3.5 Identify Bands in Size-Standard Lanes

After the lane boundaries have been delineated, you may identify the bands in the size-standard lanes. You will first allow the program to automatically detect what it can; then, you will edit the results so that the number of detected bands matches a pre-defined template. An example template, and the one used for image k102.avg, may be found in Appendix A.4. The purpose of the template is to have a standard against which images can be "warped" into alignment so as to have some basis of comparison with one another.

• Pointer to Operations Submenu, click [Std Bands] .

• Before attempting automatic band detection in the standard lanes, the Template Edit Dialog (Figure 3.7) will ask if you would like to "edit" the template. You may learn more about this in Chapter 5, Section 5.2. At this point, you will not; click [No] .

Figure 3.7: The Template Edit Dialog.

• Automatic band detection commences and short horizontal bars appear on the image where bands have been detected. On color displays, these are red. Notice that not all bands in the 3 marker lanes of image k102.avg have been detected.

• After automatic detection, you may manually add missing bands and delete false-positive bands. The Status Information Region reminds you that MB1 is used to insert a band, MB2 is used to delete a band, and MB3 is used to indicate the end of manual editing.

• Note also the appearance of a small popup (Figure 3.8) which is a toggle for editing with the semi-automatic mode turned off or on (default). The toggle button label is set appropriately:
  - Semi-automatic: when MB1 is depressed in a marker lane, a band is drawn at that location; when MB1 is released, the band is moved to the spot of highest intensity within a window of several pixels of the original spot.

  - Manual: when MB1 is clicked in a marker lane, a band is drawn at that location.

  You may switch at any time you are editing and are encouraged to experiment with both options.

Figure 3.8: The Semi-automatic band-insertion toggle ("On" or "Off").

• Notice that while inserting bands, either in the manual or in the semi-automatic mode, as long as MB1 is depressed, a small graph appears to the lower right of the screen. This graph is a vertical profile of intensity values along the center of the marker lane in which the band is being placed. A small circle tracks the movement of the pointer in the lane in order to help visualize where the pointer is in relation to intensity peaks. The profile graph disappears when MB1 is released.

• To delete a band, move the pointer over the band to be deleted and click MB2. A warning message apppears in the Status Information Region if MB2 is clicked in an area where there is no band or if the pointer is not in a marker lane. Caution; where two or more bands are close together, some care must taken when clicking MB2. You should experiment by adding and deleting bands.

• Important: The bands identified in the size-standard lanes must correspond 1:1 with those currently defined for the template for spatial normalization and fragment sizing to function correctly. If the number of bands identified in each size-standard lane does not match the number of bands defined for the template you will be informed of which lanes are discrepant.

• When the number of detected bands does not match the template, re-enter by clicking [Std Bands] . Editing is as before.

  Figure 3.9 shows the superimposed band bars in the size-standard lanes.

Figure 3.9: The red band bars in the size-standard lanes indicate band detections.