Sequencing settings (base calling)
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.
The default file name convention determines how the data files (AB1 or FSA)
associated with a plate are named.
The default file name convention is:
Application
Default settings
Fragment/HID analysis
<well>_<sample name>_<sample type>_<date and timestamp>.fsa
Sequence analysis
<well>_<sample name>_<date and timestamp>.ab1
Sequence analysis with the
Sanger variant analysis
option selected
<well>_<sample name>_<amplicon>_<specimen>_<date and
timestamp>.ab1
Modify the default
file name
convention
Chapter 10
Manage the software (instrument)
Manage run settings (instrument)
10
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SeqStudio
™
Genetic Analyzer Instrument and Software User Guide
