This screen shows the alignment of the marker bands, which is used
to normalize the positions of the sample in relation to one
master marker lane pattern, the standard lane.
The display looks similar to the one used in Step
3 but now it shows only marker lanes.
The bands in each marker lanes are connected up by lines that link up the first band of each lane with the first band any other lane etc...
At the top you see the familiar trace plot display as used in Step 3, with an additional feature.
Each band in the current lane is lined up with the bands from the standard lane as described in the second column of the standard file, which contains the actual positions of the bands using the current chemistry, rather than the original band positions of the standard lane on the master gel, to which all positions are mapped back to.
The aim of this alignment editor is to get a perfect line up of each each marker lane with the standard lane. This can be controlled in the trace plot window, 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 image window 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, we're done.
The automatic standard locking module should do a pretty good job of lining up the patterns, but to get it right it is often necessary to help it a bit :
- choose a lane, where the line up looks best
- work your way through from the left to right (on ARGs and FLIs) or from right to left (on ABIs), and check the pattern that line up.
- if standard bands locked to the wrong marker bands delete these
- add in extra bands, if they weren't called and weren't inserted by the locking module.
- If you've got one lane locked perfectly, click on [re-lock on lane].
This will lock the band positions of the current lane onto the bands in all other marker lanes, and possibly get a better line up than using the positions from the standard file.
- [re-lock whole] will discard all changes the locking module made (in terms of inserting (brown) and deleting (orange) bands) and only lock on green bands all over again.
This screen is divided into three areas (from top to bottom) :
- the usual control buttons
- the trace plot window, showing an x-y plot the pixel
values scanned along the selected marker lane
- The trace image window, showing each marker lane as
a straight strip of pixels, and the lines connecting corresponding marker pattern with each other
The control buttons
On the top row, you find the usual navigator buttons.
Below is a set of buttons to zoom the trace plot. You can specify a zoom percentage
explicitly or type it in the yellow box (make sure the mouse pointer in "hovering" over that part of the window).
[100 %] zooms the plot to the same length as the trace image
below, which makes it possible to see exactly which region in the
plot corresponds to which region on the gel.
NOTE: The zoom functions apply to both the trace plot and the trace image in this step.
The trace plot sub window
The pixel values scanned along the middle of the lane are displayed
as an x-y plot. Furthermore a magnified version of the trace picture of the
selected 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.
The current band is marked by a cursor between the triangle markers and
a red dot on the plot.
The trace image sub window
Each lane is a pixel strip which has been scanned along the curved
lane as seen in Step 2 and straightened out to
get this picture of a gel as a block of lanes.
Each lane has a number shown in a black box and a text entry field for
its clone name.
This picture cannot be magnified, it is just there to give the user an
overview over the current state of the editing - which lane is
selected and the position of the currently selected band
At the very left there is a scrollbar to scroll this picture up and
down, but the selected lane will always be centered. The bottom
sample lane is selected, when you first go to this screen.
Editing the bands
When you enter Step 3, the bottom sample lane and it's first (leftmost)
band are selected.
If you want to edit the bands with the mouse, place the mouse pointer somewhere over the plot sub window.
All the editing keystrokes are also active in the trace image sub window.
to edit the clone names, the trace image sub window has to be active.
Navigate the cursor
- keys f and b to step one band forward and backwards.
- a left mouse click on a band in the plot window selects it.
- keys F and B to step through the lanes (backwards and forwards)
Add / Delete bands
- a left mouse click on a place on the plot, where there isn't already a band, will create a new one there.
- key s to split a band, the old band is shifted slightly to the left and a new band is created to the right.
- key d to mark a band as deleted
- space key to mark band as good (e.g. if it has been marked as deleted with key 'd' before)
- key D to delete a band for good, it is lost now.
- a middle mouse click on a band deletes a band for good.
- drag the left mouse button to mark an area - answer 'Yes' in the dialog box to remove all band within that area (marked in grey with "doomed bands" in red) from all lanes. It will spare the well band (the first band of each marker lane), so this is an efficient method to delete all bands that are actually background smears before all "real" bands start - position your mouse pointer slightly left of the first real band, press the left mouse button and hold it down, while moving the mouse to the left end of the trace image window until its over the area with the clone name boxes - then release the mouse button and click on "Yes" in the upcoming dialog box.
Shift current band
- left and right cursor keys move band by a small distance. Higher zoom modes allow more accurate positioning.
- key r will roll the band to its nearest peak - if it was positioned on the side of a band, it'll jump to its top.
Other functions that may prove useful can be accessed by these keystrokes:
- key S to save current band data. Saving the data from time to time might be useful if the machine or the network is unstable and prone to crash, or the program itself decides to present a bug to you and crashes without warning. You can save your work during an editing session with this function.