1. How do I create an Iobion Array Layout File?
2. Trying to load my two-color data I am receiving the message: "Data file does not match array layout file." Why?
3. How do I load custom annotations (or any annotations other than human, rat or mouse)?
4. What data types are accepted and what files are required?
5. What software technology is used by Iobion GeneTraffic?
6. What is the order of probe-level analysis, baseline calculation, etc. in GeneTraffic Uno?
7. How are the flags imported and used within GeneTraffic?
8. Sometimes I get a blank white screen when I am loading data. Sometimes the progress bar will not go away while using GeneTraffic. Has my browser crashed?
9. Can I NFS-mount the root file system?
10. I am having trouble viewing scatter plots or clusters; I receive an OpenGL error message. The following error message (or a similar one) appears when trying to view a scatter plot or cluster visualization.
11. When I see values for Lex.E and Lex.R, are they normalized values or the raw intensities?
12. What is the definition of 'Fold Change' in GeneTraffic?
13. How can I change the name of my project?
14. Why is it that Lex.R normalized values can be seen in the spot view but not the Lex.E normalized values?
15. Can I log out of GeneTraffic while certain processes are running, such as backing up, project normalization, etc.?
16. Can I make changes to a project after the data has been loaded and the normalization has been calculated?
17. Everything in my project is running very slowly and it takes a long time to load anything or to create any data objects.
18. Can I copy my GeneTraffic project?

1. How do I create an Iobion Array Layout File?

The Iobion Array Layout Files is a tab-delimited text file, containing columns of data in the following order: Grid, Column, Row, Sample ID, Gene ID, Gene Description (optional), Flags (optional). See page 66 of the GeneTraffic Duo Manual for more information.

If you have layout information for something other than 3-coordinate data, you can easily convert your 4-coordinate data to the 3-coordinate system. This is done by creating a Grid column which replaces the GridRow and GridColumn columns. The value in the Grid column equals (GridRow – 1) * (number of grid columns on the array) + GridColumn. See Excel Worksheet (4to3Coord.xls) for an example of the conversion.

If loading the array layout results in an error message, there may be a problem with the format of the file. Ensure that the order is exact (Grid, Column, Row, etc.) and that there is no duplication in the spot layout.

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2. Trying to load my two-color data I am receiving the message: "Data file does not match array layout file." Why?

Make sure that the layout, number of rows, and identifiers in the quantified data file match those in the array layout file. It may be easier to create an array layout file directly from the quantified data file.

One example in which the number of spots may not match between the array layout file and the quantified data file is when the quantified data file does not include missing spots that are present in the layout file. This can happen with GenePix .gpr files.

Make sure your file type is supported by GeneTraffic (see below for supported file types).

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3. How do I load custom annotations (or any annotations other than human, rat or mouse)?
A custom annotation file is easy to create. A few simple guidelines must be followed:

• The file must be a tab-delimited text (.txt) file;
• The first row must contain the column headers;
• Subsequent rows contain annotation data;
• The first column of the file must be the gene ID of the type used by the project's array layout;
• There must be no duplication in the first column;
• The column headings must match exactly Iobion’s annotation column headings, as in the following example:

When annotating genes from a file, it is not necessary to load annotation for every item in the array layout; do it only for those genes that you wish to annotate.

See example tab-delimited text file (ExpAnnotFile.xls).

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4. What data types are accepted and what files are required?

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5. HOW DO I CHANGE THE BASELINE OR NORMALIZATION OPTIONS AFTER THE PROJECT HAS BEEN LOADED AND ANALYZED?

In GeneTraffic DUO:
The normalization can be changed in the Hybridization Navigator by going into the Normalization data view. Normalization options may be changed either for a single hybridization or for the entire project.

In GeneTraffic UNO:
The probe-level analysis method and the baseline definition can be changed in the project definition section. Once the new options have been selected, click the Process button in the Batch Load Queue panel to recalculate the expression values.

Note: Renormalizing a project or changing the baseline will take several minutes (depending on the size of the project) and during this time the project will be unavailable.

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6. What is the order of probe-level analysis, baseline calculation, etc. in GeneTraffic Uno?

The probe-level analysis calculation is the first operation performed on an UNO (Affymetrix) project. Baseline selection does not affect RMA and MBEI calculations. Once the signal data have been obtained for each probe set, the baseline definition is used for ratio calculations.

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7. How are the flags imported and used within GeneTraffic?

