Tutorial

After you've completed the installation of the software and all its components and also activated the settings to the shell environment you are ready to go. Using this tutorial you will be guided through all stages of the program and learn about the basic functions to process fingerprint gels.

However the on-line help pages are a more complete guide on all functions and should be use as a reference when studying this tutorial or when using the software.

1: Download the example files

  • Download the gelimage file (5760 KBytes) and save it somewhere on your filesystem. If you are using Netscape, hold down shift and click on the link. You do not want Netscape to display or process the file in any way - just to save it for you!
  • You will also need a standard file to normalize the band positions back to where they would have been on the master gel. A standard file that works with this example gel image is provided. Download the standard file and save it in the $IMTOP_DIR directory. You can put the file anywhere, provided that you set the location correctly when setting up the defaults later.

2: Create a gel project from this image

The script imimport does all the work for you when it comes to creating directories and copying files, if you want to import and existing gel image file into the image processing system's file and directory structure.

Type:

  imimport testproject testgel /where/you/saved/example.tif

This will copy the TIFF image to $IMTOP_DIR/testproject/INCOMING/testgel/image.tif

3: Run the interactive Image program im3

Note: before running image you have to configure the im3 binary.

The analysis is split up into several stages, each step has its own customized screen display to represent the data created by the external analysis modules.

Type :

  im3

and wait for the startup window to pop up snapshot.

double click on testproject. A dialog box snapshot pops up to tell you that this project has never been opened before and ask you to set the default parameters for this project in the following window click [Yes].

4: Set parameter defaults for testproject

When the defaults window first comes up it contains the program built-in defaults snapshot.

Change these values to fit the example gel image snapshot.

(You should only have to change the following)

  lane number :          49
  marker repeat :        4
  zone number :          15
  component order :      1 2 3 4
  digest order :         BamHI:EcoRI:HindIII
  standard file :        (the location of the file example.std)
  normalisation method : non linear
  pixel ratio x/y :      1.00

(leave the rest unchanged)

Then click [OK].

The gels for this project will be listed in the right chooser window snapshot. Double click on the testgel to start analyzing this gel.

5: Set gel parameters for testgel

First you need to enter gel specific parameters for this particular gel snapshot. All you have to enter here is the gel number and a clone name pattern, all the other parameters have been initialized from the project defaults, which you set earlier, when you opened the project for the first time.

  • enter the gel number - In order to recognize which gel a clone came from a gel number is attached to each clone. It also encourages a proper archiving system for your gels. This number is an integer and can't contain letters. For this example gel type 1. Note: If you hit RETURN in this entry field the cursor jumps to the entry field for the first clone name.
  • enter the first clone name - this a pattern and depending on the numbering method chosen by the two boxes at the left, the clone names will be first initialized based on that first name. Enter clone1 for example. The subsequent clone names will be initialized clone2, clone3 etc. This'll do for this test project anyway.

Having entered the gel parameters snapshot clicking [OK] image moves to the next stage. As you have downloaded a TIFF image as example gel, and it has to be converted to the hidump format first. This is done automatically, so you will see that the program is busy for a moment and the following message is printed on the parent window

  running imageconversion ...

6: Step #1 Preview/Edit Gel Image

In step1 the gel image snapshot is displayed. Any editing or rotation of the image is done at this stage.

For this example no editing is required. Click on [right arrow] to proceed to the next stage.

7: Step #2 Edit Lanetracking

In step 2 image displays the results of the lane tracking, and for this test gel it should have got it right : snapshot.

As the lanes are recognized correctly, there's not much else we want to do here. The help-page on step 2 describes the in full the functionality of this screen - all the ways to edit the lanes and the image, and what all the buttons mean.

For now just hit [right arrow] to proceed to Step 3. The external band calling module is invoked. This may take a while, until the program comes back and presents Step 3. So be patient, especially on large gels and slow or overloaded machines.

8: Step #3 Edit Bandcalling

Step 3 provides a powerful editor for the sample bands snapshot. Most of the manual editing time will be spent in this stage. So it is important to become familiar with all the functions to use its full potential.

This tutorial won't go into every detail of the editor, I'll just demonstrate some basic functions to get first results and refer again to the help-page on step 3 for an in-depth reference.

The display consists of three parts :

  • The control buttons at the top
  • The trace window - shows an x-y plot of the current lane and a magnified image of a straightened out trace along the lane with the bands indicated by the green ball.
  • The gel image window - shows the gel how it would look like with straight uniform lanes, the small green triangles correespond to the band positions

You can magnify the trace window by using the [ + ] and [ - ] buttons. The scroll bars are operated by the middle mouse buttons as in the previous screens as well.

These are some basic band editing functions:

  • turn off bad lanes by clicking the middle mouse in each lane in the bottom panel, the gel image window.

    Note: Do not do this in the name entry box to the left of each lane, since you can edit the lane name on some systems the middle mouse will paste the clipboard contents into the name entry box.

