Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide
21
Raw Data Signal that is Too High
In a few cases, the signal strength can be so high that it saturates the detector. This can
lead to an erroneous base call where the software will artificially estimate peak height
and position. In this case, the software inserts extra bases into the base sequence. By
setting the raw data to full scale (137,000 counts) and looking at the peak shapes the
user can determine if peaks are “over-ranged”. If the peaks are “squared-off” at the top
(see the blue peaks in Figure 15), then the detector is saturated and the peaks are
“over-ranged”.
Corrective Action:
1.
If the peaks are too high, the simplest solution is to rerun the same sample using
a shorter injection time (for example: 7.5 seconds instead of 15 seconds).
2.
Use less template DNA or less thermal cycles to decrease the amount of
fluorescence signal generated by the sequencing reaction.
Figure 15: Over-ranged Peaks Causing Inserted Bases
Over-ranged
“T”peaks
Inserted “G” bases
caused by over-ranged
“T”peaks
