Exercise 1 - Ladders

Before any alleles can be called, you need to check that the ladders have been called correctly and edit any that haven't. The correct ladder should automatically be fitted once all the ladder peaks are called correctly.

Select all of the provided documents and the Traces tab. Set spacing to 80, uncheck Allow Vertical Overlap and Scale X Axes, set sizing method to 3rd Order Least Squares. Make sure Loci says New Loci so you're starting fresh. Uncheck all dyes except the ladder (LIZ in this case), and turn on show traces, show peak calls, and show peak labels. Peak calls are shown by the vertical line below the trace. Not all of the peak labels (at the bottom of the peak calls) may show up, depending on the width of your screen. Mouse Co-ordinates, Document Names, and Y Axes Scale can be turned on too. You should end up with the view looking like this:


Examine the selected files looking for ones without a ladder or where the ladder hasn't fitted correctly. Select each one in the document table: A01 and A03 initially. Delete them from the document table.

Once you've deleted them, select all again and carry on checking for others that don't look good. Check the ladder starts at 60 and finishes at 600. Some may finish at less than 600.

Notice how A06 has ladder peaks at the end which look wrong. Select only the A06 sequence and check the Ladder tab - it should be obvious that the peaks have diverged from the ideal:


Return to the Traces tab. Select A05, A06 and A07 all at once so the ladders should all roughly line up. A06 appears to have some missing peak calls. You can either manually call these peaks or just discard the peaks after 480. In this case there aren't any alleles after 480 (as you'll see if you turn on the other dyes and turn off LIZ, so we can remove the peaks by selecting that region and hitting Remove Peak. If you don't remove these incorrect peaks the sizing algorithm may not work correctly.

Save your changes and check the Ladder tab again to see that the remaining peaks fit the ideal properly now. Return to the Traces tab and select all again.

A10 is over-trimmed so you should select that document and move the trimmed region. Drag it left until the first peak called in LIZ is 60. This ladder should be recognized as GeneScan 600. Save that document and then select all again.

With all the trims and ladders correct, you can turn off the ladder (LIZ) and just see the other dyes. Hit the scale X button to draw in base coordinates scaled according to the ladder instead of the electropherogram machine coordinates.

Now that you've turned the ladder (LIZ) off, notice that A10 appears to be a null data set too as there are only PET peaks and no others. If you increase the Y scale you will see peaks appearing but these are perfectly aligned with the ladder so are likely to be pull-up artefacts. Delete A10 and reselect all remaining traces.

You're now ready to move on and set the loci.


Exercise 2: Setting the Loci
Exercise 3: Binning