    In this example lanes 29-31 and 34-35 are bad lanes, turn them off.
    Note: the number/name of the deleted lanes is now blank snapshot.

  • remove a range of unwanted bands across the whole gel. The automatic band caller attempts to find the location of the well and only looks for bands away from the well. In this case the well was not detected, possibly because of the bad lanes. You can remove the well collectively across the whole gel

    Click in the gel image window at the start of a lane and slide the mouse to a position to the right of the well but to the left of the first band and release it. The area will be highlighted and all affected bands are drawn red and a dialog box pops up asking whether you want to delete these bands snapshot. Click on [Yes] and all bands in this area in all lanes (even the ones you can't see) will be removed.

  • delete a range of bands in a single lane. Consider the bands to near the end of lane 1, you can remove all these bands in a single operation.

    Click in the trace window to the just to the left of first band you want to delete and slide the mouse to the right of the last band. As you slide the mouse a rectangle appears on the display this defines the selected area. When the mouse is released the bands in the selected region are drawn red and a dialog box pops up asking whether you want to delete these bands snapshot. Click on [Yes] and the bands will be deleted.

  • to navigate around the gel you can scroll the lanes up and down with the scroll on the left (drag it using the middle mouse button) and then click on the lane you want to edit.

  • to select a band click on the corresponding green ball in the trace window.

    Note if you click away from an existing band Image will insert a new one.

  • You can also select a band by clicking in the the current lane in gel image window, band positions are indicated by the small green triangles at the top and bottom of each lane. The band nearest to the mouse click will be selected.

    Note if you click in a different lane that lane becomes the current lane.

  • to remove bands click on the band with the middle mouse button on the trace window, or select the band first and press 'D' (for strong delete). The key 'n' (for nuke) does the same. If you just want to mark a band as deleted (maybe decide later about its fate) press 'd' (for soft delete).

  • to add a band move the mouse in the trace window and click in an area with no bands - a new band will be created there and made active.

    If you want to add a band very close to an existing one, it might just activate the nearby band instead of adding a new one. You can either zoom in to the trace display using the [ + ] button at the top, or split the existing band using 's' (for split) or 'm' (for multiply).

The band calling may be the weakest part of the program, because you'll have to edit every single gel by hand, but it is also the most complex task for a machine to do, if you think about it. The bandcalling algorithm just "sees" a single trace as shown in the x-y plot and decides on where to place a band using complicated mathematical models. And even you have been unsure about some bands - call it or not ?

Before you proceed to Step 4, delete the obviously over called bands in all lanes and click on [right arrow].

9: Step #4 Edit Marker-lock

Step 4 is designed to deal with the normalization of the sample bands. To make sure that this works OK, we have to line up each marker lane with the standard lane.

The automatic line-up snapshot has problems because of the extra bands that have been called. We're going to fix the problems now. The band editing functions are largely the same as in step 3.

Lets try and get lane 0 locked correctly then match this pattern to every other lane. All we need to do for this example is delete the extra bands. Just step through the bands using the f/b keys turning off the extra bands using lower case d.

Turn off the extra bands until the display looks like this snapshot.

Lane 0 is now locked correctly. Select lane 0 and click on [re-lock on lane]. The locking module will now take every band position in lane 0 and match the pattern to every other lane. Note: that when re-locking is completed the extra bands we turned off in lane 0 have been deleted, extra bands in the other lanes are also deleted snapshot.

Although re-locking generally places the bands in the correct positions you should check all the marker lanes to ensure that the bands are located at the tops of peaks.

10: Finishing a gel

To finish the gel click the [right button] which pops up a dialog box snapshot confirming that all the marker lanes match the standard and asking if you want to proceed, select Yes. When you finish the gel the external normalization module, calculates the normalization factors and writes the normalized bands and the normalized traces. In this example a fragment size file is also written because the standard file contained size information. These files will be used by fpc.

Once this is completed a second dialog box snapshot asks whether you want to transfer this gel to fpc. If you have an fpc project set up with the testproject go ahead and click on [Yes]. But I'd suggest you don't transfer and archive the gel just yet, and select [No]. Another dialog box snapshot asks whether you want to stay in step 4 or return to the startup screen. Select [Yes] and go back to the startup screen.

11: Back to the start after finishing a gel

Back in the start screen, you'll notice that the gel chooser display has changed. The table on the right is now filled with ticks. The columns are labelled i, l, b, s at the top and stand for info (step 1), lanes (step 2), bands (step 3) and standard locking (step 4) respectively. All these data files have been created by the external analysis modules during the editing cycle. Each existing data file is marked with a tick. The arrow on the far right means that the gel is finished (i.e the fpc files have been written) and is ready to be transferred to the database. snapshot

Note : a gel that is transferred is also archived, so it doesn't show up in the gel chooser anymore. This means, that you should be very sure about a gel when you transfer it and there shouldn't be a need to edit it any further.

The database program fpc is available via anonymous ftp.

* quick link - http://q.sanger.ac.uk/8weztk48