There are tools available outside HelixTree to visualize your copy number segmenting analysis results. Using the optional wiggle track file output or the CSV export of the segments data set (25.4), you can directly import your results into a genome viewer.

25.8.1 BeadStudio Genome Viewer

The comma separated value (CSV) bookmark file can be exported from CNAM (see 25.4.2) to be directly imported into BeadStudio’s Genome Viewer. To enable this feature in BeadStudio, you must install the HelixTree Segment Importer plug-in. For instructions on how to install the plug-in, see D.3.

To visualize the segmenting results in BeadStudio, export the Log2Ratio Segments Dataset using the File->Save as Comma Delimited Text File feature of the spreadsheet view. In BeadStudio, open a project containing the same data that was segmented in CNAM. From the toolbar in BeadStudio, open Tools->Show Genome Viewer.... (Note: If using an older version of BeadStudio, the Genome Viewer feature may be found in the Analysis drop down menu.) From the toolbar of the Genome Viewer, open Analysis->Run Auto-Bookmark.... If the plug-in was installed correctly the HelixTree Segment Importer analysis algorithm should be available in the window that opens. Select the HelixTree algorithm, give the bookmark analysis a name, and click Next.

Figure 25.9: Bookmark Importer in BeadStudio showing the available HelixTree Segment Importer algorithm.

Continue through the bookmark analysis importer selecting the desired samples and chromosomes. At the last screen of the importer click Start and a small dialog will prompt you for the CSV bookmark file that was created in HelixTree.

After importing the CSV bookmark file, return to the BeadStudio Genome Viewer and select View->Chromosome Browser. You may need to go to the Plot Settings tab, found either on the left or bottom of the screen, and select the plot to be viewed. Change the chromosome in the toolbar directly above the plot window. The segments found in CNAM will be visible as red and green bands behind the data points being viewed. These bands show the locations of the segments.

Figure 25.10: Chromosome Viewer in BeadStudio showing the CNAM segmenting results in green and red.

To view the details of the imported bookmark file, select from the Genome Viewer View->Bookmark Viewer. Select the imported bookmark analysis to view the data in the lower portion of that window.

25.8.2 UCSC Genome Browser

The UCSC Genome Browser is an online tool created by the University of California, Santa Cruz ( http://genome.ucsc.edu/cgi-bin/hgGateway). The genome browser is used to view genome data formatted in the Wiggle track format (WIG).

To output segmenting results as a WIG file, use the Optional Bookmark File Output option of the Copy Number Analysis Segmentation tool (25.3). In the online genome browser, select the add custom tracks button to open the Add Custom Tracks page. From this page browse for a WIG file created in CNAM and select Submit. The next page will have a table to view the tracks found in the specified WIG file. To add more data tracks (samples), select add custom tracks. To view the tracks in the genome browser, select go to genome browser.

Figure 25.11: UCSC Genome Browser showing CNAM segmenting results.

There are some restrictions on the data that can be imported into the UCSC Genome Browser because it is an online tool. When performing univariate segmenting analysis in CNAM, the results may contain segments that are only one marker in length. The UCSC Genome Browser does not accept data files containing segments with length equal to one. To avoid this conflict, set the Min #Markers per segment parameter in the Copy Number Analysis Segmentation tool (25.3) to a value greater than one.

The second conflict when using the USCS Genome Browser to view CNAM segmenting results occurs when the difference between a segment’s chromosome start position and chromosome end position is found to be larger than 10,000,000. Currently the best way to handle this issue is to manually separate the large segments into multiple adjacent segments with the same segment mean. The WIG file can be opened and edited in a text editor. Following the format outputted by CNAM, insert rows to divide the long segment into multiple shorter segments.

25.8.3 Affymetrix IGB

An alternative to the UCSC Genome Browser is the Affymetrix Integrated Genome Browser (IGB). Affymetrix IGB is a Java based tool that can be downloaded from http://www.affymetrix.com/support/developer/tools/download_igb.affx. IGB supports the Wiggle track format (WIG) as well as other data formats created using Affymetrix software. IGB does not require the GCOS database or libraries.

To use the Affymetrix IGB, output a WIG track file while running a segmenting analysis in CNAM (25.3). In Affymetrix IGB, open File->Open file. Select to open "Files of type: Graph Files" and browse for the WIG file created in CNAM. Select Open and the segmenting data should become visible in the plot window. You may change the appearance of the plot using the Graph Adjuster tab at the bottom of the window.

Figure 25.12: Affymetrix IGB showing CNAM segmenting results (in yellow) plotted with log2 Ratio data.

Affymetrix IGB also has features to import annotations data, sequence data, and other graph data that can be viewed together in the same plot window. To use IGB to view log2 Ratio data, you may export copy number CNT file data from the Affymetrix CNAT Viewer into the SGR file format. If you do not have CNT files of your log2 ratio data, you may create your own SGR file. An SGR file is a tab separated ASCII file with three columns: chromosome, chromosome position, and data value. The file should not contain column headings. Rename the text file to have the *.sgr extension to be recognized by IGB. An example SGR file is as follows: