Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 78)

Section 6 Data Analysis Module
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The spreadsheet displays the following information for each peak:
• Peak ID: A unique identification number in the format RCC.N, where R is a row letter, CC
is a column number, and N is a number beginning with 0, 1, 2, etc. In Figure 6.27, the first
peak for well E3 is identified as E03.0 and the second peak as E03.1
• Melt Temp: The temperature at the highest point of the melt peak
• Peak Height: The highest point of the melt peak
• Begin and End Temp: Starting and ending point for melt peak. Area calculations are
based on these starting and ending points. These values may be edited, but not in the
spreadsheet
• Area: The area beneath the melt peak curve, bounded by the default peak begin and end
temperatures and the default position of the threshold bar. Because this area calculation is
defined by the default values of begin and end temperature and threshold bar, it does not
change as the threshold bar is moved or if the begin or end temperatures are edited
• Area Fraction %: When there is more than one peak associated with a well, the
contribution of each well to the total area beneath all melt peaks for that well is calculated.
The iQ5 software makes this calculation using default begin and end temperatures and the
default position of the threshold bar, so data are not affected by edits to any of those
parameters
• Edited Begin Temp: When the peak begin temperature bar is dragged to a new position,
it is reflected here
• Edited End Temp: When the peak end temperature bar is dragged to a new position, it is
reflected here
6.7 Allelic Discrimination Module (For Multiplex Data Only)
The Allelic Discrimination module is useful for assigning genotypes to unknown samples by making
comparisons to known genotypes. It can be used to distinguish among homozygous wild types,
homozygous mutants and heterozygous individuals based either on the C
T
or RFU value. RFU data
may be chosen from any cycle in the experiment. The assignments can be made automatically if
controls are specified, or you can make the assignments manually.
NOTE: The Allelic Discrimination module is not available with working with single color data files or
any data file generated by the MyiQ Real-Time PCR Detection System.
6.7.1 Allelic Discrimination Plot
The Allelic Discrimination plot shows either RFU (Figure 6.28A) or C
T
(Figure 6.28B) data from
two different fluorophores at the same time. Choose which plot to view using the RFU or
Threshold Cycle radio button in the Display Mode box.
In Automatic Call mode, two bars, one vertical and one horizontal, divide the plot into four
sections: one for each homozygous state, one for the heterozygous state and a non-reactive
section. The positions of these bars may be adjusted in the Automatic Call mode. The bars do not
appear in Manual Call mode.