Step 3 - Edit bandcalling

Show the sample bands and provided functions to edit them on an artificial gel display. In accordance with the lane grid (see Step 2) an thin area either side of the lane centre-line has been scanned across the lane to get a straight lane-trace. These image strips are assembled into an artificial gel image which is perfectly square with straight lanes.

The screen has two main displays :

  • the trace plot display, showing an x-y plot of the pixel values scanned along the active lane.
  • The trace gel display, showing a composite of a thin strip traced along each lane on the original gel to make up an artificial gel display with straight lanes.

Menubar

  • File
    • Save bands - saves all changes to disk.
    • Print plot - brings up print dialog to print the trace plot display of the active lane.
    • Print traces - brings up print dialog to print the trace gel display of the whole gel.
    • Quit - saves all current changes and quits the program.
  • Edit
    • Clear lane - removes all bands (but one) in the active lane. Do not use this function if this clone is empty anyway and should be excluded from the database. You should switch the lane off completely using the middle mouse button (see below).
    • Re-load names - discards the current names and re-initializes the names by creating them from a template or by reading them from an external program.
    • Re-run bandcalling - calls the external bandcalling program again, to calculate the bands from scratch. The current bands will be lost.
  • View (change magnification of the trace plot only)
    • 100 % - display the trace plot to fit the display section at the top.
    • 200 % - display the trace plot at 2x magnification.
    • 300 % - ...
    • 400 % - ...
    • Set zoom - Lets you enter the horizontal magnification factor for the trace plot.
    • Show bands on trace - If there are too many band on the gel it may be useful to turn them off in the trace gel display to speed up the drawing time for that window. The program will automatically determine when that is a good idea, so this toggle menu item is a way to override the choice that was made.
  • Help (context sensitive help)
    • current - brings up this help page.
    • about Shortcuts - brief reminder about the mouse and keyboard actions in this display.

Toolbar

Zoom trace plot :
Image of toolbar zoom buttons
change magnification of the trace plot to zoom in or out. This display can only be zoomed horizontally.
grey ramp tool
modify contrast using the greyramp tool.
The arrow buttons
Image of toolbar arrows

Keyboards shortcuts

Most menu items will have accelerator keys associated with them, which are shown next to the menu item, when you browse through the menu bar.

As well as those accelerators, there are a few more key-strokes which perform useful functions.

Note : The mouse pointer has to be inside the trace plot or the trace gel display area to guarantee that these key-strokes will work.

Most keystrokes operate on the currently selected band (marked in RED) and then step forward. If you are already on the last band in one lane it will step to the first band in the next lane.

SPACE
accept the band. In most cases this will be used to move the cursor forward, but it will also un-mark a band which has been marked as deleted using the 'd' key-function.
d
mark the band as deleted. The band will be drawn in BLUE from now on, and it will not be included in the final output data. The SPACE function will mark this band as "good" again.
D or n
Delete or nuke the band. Most commonly used to remove a band. The band disappears totally and you would have to use another function to re-create the band at this position.
x
Expunges all bands currently marked for deletion. This will apply the nuke function to all blue bands. The nuke function can be slow sometimes, especially when all bands are also shown in the trace display. For faster editing one can mark a large number of bands for deletion and then nuke them all at once.
m or s
to multiply (i.e. split) the band. Useful if a single band was called on a wide peak. This function make two bands out of the selected band and position them either side of the old band position. The cursor will jump on the right of those two bands.
left / right cursor keys
move selected band left or right by a small amount. Higher magnification in the trace plot display allows finer positioning.
r
roll band to the nearest peak. A highly useful function which is in fact a specialized "move" (as performed by the left/right cursor keys). This function will determine whether the band sits on the slope of a peak and roll up to the top of that peak. If the band sits on a flat part of the trace nothing will happen.
f and b
simply moves the cursor forward or backwards. no action is performed on the selected band; it is simply skipped.
F or up cursor key
move to the next clone (up the gel), and position the cursor on the first band in that lane.
B or down cursor key
move to the previous clone (down the gel), and position the cursor on the first band in that lane.
RETURN
moves the keyboard focus to the clone-name text-box in the active lane. See below how to edit the clone names. When a text-box is active the key-strokes here will not work as such, because the characters will be typed into the text-box.
C
performs the same action as the "Clear lane" menu item.
S
performs the same action as the "Save bands" menu item.
R
performs the same action as the "Re-run bandcalling" menu item.

