Tutorial:Domain Graph

From OpenTutorials
Jump to: navigation, search


Slideshow Domain_Graph (about ?? minutes)
Handout Domain_Graph.pdf (?? pages)

Tutorial Sources
Tutorial Curators Anna Kuchinsky


Biological Use Case: Visualize the domain interactions that actually form a protein interaction network. Integrate exon expression data to visualize occurrences of alternative splicing events and the impact on the interactions.

Each of these procedures begins with installing DomainGraph and a database. To install DomainGraph...

  1. Go to the plugins manager by selecting Plugins->Manage Plugins->Network Inference->DomainGraph.
  2. Start DomainGraph via Plugins->DomainGraph.
  3. Upon first usage, you need to register and accept the DomainGraph license.

Next, install a database to use...

  1. Go to Plugins->DomainGraph->Manage DomainGraph database->Import data for selected species into database.
  2. Choose a local folder to install the database. Make sure you have write permission for this folder.
  3. Select Homo sapiens and click Import.
  4. Wait until a window pops up telling you the database is complete.

Analyzing the statistical results of AltAnalyze

After installing DomainGraph and the database, you can use DomainGraph to analyze the statistical results of AltAnalyze.

General analysis of a differential expression computed by AltAnalyze

This step requires a probeset statistics file produced by AltAnalyze (the appropriate file can be found in the results folder of AltAnalyze, called YOUR_DATASET_NAME-DomainGraph.txt). You can download a sample file via LINK. This sample file compares human embryonic stem cells (control group) to cardiac progenitor cells (experimental group). Save the file to your disk.

  1. Go to Plugins->DomainGraph->Start Domain Graph.
  2. Select Import AltAnalyze results for further analysis.
  3. Click the Start button.
  4. Select the AltAnalyze results file and click Open.
  5. Wait for the file to be processed. The resulting table should look like this.
AltAnalyze Workflow Table

The table shows the differentially expressed probesets detected by AltAnalyze. The table is enriched with information on the genes containing these probesets, the Splicing Indices (SI), the SI p-values, the MiDAS p-values, the Reactome and WikiPathway pathway(s) the genes participate in, miRNa binding sites associated with the probesets and alternative splicing (AS) annotations.

Analysis of a single gene

  1. Sort the table according to gene names by clicking on the header of the GeneID column.
  2. Scroll to the entry FYN in the gene column.
  3. Click on FYN. Wait for the graphics to be created.

You will receive a 'single gene'-domain graph. This network consists of the gene, all encoded proteins and their domain compositions. Nodes highlighted in yellow indicate the presence of differentially expressed probesets. The data panel shows the graphical representation of the protein domain architecture, the exon structure, the probeset and miRNA annotations. The results should look like this.

FYN

Please note exon no. 14 in the graphics: it shows significant down-regulation in the experimental group (cardia progenitors) compared to the control group (human embryonic stem cells) as can be seen by the annotated probesets colored in green. This result indicates that transcripts containing exon no. 14 are not favored in cardiac progenitor cells.

  • Move the mouse over a domain, exon, probeset, miRNA block in the graphics. The tooltips show some additional information on the domain.
  • Move the mouse over the probesets below exon no. 14. The tooltip shows e.g. the Splicing Index (dl), alternative splicing annotations and cross-hybridization information.

Analysis of a Reactome pathway

  1. Sort the table according to Reactome pathways by clicking on the Reactome pathway(s) header.
  2. Scroll to an entry containing Apoprosis in the Reactome column.
  3. Click on Apoptosis.
  4. A new window opens displaying all pathways associated with the probeset's gene. Click on Apoptosis in this window and then on Submit.
  5. Wait for the Reactome pathway to be loaded. Please note that you need to have an internet connection to load the pathways! The resulting pathway should look like this.
Reactome Apoptosis Pathway

Pathway nodes associated with differentially expressed probesets are highlighted. The table in the results panel shows the node ids, UniProt proteins and gene names for regulated nodes. Please note that you can import this pathway into DomainGraph if you want to further analyze it.

