Run A Spectral Calibration; Overview Of Spectral Calibration - Thermo Scientific appliedbiosystems SeqStudio Flex Series User Manual

Chapter 12 Run calibrations and install checks
12

Run a spectral calibration

Run a spectral calibration
This section includes the following topics:

• Overview of spectral calibration

• System dye sets versus custom dye sets
• "Using BigDye
• Estimated spectral calibration run times
• When to perform a spectral calibration
• Prepare for custom dye set spectral calibration
• Prepare for spectral calibration
– Prepare the instrument
– Prepare the spectral calibration standard
– Prepare the standard plate
– Load a plate into the instrument
• Perform a spectral calibration
• View spectral calibration results
– Spectral QV (quality value) and Condition number definitions and limits
– Spectral calibration sharing between capillaries
– Spectral calibration with Allow borrowing disabled
– Spectral calibration with borrowing enabled
• Example spectral calibration data
• Export spectral calibration logs or report
• View and export the spectral calibration history
Overview of spectral calibration
During data collection, the instrument detects the signal of fluorescent dyes that are attached to DNA
amplicons. Although each dye emits its maximum fluorescence at a different wavelength, there is
some overlap in the emission spectra between the dyes. A spectral calibration creates a reference
deconvolution matrix (also referred to as a dye matrix) that corrects for the overlapping fluorescence
emission spectra of the dyes. A deconvolution matrix is created for each capillary in a capillary array.
A dye matrix is created when you run the manual spectral calibration function. The dye matrix is then
optimized and updated by the auto-spectral calibration function, which is performed automatically for
each capillary during each injection.
In the auto-spectral calibration process, the instrument uses spectral data from the samples to optimize
and update the dye matrix from the manual calibration, which generates an auto-spectral dye matrix.
This feature optimally reduces pull‑up (false secondary peaks under a true peak) that can be caused by
changes in the spectral characteristics from the original manual calibration dye matrix. The instrument
performs an auto-spectral calibration for system and custom dyes.
For more information, see "Auto-spectral calibration during an injection"
An example of the dye emission spectral overlap in the DS-33 Matrix Standard Kit (Dye Set G5) is
shown below.
310
™
Direct Cycle Sequencing Kit chemistry"
SeqStudio ™
on page 312
Flex Series Genetic Analyzer with Instrument Software v1.0 User Guide
on page 150.