The default settings are optimized for sequencing of PCR amplicons from diploid
genes, which are expected to have Mixed bases and which should end At PCR Stop.
For sequencing from plasmid templates, which are of pure sequence (no Mixed bases)
and have longer sequence read times than PCR products, create new analysis settings
and disable (uncheck) At PCR Stop and Mixed base threshold checkboxes.
Setting
Description
Quality Threshold
• Basecall assignment (ambiguous bases):
– Do not assign Ns to basecalls
– Assign Ns to basecalls with QV<5— Bases with a QV less than the threshold
display N instead of the base letter
• End base—Last base on which to perform basecalling:
– At PCR Stop
– After X number of bases
– After X number of Ns in X number of bases
– After X number of Ns
Note:
If you have PCR products with sequences that end while data is still being
collected, select the At PCR Stop checkbox.
Mixed bases threshold
When enabled, determines the secondary peak height ratio where the secondary peak
is considered a potential mixed base. Reaching the threshold is a necessary but not
sufficient condition for the basecalling algorithm to call a mixed base.
Clear range methods
• Use quality values—Sets a window with a specified number of allowed low-quality
bases by removing bases until there are <X number of bases per Z number of
bases with QV <Y.
• Use base positions—Specifies the first and last base in the range to consider, or
trims the specified number of bases from the 3
¢
end.
• Mask base positions before—Specifies the base position before which to disregard
bases.
Sequencing
settings (base
calling)
Chapter 9
Manage the software (Plate Manager)
Manage analysis settings
9
SeqStudio
™
Genetic Analyzer Instrument and Software User Guide
129
