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

Algorithmic
Manipulation of
Raw Allelic
Discrimination Data
The SDS software can analyze raw data immediately upon completion of an allelic
discrimination run. The term ‘raw data’ refers to the spectral data between 500 nm to
660 nm collected by the SDS software during the plate-read. During the analysis, the
software employs several mathematical algorithms to generate from the raw data a
more direct measure of the relationship between the spectra changes in the unknown
samples.
The first mathematical algorithm involves the conversion of the raw data, expressed in
terms of Fluorescent Signal vs. Wavelength, to pure dye components using the
extracted pure dye standards. After the dye components have been identified, the
software determines the contribution of each dye in the raw data using the
multicomponent algorithm. See “Multicomponenting” on page A-5 for a complete
description of the process.
Cluster Variations The SDS software graphs the results of an allelic discrimination run on a scatter plot
contrasting reporter dye fluorescence (Allele X R n versus Allele Y R n ). The software
represents each well of the 384-well plate as a datapoint on the plot. The clustering of
these datapoints can vary along the horizontal axis (Allele X), vertical axis (Allele Y),
or diagonal (Allele X/Allele Y). This variation is due to differences in the extent of PCR
amplification, which could result from differences in initial DNA concentration.
The example below shows the variation in clustering due to differences in the extent of
PCR amplification.
Allele Y homozygotes
Allele X/Allele Y
heterozygotes
Outliers
Allele X homozygotes
No amplification
End-Point Analysis 5-5