via links | ||
| content: 'Import files from <a href="https://zenodo.org/record/1001880">Zenodo</a>.' | ||
| backdrop: true | ||
| - title: Uploading the new data | ||
| element: '#tool-panel-upload-button .fa.fa-upload' | ||
| content: We need to upload data. Open the Galaxy Upload Manager | ||
| placement: right | ||
| postclick: | ||
| - '#tool-panel-upload-button .fa.fa-upload' | ||
| - '#btn-reset' | ||
| - title: Uploading the input data | ||
| element: '#btn-new' | ||
| content: Click on Paste/Fetch Data | ||
| placement: right | ||
| postclick: | ||
| - '#btn-new' | ||
| - title: Uploading the input data | ||
| element: .upload-text-column .upload-text .upload-text-content.form-control | ||
| content: >- | ||
| The corresponding <a | ||
| href="https://zenodo.org/record/1001880/files/CuffDiff_SQLite_database.sqlite">link</a> | ||
| should be provided in this field | ||
| placement: right | ||
| textinsert: 'https://zenodo.org/record/1001880/files/CuffDiff_SQLite_database.sqlite' | ||
| backdrop: false | ||
| - title: Uploading the input data | ||
| element: '#btn-start' | ||
| content: Click on "Start" to start loading the data to history | ||
| placement: right | ||
| postclick: | ||
| - '#btn-start' | ||
| - title: Uploading the input data | ||
| element: '#btn-close' | ||
| content: >- | ||
| The upload may take a while.<br> Hit the close button to close this | ||
| window. | ||
| placement: right | ||
| postclick: | ||
| - '#btn-close' | ||
| - title: Rename the input data | ||
| element: '.history-right-panel .list-items > *:first' | ||
| content: >- | ||
| The uploaded datasets is in the history, but its name corresponds to the | ||
| link. We want to rename them it to something more meaningful, e.g. | ||
| "RNA-Seq SQLite result data"<br><br> <ul> | ||
| <li>Click on the pencil icon beside the file to "Edit Attributes".</li> | ||
| <li>Change the "<b>Name:</b>" accordingly.</li> | ||
| </ul> | ||
| position: left | ||
| - title: Extract CuffDiff results | ||
| content: >- | ||
| CuffDiff’s output data is organized in a SQLite database, so we need to | ||
| extract it to be able to see what it looks like.<br><br> For this | ||
| tutorial, we are interested in CuffDiff’s tested transcripts for | ||
| differential expression. | ||
| backdrop: true | ||
| - title: Extract CuffDiff results | ||
| element: '#tool-search-query' | ||
| content: Search for 'Extract CuffDiff' tool. | ||
| placement: right | ||
| textinsert: Extract CuffDiff | ||
| - title: Extract CuffDiff results | ||
| element: '#tool-search' | ||
| content: Click on the 'Extract CuffDiff' tool to open it. | ||
| placement: right | ||
| postclick: | ||
| - >- | ||
| a[href$="https://galaxy.uni-freiburg.de/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fdevteam%2Fcummerbund_to_tabular%2Fcummerbund_to_cuffdiff%2F1.0.1"] | ||
| .tool-old-link | ||
| - title: Extract CuffDiff results | ||
| element: '#tool-search' | ||
| content: >- | ||
| Run the tool with the following parameters:<ul> | ||
| <li>“Select tables to output” to `Transcript differential expression | ||
| testing`</li> | ||
| </ul> | ||
| position: right | ||
| - title: Extract CuffDiff results | ||
| element: '.history-right-panel .list-items > *:first' | ||
| content: >- | ||
| Inspect the table by clicking in the eye (“View data”) on the right of the | ||
| file name in the history. | ||
| <br>Each entry represents a differentially expressed gene, but not all are | ||
| significant. We want to keep only those that are reported as significant | ||
| differentially expressed. | ||
| position: left | ||
| - title: Questions | ||
| content: |- | ||
| <ul> | ||
| <li>How to retain only the significant differentially expressed genes?</li> | ||
| <li>Which column stores this information?</li> | ||
| </ul> | ||
| backdrop: false | ||
| - title: Filtering and sorting | ||
| content: >- | ||
| We now want to first highlight the most significant differentially | ||
| expressed genes in our analysis, and then obtain informative | ||
| visualizations. | ||
| backdrop: true | ||
| - title: Extract CuffDiff’s most significant differentially expressed genes | ||
| element: '#tool-search-query' | ||
| content: Search for 'Filter' tool. | ||
| placement: right | ||
| textinsert: Filter | ||
| - title: Extract CuffDiff’s most significant differentially expressed genes | ||
| element: '#tool-search' | ||
| content: Click on the 'Filter' tool to open it. | ||
| placement: right | ||
| postclick: | ||
| - >- | ||
| a[href$="https://galaxy.uni-freiburg.de/tool_runner?tool_id=Filter1"] | ||
| .tool-old-link | ||
| - title: Extract CuffDiff’s most significant differentially expressed genes | ||
| element: '#tool-search' | ||
| content: |- | ||
| Run the tool with the following parameters:<ul> | ||
| <li>“Filter” to the extracted table from the previous step</li> | ||
| <li>“With following condition” to `c14=='yes'`</li> | ||
| </ul> | ||
| position: right | ||
| - title: Questions | ||
| content: |- | ||
| <ul> | ||
| <li>What column stores the information of significance for each record?</li> | ||
| <li>Which conditional expression has to be set to filter all records on the selected column?</li> | ||
| <li>What happened to the records in the original table?</li> | ||
