ComboScreenJ

A brief overview of how to use the ComboScreenJ program.

Accessing ComboScreenJ

• Open http://rice.genomics.purdue.edu in your web browser
• Click on "Lab Resources"
• Click on "ComboScreenJ" link

Contact Brian Abernathy (496-7208, room 206D) if you need further assistance. Contact James Kupke for bugs or suggestions.

Loading images in ComboScreenJ

Once you have started ComboScreenJ, you need to log in to ComboScreenJ itself.  Choose Log In from the User drop-down menu; a dialog box will appear.  In this box, enter the name of the database you will be scoring images into in the Database area.  The most commonly used databases are "comboscr" and "training," although others such as "single_overgo_pools" and "rescore_pools" have been created.  Username should be your userid from your email address, and you should have created your Password with Brian Abernathy. Hit Enter on your keyboard.

You are now ready to actually do things with your data.

The main ComboScreenJ window looks like the screen shot you see above.

Start by choosing the Pool of the image you want to score.  Next, choose the Hyb Bottle for the image, which is the filter set/BAC library from which you want to score a filter.  If the pool or hyb bottle you need to score does not appear in ComboScreenJ, contact Brian Abernathy (496-7208, room 206D).

In some cases, we have filter sets of the same BAC library from different sources.  For example, we have filters for Nipponbare that were printed at Purdue and filters for Nipponbare that were printed at Arizona.  It is critical that you choose the right filter set, as Purdue prints the BAC clones on the filters in a different order than Arizona.  Choosing the wrong filter means ComboScreenJ will identify the positive clones incorrectly and totally mess up the results!  Purdue's filters have 4-digit serial numbers, while Arizona's filters have 6-digit serial numbers.  If you are not sure which filter to select, ask for help!! 

Next, in the Filter area, choose the number of the filter you are scoring.  Sorghum (YRL) filters are numbered YRL_01 (filter 1) and YRL_02 (filter 2).  Filters for all other library sets are given letters, so for example, Nipponbare A will be filter A, Nipponbare B will be filter B, and so on.  In the example below, OA_ABaB from pool 141 will be scored.

Now you need to select the image you will be scoring.  Click the Open button (at the bottom of the Plates area).  When the File Selection window appears, the default directory is /Users/lab/Images-local/*, where * is the pool number.  Using the above example, the default directory will be /Users/lab/Images/141.  Most of the time, the file you are looking for will automatically appear in the window.  In the above example, the file OA_BcB_0022_9-5-04_141.tif appears (note OA_Bc is the old name of the OA_ABa library).  If the file you are looking for does not appear in the window (for example, if an filter was rescanned, the image may be from the wrong date), the image you are looking for should be in the list.  If you still can't find the file you are looking for, ask for help.

Note: ComboScreenJ does not care about the image itself.  The program uses the information in the Pool, Hyb Bottle, Filter, and Panel areas, along with the grids you create on top of the image (discussed below), to identify the names of positive clones.  If the image you load does not match the Pool, Hyb Bottle, and Filter you chose earlier, the computer will think the positive clones you mark belong to a different pool or hyb bottle or filter and interpret the data incorrectly.  Therefore, it is critical to make sure the Pool, Hyb Bottle, and Filter chosen match the image you loaded.  You need to be smarter than the computer!

Orienting images and using the grid

You will spend most of your time in ComboScreenJ working with the grid, so we will discuss the creation of grids before we go any further. To choose a panel, see step 1 under "Entering data" below. 

The first thing you should notice is this tool bar just below the loaded image:

The tool bar allows you to change the appearance and orientation of the loaded image. The left-hand slider changes the brightness of the image, while the right-hand slider changes the contrast.  The arrow buttons rotate the image left or right by 90 degrees each time you click them.  Always orient the image so that the filter name or serial number is somewhere in the upper left corner of the screen.  The only exception to this is the Sorghum (YRL) filters, which should be oriented so that the name/serial number is in the bottom right corner of the screen. By default, ComboScreenJ will try to autorotate the image in the correct position.  If you make a mistake, the "Reset" button resets the image to how it was when you first loaded it.  Once your image is correctly oriented and adjusted so that you can see the background grid pattern and/or the white spots where the filter was hole-punched, you are ready to create a grid.