Analysis of a WikiPathway pathway

  1. Sort the table according to wikiPathway pathways by clicking on the Wikipathway(s) header.
  2. Scroll to an entry containing Apoptosis in the Wikipathway column.
  3. Click on Apoptosis.
  4. A new window opens displaying all pathways associated with the probeset's gene. Click on Apoptosis in this window and then on Submit.
  5. Wait for the Reactome pathway to be loaded. Please note that you need to have an internet connection to load the pathways! The resulting pathway should look like this.
WikiPathway Apoptosis

Pathway nodes associated with differentially expressed probesets are highlighted. The table in the results panel shows the node ids, UniProt proteins and gene names for regulated nodes. Please note that you can import this pathway into DomainGraph if you want to further analyze it.

Analyzing a protein interaction network

After installing DomainGraph and the database, you can use DomainGraph to analyze a protein interaction network.

Importing a protein interaction network into DomainGraph

This step requires a protein interaction network, given with Ensembl or UniProt ids. You can download a toy protein interaction network containing human proteins via LINK. Save the file to your disk.

  1. Go to Plugins->DomainGraph->Start DomainGraph.
  2. Select Create domain graph for gene or protein interaction network.
  3. Click Start.
  4. Select Visualize protein interaction network.
  5. Click on the drop-down menu to select/import protein interactions.
  6. Select import own network.
  7. Choose the location of your protein interaction network.
  8. Click on the second drop-down menu to select domain interactions. Choose InterDom as domain interaction dataset.
  9. Leave the network view on extended and identified on UniProt.
  10. Click Submit.

The resulting domain graph consists of the input proteins and protein interactions and is enriched by the domain compositions and domain interactions occurring according to InterDom. Your resulting network should look like this.

Toy Protein Interaction Network

Integrating Affymetrix Exon Array data for Single Experiments

This step requires two preprocessed files, an expression data file and a p-value file, created with APT or Expression Console. You can download a sample expression data file via LINK. You can download the corresponding p-value file via LINK. THese fample files describe the expression in testis tissue. Save the files to your disk.

  1. Go to Plugins->DomainGraph->Integrate Affymetrix exon expression data.
  2. Select the Single Experiment tab.
  3. Click the Import button right of choose expression file.
  4. Select the expression sample data for import.
  5. Once the data is imported, it is shown in the drop-down menu.
  6. Click the Import button right of choose p-value file.
  7. Select the p-value sample data for import.
  8. Type in a name for this data set and click Import.
  9. Once the data is imported, it is shown in the drop-down menu.
  10. Click OK and wait for the data integration.

In the resulting domain graph, domains partly or completely not expressed (=spliced out) are highlighted in pink. Orange domains are those being normally expressed but would form interactions with a spliced-out domain. Your resulting network should look similar to this.

Toy Network Protein Single Experiment

In the results panel on the right you can see the proteins, domains, and probesets that are not expressed in the sample according to their DABG p-values. Click on a row in the results panel to select the respective node in the network.

Viewing proteins, domains, exons, probesets, and miRNA binding sites as graphics

  1. Double-click on the protein node labelled NTNG2 (you might have to zoom in to be able to read the node labels). The graphics are displayed in the data panel and should look like this.
Toy Network Protein Single Experiment Features
  • The top row of an entry shows the protein with its contained domains (colored rectangles).
  • Below is the exon structure including 5' and 3'UTRs.
  • Below are the probesets. Pink coloring indicates that the probeset is not expressed. A color gradient from yellow to red is applied for expressed probesets representing expression strength.
  • Please note the absent probesets (colored in pink) covering most of the sequence of the Laminin-N domain and the 3rd Laminin-EGF domain.
  • The blue rectangle below one probeset indicates a microRNA binding site.
  • Move the mouse over the domains, exons, probesets, and miRNAs. Tooltips providing additional information are shown.
  • Move the mouse over the pink probesets. The tooltips display the p-values computed by DABG.
  • Right-click somewhere in the data panel. A menu will pop up for customizing and saving the graphics.
  • Click on an exon. This will open the corresponding Ensembl website for further information.

Analyzing a gene interaction network

After installing DomainGraph and the database, you can use DomainGraph to analyze a gene interaction network.

Importing a gene interaction network into DomainGraph

This step requires a gene interaction network, given with Ensembl or Entrez gene ids. You can download a toy gene interaction network containing human genes via LINK. Save the file to your disk.

  1. Go to Plugins->DomainGraph->Start DomainGraph.
  2. Select Create domain graph for gene or protein interaction network.
  3. Click Start.
  4. Select Visualize gene interaction network.
  5. Click on the drop-down menu to select/import gene interactions.
  6. Select Import own network.
  7. Choose the location of your gene interaction network.
  8. Leave the network view on extended and identified on Ens Gene.
  9. Click Submit.

The resulting domain graph consists of the input genes and gene interactions and is enriched by the protein isoforms encoded by the genes together with their domain compositions. Your resulting network should look like this.

Toy Gene Interaction Network

Integrating Affymetrix Exon Array data produced by AltAnalyze for analyzing differential exon expression

This step requires a results file produced by AltAnalyze (in the results folder of AltAnalyze, called "YOUR_DATASET_NAME-DomainGraph.txt"). You can download a sample expression data file via LINK. This sample file compares human embryonic stem cells (control group) to cardiac progenitor cells (experimental group). Save the file to your disk.

  1. Go to Plugins->DomainGraph->Integrate Affymetrix exon expression data.
  2. Select the AltAnalyze data tab.
  3. Click the Import button.
  4. Select the sample data for import.
  5. Once the data is imported, it is shown in the drop-down menu.
  6. Click the OK button and wait for the data integration.

In the resulting domain graph, all genes, proteins and domains containing differentially expressed probeset are highlighted in yellow. Your resulting network should look similar to this.

AltAnalyze Toy Gene Network Data

In the results panel on the right you can see the genes, proteins, domains, and probesets that are differentially expressed in the two samples together with their splicing indices. Click on a row in the results panel to select the respective node in the network.

Viewing proteins, domains, exons, probesets, and miRNA binding sites as graphics

  1. Double-click on the protein node labelled FYN (you might have to zoom in to be able to read the node labels). The graphics are displayed in the data panel and should look like this.
Features of Toy Gene Network
  • The top row of an entry shows the protein with its contained domains (colored rectangles).
  • Below is the exon structure including 5' and 3'UTRs. Identical exon numbers in different transcripts indicate identical exons.
  • Below are the probesets. White coloring means the probeset was not statistically analyzed by AltAnalyze due to pre-filtering steps. Graph probesets are expressed at similar levels in both groups of samples. Differentially expressed probesets are showin in red (up-regulated in the experimental group) and green (down-regulated in the experimental group).
  • Scroll down the graphics. Please note the down-regulated (highlighted by green-colored probesets) exon no. 14. This result indicates that protein isoforms containing exon no. 14 are down-regulated in cardiac progenitor cells.
  • Blue rectangles below some probesets indicate microRNA binding sites.
  • Move the mouse over the domains, exons, probesets, and miRNAs. Tooltips providing additional information are shown.
  • Move the mouse over the green-colored probesets annotated to exon no. 14. The tooltips display that exon no. 14 is knows to be a cassette exon, supporting the potential alternative splicing event. The Cross-hybridization information shows that both these probesets are "unique." This means, the probesets were specifically designed for this genomic locus and (at design-time of the microarray) did not align to any other transcript.
  • Right-click somewhere in the data panel. A menu will pop up for customizing and saving the graphics.
  • Click on an exon. This will open the corresponding Ensembl website for further information.

Additional Information

A PowerPoint presentation for the visual analysis of the biological effects alternative splicing is available here:

File:Domain graph.ppt