| </ul> | ||
| backdrop: false | ||
| - title: Extract CuffDiff’s most significant differentially expressed genes | ||
| element: '.history-right-panel .list-items > *:first' | ||
| content: >- | ||
| Look at your data. The differential expression values are stored on column | ||
| 10, we will sort (descending) all records on the basis of their value at | ||
| the 10th column | ||
| position: left | ||
| - title: Extract CuffDiff’s most significant differentially expressed genes | ||
| element: '#tool-search-query' | ||
| content: Search for 'Sort' tool. | ||
| placement: right | ||
| textinsert: Sort | ||
| - title: Extract CuffDiff’s most significant differentially expressed genes | ||
| element: '#tool-search' | ||
| content: Click on the 'Sort' tool to open it. | ||
| placement: right | ||
| postclick: | ||
| - >- | ||
| a[href$="https://galaxy.uni-freiburg.de/tool_runner?tool_id=sort1"] | ||
| .tool-old-link | ||
| - title: Extract CuffDiff’s most significant differentially expressed genes | ||
| element: '#tool-search' | ||
| content: |- | ||
| Run the tool with the following parameters:<ul> | ||
| <li>“Sort Dataset” to the filtered table</li> | ||
| <li>“on column" to 'c10'</li> | ||
| <li>"with flavor" to 'Numerical sort'</li> | ||
| <li>"everything in" to 'Descending order'</li> | ||
| </ul> | ||
| position: right | ||
| - title: Questions | ||
| content: |- | ||
| <ul> | ||
| <li>Since the start of our filtering process, how many records now represent the significant subset for extracting informations?</li> | ||
| <li>What does this shrinking of the number of lines represent?</li> | ||
| </ul> | ||
| backdrop: false | ||
| - title: CummeRbund | ||
| content: >- | ||
| With CummeRbund we can visualize our RNA-Seq results of interest.<br><br> | ||
| CummeRbund generates always two outputs:<ul> | ||
| <li>the plot</li> | ||
| <li>the R script responsible for generating the plot</li></ul> | ||
| We are interested in visualizing all expression values of all transcripts | ||
| relative to the most significant differentially expressed gene we found in | ||
| the previous section. | ||
| backdrop: true | ||
| - title: Visualization | ||
| element: '#tool-search-query' | ||
| content: Search for 'CummeRbund' tool. | ||
| placement: right | ||
| textinsert: CummeRbund | ||
| - title: Visualization | ||
| element: '#tool-search' | ||
| content: Click on the 'CummeRbund' tool to open it. | ||
| placement: right | ||
| postclick: | ||
| - >- | ||
| a[href$="https://galaxy.uni-freiburg.de/tool_runner?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fdevteam%2Fcummerbund%2FcummeRbund%2F2.16.0"] | ||
| .tool-old-link | ||
| - title: Visualization | ||
| element: '#tool-search' | ||
| content: >- | ||
| Run the tool with the following parameters:<ul> | ||
| <li>"Select backend database (sqlite)" to the corresponding result | ||
| file</li> | ||
| <li>Click on “Insert plot”</li> | ||
| <li>"The width of the image" to `800`</li> | ||
| <li>"The height of the image" to `600`</li> | ||
| <li>"Plot type" to `Expression Plot`</li> | ||
| <li>"Gene ID" to 'NDUFV1'</li> | ||
| </ul> | ||
| position: right | ||
| - title: Visualization | ||
| element: '.history-right-panel .list-items > *:first' | ||
| content: |- | ||
| Inspect the output "Expression plot":<ul> | ||
| <li>The Expression Plot represents the expression of all isoforms of a single gene (NDUFV1) with replicate FPKMs exposed.</li> | ||
| <li>Our plot has a modest number of isoforms, and is therefore already readable. However, in case of 5 or 6 isoforms, the plot can look very busy. We can therefore change the visualization type by selecting another type of plot.</li><ul> | ||
| position: left | ||
| - title: Visualization | ||
| element: '.history-right-panel .list-items > *:first' | ||
| content: Re-run the tool again by clicking on "Run this job again" button | ||
| position: left | ||
| - title: Visualization | ||
| element: '#tool-search' | ||
| content: |- | ||
| Re-run the tool changing the parameter:<ul> | ||
| <li>Click on “Insert plot”</li> | ||
| <li>"Plot type" to `Expression Bar Plot`</li> | ||
| <li>"Expression levels to plot" to 'Isoforms'</li> | ||
| </ul> | ||
| position: right | ||
| - title: Visualization | ||
| element: '.history-right-panel .list-items > *:first' | ||
| content: >- | ||
| Expression Bar Plot of a single gene (NDUFV1) with replicate FPKMs | ||
| exposed. | ||
| position: left | ||
| - title: Conclusion | ||
| content: >- | ||
| Visualization tools help researchers making sense of data, providing a | ||
| bird’s-eye view of the underlying analysis results. In this tutorial we | ||
| overviewed the advantages of visualizing RNA-Seq results with CummeRbund, | ||
| and gained insights on CuffDiff’s big-data output by plotting informations | ||
| relative to the most significant differentially expressed genes in our | ||
| RNA-Seq analysis. | ||
| backdrop: true | ||
| - title: Key points | ||
| content: >- | ||
| <ul><li>Extract informations from a SQLite CuffDiff database</li> | ||
| <li>Filter and sort results to highlight differential expressed genes of | ||
| interest</li> | ||
| <li>Generate publication-ready visualizations for RNA-Seq analysis | ||
| results</il></ul> | ||
| backdrop: true |