• If you double-click in the Plates pane, it will create an average-sized grid where you clicked.  Or...
• If you hold down control and click and drag (if you are on a Mac) or right-click and drag (if you are not), it will create a grid in the area you drag over.
• If you hold down z and click and drag in the Plates pane, you can move the grid.
• If you use the arrow keys normally, it will move the grid one pixel at a time.
• If you hold down x and click and drag in the Plates pane, you can resize the grid.  Note that the grid resizes by moving the right edge or the bottom edge; the top and left edges are stationary.
• If you hold down x and use the arrow keys, it will resize the grid one pixel at a time.
• If you hold down c and click and drag in the Plates pane, you can rotate the grid.  Note that the grid rotates around its upper left corner.
• If you hold down c and use the arrow keys, it will rotate the grid one pixel at a time.
• If you click normally anywhere in the grid, it will shade the square blue.
• To copy a grid from one panel to another, you can use the Grid menu to copy and paste. Select the panel of the grid you want to copy. Click on Copy, and then select the new panel and click on Paste. Then, adjust the grid to the new panel. The keyboard shortcuts for copy and paste are listed next to the command.
  

Entering data

To enter a positive BAC clone into the database, do this for each clone by following these steps:

1. Select the panel - the one-sixth chunk of the image you are going to be working in - by clicking the corresponding button in the Panel area.  It does not matter what order you score the panels in, but I suggest starting with the panel that you think will be easiest to align a grid over.  Note that the order of the panels will sometimes change, as the panels are ordered differently on some filters.  Just make sure the image is oriented correctly and then choose the panels based on their position (ie. upper left, middle right, lower right, etc.), not their number.
2. Create a grid.  I suggest you do this by double-clicking in the upper-left corner of the panel you want the grid to fill, and adjusting its rotation and size with the z, x and c keys as described above, until it covers the panel exactly. If you cannot see a background grid on the filter image, you can use the white spots where the filters were hole-punched to align the grid.  The corners of the grid should touch the inside "corners" of the white spots.  Use the position of the positive hits within each square to double check the sizing and alignment of the grid squares.
3. Score the panel:
   1. If there are no hits on the filter, click the No hits box in the Hits menu.
   2. If there are hits on the filter, score by clicking on a square with a positive signal in the grid. A blue X will appear in that grid square.
   3. Click on the image of the pattern the signal matches from the list at the bottom of the screen.
   4. If the signal is really weak or really strong, click the Strong or Weak button in the Clones panel.
   5. Click Add, or hit the space bar. The square will turn red and the clone name will appear in the clone list area.
   6. Repeat steps b-e until all positive signals in the panel have been marked and added.
   7. When you're sure you've marked all the clones, go on to step 4.
      
4. Click the button for the next panel you want to work on.  Or copy the grid for use in the next panel so that you don't have to mess with the sizing again (refer to "Orienting images and using the grid" section above).  Repeat steps 2 and 3.  Do this for each of the six panels in the image.  Obviously, you don't have to create a grid for or score panels that have no hits in them.
5. When you have finished scoring the image, click the Close button in the bottom right corner of the screen.  You can now load and score another image.
   
6. When you have finished using ComboScreenJ, choose Exit in the User menu.
   

Viewing and removing data

Once you have added a BAC clone with the Add button, it will appear in the list as shown at right and stay there for as long as the panel the clone was scored in is still selected.  Signal strength will also be shown along with each entry if signal is strong [S] or weak [W].  If you click on an entry in the list, the square for that clone in the grid will turn purple.  If a clone is added in error, you can delete it by selecting it in the clone list and clicking Delete.

ComboScreenJ will remember all the clones that you add for a given combination of Pool, Hyb Bottle, Filter, and Panel.  If you select a different Pool, Hyb Bottle, Filter, or Panel, the image will close and the grid data will be saved. You can also see all the clones that have been entered through the web interface.  ComboScreenJ will remember the positions of the grids for each panel of each image you score.

If, after scoring, you discover that something was entered in error, open up the image file it was scored from, select the Pool, Hyb Bottle, and Filter corresponding to the image, and select the Panel that has the incorrect information. You will be able to select the incorrect clone from the list and delete it, or add new clones.