Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 79)

Section 6 Data Analysis Module
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A B
Fig. 6.28. Allelic Discrimination Plot; RFU View (A) and Threshold Cycle View (b).
Genotype assignments for unknown samples are determined by plotting the RFU for one
fluorophore (allele 1 on the x-axis) against the RFU for the other fluorophore (allele 2 on the y-
axis) on the allelic discrimination plot.
•
If the unknown RFU values are greater than the horizontal and vertical bars, then the
genotype is heterozygous
•
If the unknown RFU values are greater than the horizontal bar and less than the vertical
bar, the genotype is homozygous for allele 2 (allele 2 RFU is plotted on the y-axis)
•
If the unknown RFU values are less than the horizontal bar and greater than the vertical
bar, the genotype is homozygous for allele 1 (allele 1 RFU is plotted on the x-axis)
•
If the unknown RFU values are less than the horizontal and vertical bars, then no
genotype can be assigned
Genotype assignments for unknown samples may also be determined by plotting the threshold
cycle for one fluorophore (allele 1 on the x-axis) against the threshold cycle for the other
fluorophore (allele 2 on the y-axis) on the allelic discrimination plot.
•
If the unknown threshold cycle values are greater than the horizontal and vertical bars,
then the genotype is “none”, or not assigned. Samples not crossing threshold during the
protocol are placed in this quadrant
•
If the unknown threshold cycle values are greater than the horizontal bar and less than
the vertical bar, the genotype is homozygous for allele 1 (allele 1 threshold cycle is
plotted on the x-axis)
•
If the unknown threshold cycle values are less than the horizontal bar and greater than
the vertical bar, the genotype is homozygous for allele 2 (allele 2 threshold cycle is
plotted on the y-axis)
• If the unknown threshold cycle values are less than the horizontal and vertical bars, then
the genotype is heterozygous