Integrated Genome browser (IGB) installation

Transcription

1 Integrated Genome browser (IGB) installation Navigate to the IGB download page You will see three icons for download: The three icons correspond to different memory (RAM) requirements but the software is the same. If your computer has a lot of memory (8GB or more), you can try downloading the large memory version. Otherwise, use the icons with less memory. 1 GB will be fine too, except that certain operations will take a little longer. If you click an icon, a Java virtual machine (JVM) will be launched on your computer. The Java virtual machine (JVM) will try to reserve the amount of memory that corresponds to the icon you clicked on. If sufficient memory cannot be reserved, you will receive an error message. In that case, try clicking an icon that specifies less memory. If your computer doesn t have the Java runtime installed, go to

2 and follow the download instructions. If the JVM was launched successfully, the application will now be downloaded. Upon completion of the download, the application will be launched. A security warning will appear:

3 Click on I accept the risk and want to run this application. Then click Run. The application Integrated Genome Browser will now be launched:

4 In the upper right corner, select Species Homo sapiens and Genome Version H_sapiens_Feb_2009 After loading the genome, in the lower left corner on the Data Access tab you will see two registered servers:

5 These servers provide basic annotation and sequence information to the IGB. However, both of these servers are located in California and, therefore, loading the data takes some time. The has set up a DAS/2 server that you can use as an alternative. To register this server in IGB do the following. On the Data Access tab click Configure (the blue link in the image above). A screen opens up that permits registering other servers in IGB

6 To register a new server, click Add. Fill in the fields as shown below: Name: IIT_Milano (or some other name you like) Type: DAS2 URL: Click Add server. An authentication dialog will be displayed. Provide your username and password and click Login. On the Data Access tab you should now see the following server after opening up the trees:

7 You are now ready to use IGB. It is suggested to inspect the IGB manual. You can find the IGB manual here: IIT Quickload server:

8 Tips and tricks IGB loading genomes IGB loading data, from file, URL, server Navigating in IGB The zoom slider bar (purple box/arrow) Use control+mouse wheel (Windows) or command+mouse wheel (Mac) (yellow box/arrow) Double click on a feature (orange box/arrow) Enter a coordinate range (green box/arrow) Click and drag within the coordinate axis (blue box/arrow) Search and click (red box/arrow) To jump-zoom to a feature: Double click on it To jump-zoom to a region: Use the selection tool: Click the selection tool icon to activate the 'arrow' cursor is active Click-drag across the coordinates axis; you should see a blue box indicating the region you want. Release the mouse button. Enter coordinates in the coordinate range box (top left) and type ENTER To jump-zoom to a gene: Type the name of the gene in the coordinate range box (top left) and type ENTER File formats, bam, graph, bed Customize view (double click, sliders, search, stack height, colors, normalization, view sequence, arrange tracks by dragging them around) Selecting feature: click drag, shift click, shift control click Show hide tracks: click the eye icon Interbase coordinates, zooming, coordinate drag, zoom stripe plus slider Restriction sites Simplify track names

9 Setting Stack height. You can set the track height manually by using the Set Stack Height or let IGB set the optimal track height by clicking the Optimize Stack Height option View Sequence Click on a splice variant to select it. The selected variant will be enclosed in a red rectangle.

10 Click View -> View Genomic Sequence in Sequence Viewer. The Sequence Viewer will open up and display the sequence of the selected transcript.

11 Click Show cdna and the Show -> +2 Translation to display the protein sequence.

12 To display all reads, set stack height to 0: Alternative splicing Load Arabidopsis thaliana genome, assembly Jun 2009 In the Advanced Search tab, type AT1G Three transcripts will be displayed. Double click the first one to zoom into the corresponding genome region. Note that the sixth exon counted from the left has alternative boundaries because it is alternatively spliced. From IGB Quickload, load track: RNA-Seq / Loraine Lab / Mixed Cold / SM / Reads / Cold, alignments Note that there are reads that correspond to either spliceform (pointed to by the black arrows). Count them to find out, which spliceform is expressed at a higher level. You can use read selection for counting. Drag over the reads to select them. To add reads, shift-click them. To remove reads, controlshift click them. The number of selected items is displayed in the upper right corner.

13 To export a view, got to File -> Export Image or click the Export Image button on the tool bar: Choose resolution, image format, view details, and location to save the view to a file.

14 Working with graph files Load species Zea mais, genome version Z_mays_B73_Mar_2010 Under the Data Access tabbed panel, open Data Source IGB Quickload > RNASeq > SRP Reproductive > Single-map reads (SM). Load the tracks coverage SRR Ovule coverage SRR Pollen coverage SRR Embryo coverage SRR Leaves Adjust the track names by clicking on the track name field and typing a short descriptive name. Use the FG (fore ground) and BG (background) fields to customize the track colors.

15 Zoom into region 1 : 51,631,058-61,254,530 and click the Load Data button. In order to permit visual comparison of tracks, you need to scale them. Select graphs by dragging over the track handles to the left. A red arrow appears to indicate that the track is selected. Alternatively, in the Graph tab click Select All. In the Graph tab, several operations are available to perform on selected tracks. Go to Y- Axis Scale and adjust the scale by Value to Max 300. You can now compare the track heights visually to obtain an idea about differential expression of transcripts in different tissues. Note that this is makes sense only for tracks with similar sequencing depth, which is the case here. Use the vertical slider to the left to stretch the display vertically. Try the different options that can be performed on tracks, such as changing display types or numerical operations (sum, difference etc). Go to region 1:56,439,168-56,446,420. The gene in that region is preferentially expressed in ovule:

16 Go to region 1 : 141,735, ,742,038. You can see the expression of exons that are not yet annotated and might represent a new gene.

17 Supported File types