Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

Chapter 5Analyzing End-Point DataCluster VariationsThe SDS software graphs the results of an allelic discrimination run on a scatterplotcontrasting reporter dye fluorescence (Allele X R n versus Allele Y R n ). The softwarerepresents each well of the 384-well plate as a datapoint on the plot. The clustering ofthese 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 theextent of PCR amplification, which could result from differences in initial DNAconcentration.The example below shows the variation in clustering due to differences in the extentof PCR amplification.Allele Y homozygotesAllele X/Allele YheterozygotesOutliersAllele X homozygotesNo amplificationFigure 5-4Common Datapoint ClustersGenotypic Segregation of Datapoint ClustersFigure 5-4 illustrates the concept of genotypic segregation of samples within theallele plot. The plot contains four separate, distinct clusters that represent the NoTemplate Controls and the three possible genotypes (allele X homozygous, allele Yhomozygous, and heterozygous). Because of their homogenous genetic compliment,homozygous samples exhibit increased fluorescence along one axis of the plot(depending on the allele they contain). In contrast, heterozygous samples appearwithin the center of the plot because they contain copies of both alleles, and thereforeexhibit increased fluorescence for both reporter dyes.About OutliersSamples that did not cluster tightly may:• Contain rare sequence variations• Contain sequence duplications• Not contain a crucial reagent for amplification (the result of a pipetting error)5-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm