Beckman Coulter CEQ 8000, User Manual

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Sequence Analysis Procedures
154 CEQ™ 8000 Genetic Analysis System
Use Default Mobility Calibration
The analysis algorithm determines the mobility corrections (Mobility Calibration) to
be applied to each of the dye channels for every sample analyzed; stored mobility
calibrations are not used. When processing short PCR samples, the software has too
little data to determine an accurate mobility calibration from the data itself. Because of
this, we recommend using the Default Mobility Calibration now supplied with the
software. Reprocessing in such a manner may produce results with more consistent
peak spacing (especially in the first 50 or so bases).
In some cases, the Default Mobility Calibration may prove to be inappropriate. If the
bases near the beginning of the analyzed data appear to be inconsistently spaced, try
processing the data with this option unchecked.
Pre-peak Reduction
Data collected from the CEQ System may have small peaks (“pre-peaks”)
approximately one base prior to the peaks representing full-length DNA sequencing
fragments. These pre-peaks appear in the same channel and have two confirmed
sources:
• The presence of primers shorter than the full-length primer (N-1, N-2, etc.
primers) are present during the sequencing reaction, and
• The presence of a run of a single base (e.g. polyA) in the template leads to a
“copying” error.
To enable the system to attempt to identify and reduce the inclusion of pre-peaks in the
analyzed data, check the
Pre-peak reduction
check box in the
General
portion of
the
Sequence Analysis Parameters Editor
dialog box.
If the primers used for the sequencing reaction are known to contain
or suspected of containing N-1 primers as well as full-length primers,
the Pre-peak reduction check box should be checked.
Color Calibration
The values in the color matrix show the cross talk between the filters and the emissions
for A, C, G, and T. The base calling algorithm is designed to remove signal due to
cross talk. Click
Final Values
to view the computed color calibration after the run.
Click
Initial Values
to view the color calibration values prior to the run. If the color
calibration successfully removed peaks due to cross talk, it should be saved so it will
apply to data with residual cross-talk signal. If the data is going to be reanalyzed using
a known good color calibration, do not specify that the color calibration is to be
recomputed.
Check the
Compute color matrix
check box to re-compute the color calibration
the next time the data is analyzed. (This option is not available if accessed from the
Parameters Used to Compute Sequence menu option under the View menu.)