ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

Mathematical Transformations
Overview The SDS software performs a series of mathematical transformations on the raw data
during an analysis of all end-point and real-time runs. The term raw data refers to the
spectral data between 500 nm to 660 nm collected by the SDS software during the
sequence detection run. The following section describes the fundamental analysis of
raw run data performed on both real-time and end-point run by the SDS software.
Multicomponenting Multicomponenting is the term used for distinguishing the contribution each individual
dye and background component makes to the fluorescent spectra detected by the
7900HT instrument. During the multicomponent transformation, the SDS software
employs several algorithms to separate the composite spectra from the raw spectrum
and then to determine the contribution of each dye in the raw data.
First, the algorithm eliminates the contribution of background fluorescence in the raw
data, by subtracting the background component stored within the background
calibration file (see page 7-13). Next, the software employs the extracted pure dye
standards (see page 7-17) to express the composite spectrum in terms of the pure
dye components. The figure below shows one composite spectrum that represents a
fluorescent reading from a single well that contains the passive reference and two
fluorogenic probes, labeled with the FAM and VIC reporter dyes and a
non-fluorescent quencher. The example spectra demonstrate how the overlapping
component dye spectra contribute to the composite spectrum. The SDS software
multicomponenting algorithm applies matrix calculations to determine the
contributions of each component dye spectra.
500
FAM
VIC
ROX
550 600
Wavelength
650
Composite spectrum
(FAM + VIC + ROX +
Background + MSE)
Pure Dye components
Background
(from background
calibration run)
Theory of Operation A-5