Flags can be imported into GeneTraffic in four ways:
1) Through the quantified data file (B flag)
2) Through the Iobion Array Layout File (S-Z and H, C, R flags)
3) Through the flagging options available within GeneTraffic normalization (F,G, I, J, K, L)
4) Manually, by clicking on unflagged spots in GeneTraffic (A)

Spots flagged in GeneTraffic by methods 1, 3, and 4 are omitted from the calculation of aggregate statistics. The information will be retained (there is no R on the website) in the database and can be searched for in the Spot search panel, but in gene-level navigation the flagged data will be excluded from calculations. When clustering spot tables, flagged spots will be included, but when clustering gene tables, they will not.

H, C, R and S-Z flags are not omitted from the aggregate statistics. The S-Z flags in method 2 can be created by the user to mark various types of spots (exp: spots corresponding to genes in specific pathways). The H, C, R flags are used to mark housekeeping, control and reference genes, respectively. These flags can be used in various parts of GeneTraffic including the ScatterPlot view, Normalization panel, etc.

All flagged spots can be queried in the Spot Search panel by choosing Flag = “B”, “S”, etc. (make sure to use capitalized letters). The results of a spot search and the spot_flag project summary file provide flag information, in the form of letters denoting flags that are on.

When spots are manually flagged by the user, aggregate statistics are recalculated. If a very large number of spots are being flagged or unflagged, the process may take a long time.

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8. Sometimes I get a blank white screen when I am loading data. Sometimes the progress bar will not go away while using GeneTraffic. Has my browser crashed?

No. Sometimes due to the nature of HTML and how Internet Explorer loads a page, the Iobion progress bar may not show up until the end of the page loading or may show up and appear to process forever. To see whether your page is still processing simply move your mouse pointer to the blue title (where it says Microsoft Internet Explorer) at the top of the screen. If you see the hourglass beside your arrow pointer, the page is still processing and you should wait. If you see only the arrow, you may have to refresh your page by pressing F5. Should this fail to correct the problem, you may have to close the browser and start a new GeneTraffic session.

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9. Can I NFS-mount the root file system?

When you enable NFS in GeneTraffic, only the data directories are shared. This allows you to backup the important information and ignore the actual application files (operating system, Web server, configuration files, etc.)

In the event of a disaster you can simply reinstall the software from CD and use the backed up data directories.

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10. I am having trouble viewing scatter plots or clusters; I receive an OpenGL error message. The following error message (or a similar one) appears when trying to view a scatter plot or cluster visualization.

---------------------------------
OpenGLWnd.cpp
Line#100
OpenGL info
Vendor null
Renderer null
Version null
---------------------------------

Install the latest drivers available for your card from your video card manufacturer’s Web site:
www.ati.com
www.nvidea.com
www.ibm.com
www.dell.com
In most cases, this will fix the problem. If you still receive the error notification, please contact Iobion Technical Support with the exact model of your video card for further troubleshooting.

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11.When I see values for Lex.E and Lex.R, are they normalized values or the raw intensities?

When the value reads “Lex.E raw”, it is the raw intensity value and when it reads “Lex.E,” it is the normalized intensity value.

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12.What is the definition of 'Fold Change' in GeneTraffic?

Fold Change = LexE / LexR when LexE > LexR
and
-LexE / LexR when LexR > LexE.

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13.How can I change the name of my project?

When the project is open and you can view the name in the top left corner of your screen, click inside of this screen to change the name of the project.

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14.Why is it that Lex.R normalized values can be seen in the spot view by not the Lex.E normalized values?

When the normalization takes place, LexR Raw is noramlized against LexE Raw and so only LexR Raw changes.

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15.Can I log out of GeneTraffic while certain processes are running, such as backing up, project normalization, etc.?

You can log out of GeneTraffic while a project is being backed up or normalized. Situations in which you should NOT log out of GeneTraffic include: file transfer, load data (for an individual chip), before 'restore in is progress' message appears, while queries are executing, or any time you see a c reen that reads 'Processing request. This may take a moment, please wait.'

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16.Can I make changes to a project after the data has been loaded and the normalization has been calculated?

You can make changes to your project by going into the project definition. You can add or delete hybridizations, change your array layout or chip type, re-associate hybridizations with different groups, etc.

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17.Everything in my project is running very slowly and it takes a long time to load anything or to create any data objects.

Check to make sure that your project performance is optimized. The second icon in the list of options for a project is 'Test Performance.' If your performance is anything other than 'good,' you should optimize to ensure that it functions properly.

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18.Can I copy my GeneTraffic project?

You can copy by backing up a project and then restoring it. This will enable you to have two copies of a project to make various changes. You can backup a project by using the 8th icon in the actions options in the project list. Restore this project by then choosing the restore project function in the options in the left panel.