Mouse actions

The mouse actions differ for the two displays:

trace plot display at the top :

  • left click on or near a band will set the cursor on that band (i.e. make it the "selected band").
  • left click on any other point will create a new band at the mouse position.
  • left drag a bounding box around unwanted bands to delete a whole range of them in one go. The bands that fall within the area of the bounding box will be marked in RED and you can then choose to remove them.

    This "kill-box" function may be particularly useful to remove all bands below a certain vertical threshold.

  • middle click on or near a band will remove that band (i.e. do the same as the Delete-nuke keystroke).

trace gel display at the bottom :

  • left click on or near any band will jump to that band. If the band is in a lane other than the active one, the trace plot display at the top also switches to that lane.
  • left click on any other area in a sample lane or a left click on its lane number will jump to that lane and position the cursor on the first band in that lane.

    Nothing happens if you click on an empty area in the active lane (i.e. it doesn't create a new band there - you have to click on the trace plot for that.)

  • left drag left to right over an area where there shouldn't be any bands in any lane. All bands between the left and the right margin of the dragged area will be marked in RED and you can choose to delete them.
    Note: This also affects bands in marker lanes. The first band in every marker lane will be thought of as the Well band and not be deleted.
  • middle click on any lane or its lane number to switch off that lane. Such lanes will be excluded from editing in the future and that clone will not be included in the final output. If there is a blank lane on the gel, this is the preferred action to exclude it from being transferred to the database - do not use the "Clear lane" menu-function if that is what you want.

The trace plot display

The active lane is shown : the pixel values scanned along the centre of the active lane are displayed as an x-y plot. Furthermore a magnified version of the trace gel strip for the active lane is displayed beneath the plot.

Bands are marked as dots on the peaks of the plot and little triangles facing each other on the lane strip. If there is more than one band on top of a given point, a floating number will warn you about the bands that you can't see. This is only updated when the entire screen is refreshed, not on every addition or deletion of a band, for efficiency reasons. The number may also disappear at higher resolutions if the bands can be seen distinctly.

The selected band is marked by a cursor between the triangle markers and a red dot on the plot.

The trace gel display

Each lane is a pixel strip which has been scanned along the curved lane as seen in Step 2 and straightened out to create this artificial gel image with perfectly straight lanes.

Each lane has a number shown in a black box and a text entry field for its clone name. If the gel parameter "digestorder" is anything other than a '-' (hyphen), it means that different digests may be have been used for this gel. In that case a second entry box appears for each lane into which you can enter the digest name. If the "digestorder" parameter was set correctly, these names should have been correctly initialized.

This picture cannot be magnified, it is just there to give the user an overview over the current state of the editing - it marks the active lane and the position of the currently selected band.

At the very right there is a scrollbar to scroll this picture up and down, but the active lane should always be centered when you switch between lanes. When you first start up Step 3, the first sample at the bottom of this display will be activated.

Strategy to edit bands

This page provides a reference of all the functionality that the band-editor offers. It cannot, however, provide a universal recipe for what to do with the band data. It will depend on the actual gel and the individual experience of the user to decide which bands to leave and which bands to delete.

Based on your experience with the chemistry that is used, you will have to come up with a rating scheme for bands and train your users to stick to your rules. Such are decisions on partial bands or weak bands. The band calling program has no such knowledge and the initial output can only be viewed as a "rough cut".

Users have been to become very proficient on the Space and n key when they zip through lanes at zoom 400% and go over every band call to make split-second decision on the keyboard. The band-calling is certainly an area where improvement is due, but unfortunately it currently requires quite a bit of manual work which has to be done with the utmost precision.

Edit clone / digest names

To edit the clone names move your mouse pointer into the trace gel display. You can activate a text-box by hitting RETURN - the text-box for the active lane gets the keyboard focus and you can type/correct the clone name.

If digests are used, you can hit RETURN in the clone-name text-box to switch to the digest text-box. Hit RETURN again in that text-box to switch to the next lane where the cursor will be placed on the first band in that lane.

You can activate any text-box across the trace gel by clicking on the text-box. The particular lane will be activated and the selected text-box gets into focus for typing.

Note: Every time a clone name or digest text-box is active, all keystrokes typed when the mouse in in the trace gel display will go to the text-box to edit the selected name. If the mouse is moved into the trace plot display, all the usual band-editing keystrokes apply there.

The clone names and digests are stored in the names file which is located in the geldirectory.

Last modified : November 1999 Image 3.10

* quick link - http://q.sanger.ac.uk/ch9h6kzx