Section IV: Sequence Analysis FAQ Sheet
Sequence Analysis Troubleshooting Guide
37
What can be done to have the greatest chance of
accurately detecting heterozygotes?
While the default settings work well for most good sequencing results, adjustment of
the parameters may be required under the following circumstances:
•
Heterozygosities that occur before the 50th or after the 500th base
•
Raw Signal is too weak or too strong
•
Raw signal has contaminating sequence
To optimize the resolution of bases late in the run, it is recommended that the
separation method LFR-a be used. Other than this recommendation, there is no way
for the user to know how aggressive to be with the heterozygote detection settings on
his own data without experimentation. On clean data, false heterozygosities may be
absent with % Average peak spacing of 90% and Sensitivity settings as high as 0.75.
However, routinely using these settings is likely to cause many false “red” bases
(heterozygotes) to appear the sequence results.
Review the examples on the following pages.
Figure 23:
Too little signal. Noise is being recognized as heterozygosity, even at the low
Sensitivity setting of 0.1. The Raw Data is scaled from 0 to 130,000 counts.
