Step 4 - Edit markerlock

This step in the gel processing is probably the hardest to grasp. You may encounter a lot of unusual concepts and terminology. Please make sure you are familiar with the concept of normalisation before you attempt to use this display.

The screen is laid out similar to that of Step 3, but this time only the marker lanes are shown. In the gel trace display all marker bands are connected up by vertical lines to show which marker bands is thought to be the equivalent of which in every lane. The trace plot display shows the usual x-y plot and a magnified traced strip of gel image, and it shows the most important information in Step 4 - The line up of marker bands to the standard bands.

Menubar

  • File
    • Create new std - make a new standard file from the marker band pattern in the current lane.
    • Merge into std - average the marker band pattern of the current lane into the existing pattern of standard file that had been made previously with the Create new std function. The Merge and Create function is only to be used at the beginning of a mapping project when no standard file has yet been established.
    • 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
    • Re-lock on lane - Useful if the active lane has a correct marker lock. This information can then be used to adjust the band in the other lanes. The current bands will be lost, but only marker bands in lanes other than the active lane will be changed.
      Note: if there are any bands in the active lane that are marked as deleted these are lost when re-locking.
  • View (change magnification of both trace displays)
    • 100 % - display both trace displays to fit the window.
    • 200 % - display the trace displays at 2x magnification.
    • 300 % - ...
    • 400 % - ...
    • Set zoom - Lets you enter the horizontal magnification factor for both displays.
    • Edit in lockdirection - changes the notion of stepping "forward" when you edit bands with the various key-stroke commands. The lockdirection is set in the gel parameter dialog. Usually ABI gels are locked from the right, and other types of gels are locked from the left. Normally the cursor will move right after an editing key-stroke. If a gel is locked from the right (as are ABI gels) you may prefer the cursor to step left. If this menu item is checked, the cursor will always follow the lockdirection, otherwise it will edit left-to-right in the usual fashion.
      That means nothing will change on gels that lock from the left.
  • 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 both displays to zoom in or out 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 plot or the trace 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. See above how to edit right-to-left on gels that lock from the right (e.g. ABI gels). The default setting is to edit left-to-right, so when stepping forward the cursor will jump on the next band to the right.

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 used in the marker line-up. 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.
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), The cursor moves along the match lines and therefore moves to the equivalent band in the next lane.
B or down cursor key
move to the previous clone (down the gel), The cursor moves along the match lines and therefore moves to the equivalent band in the next lane.

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.
  • 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.)

Lining up the marker bands with the standard

The standard bands are loaded from the standard file. The standard bands are shown as triangles and they are linked up to their equivalent marker bands by lines.

The aim of this alignment editor is to get a perfect line up of each marker lane with the standard lane. This can be controlled in the trace plot display, where you check that each pattern in the marker lane is lined up with the corresponding pattern in the standard lane. The line up in the trace gel display is quick way to visualize the marker locking of the entire gel. If this line up is fairly straight and lines up the same pattern in each marker lane, you're done.

If the band calling hasn't done a very good job in the marker lanes, this can be a tedious process. But there are tools and tricks at hand to make it easier and save editing time.

Browse through all marker lanes and find a lane where the bands line up best with the standards. Check the trace plot display for the line-up and ignore the bottom display for now. The line up shown here may look messy but you can spot that most of the misalignment comes from the first few mis-called bands :

Good start

Good start

zoom

First delete those few bands highlighted by the first red circle. This will already dramatically change the line up. As you step through the bands it will become easy to see which ones fit into the standard pattern and which ones don't. In the other areas which are highlighted had been over-called, i.e. it called bands where there really are none. To show where the correct edits had been made - I used the 'd' key-stroke function to mark those bands as deleted. In real life you would probably delete them properly (i.e. "nuke" them!). This shows the correct line up :

Lined up

Lined up

zoom

Note that the marker lane now contains exactly as many bands as the standard lane pattern. This is absolutely essential.

When the correct line up is achieved the software will already have a fairly good idea how the actual marker bands on this gel relate to the standard bands. So it would be too tedious to make the same edits in every other marker lane. So instead, you can tell the program to figure out a good line-up in the other lanes based on the correct line up in the active lane. Now comes the time when you should inspect the bottom display. At the moment the lane you've just edited will probably not line at all with the other lanes.

Select the function [Re-lock on lane] to re-calculate the marker bands in the other lanes just based on the corrected band call of the active lane.

The result is illustrated in the following figure :

Relock result

Relock result

zoom

Note this will change the bands. Only the undeleted (red or green) bands in the active lane will be retained. Bands in the other lanes will have been re-called. In particular the number of bands in each marker lane is now the same as in the standard lane pattern.

[Re-lock on lane] button is the perfect savior, you will still have to inspect the lanes to make sure the marker lock is correct.

The gel can now be finished. Click the right arrow button. You will receive a warning if the program suspects a mistake in any lane. To be able to pass this test there have to be the same number of marker bands as standard bands in every lane. This is usually a good indication that a marker-lock has been achieved.

Last modified : November 1999 Image 3.10

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