Bio-Rad Precision Melt Analysis™ Software - Manual (Page 25)

Introduction to Precision Melt Analysis™ Software
20
Analysis Options
Use the Analysis Options Manager window to choose the settings for analyzing a melt file.
• Auto detect the melt region. Select the checkbox to enable the software to
automatically define the pre-melt and post-melt temperature ranges. To manually define
the pre-melt and post-melt ranges, deselect the checkbox and enter temperature values
by typing a number in the text box or using the up-down arrows.
• Normalized view. Select to open the melt file with the melt curve data displayed in
normalized view in Precision Melt tab.
• Temperature shifted view. Select to open the melt file with a temperature shift applied
to each normalized fluorescence curve along the temperature axis (x-axis).
• Temperature shift bar height. Specify the temperature shift bar height by entering a
number between 0 (baseline) and 1 (top of melt region). For most applications, the
default temperature shift bar height of 0.20 produces acceptable results, with the melt
curves clustered into tight groups.
Cluster Detection Settings
Set the cluster detection settings to determine the stringency used to cluster the melt curves.
M ELT CURVE S HAPE S ENSITIVITY
Clustering shape sensitivity determines the stringency used to classify melt curves into
different clusters. To refine clustering results, increase or decrease the sensitivity of clustering
based on the shape of the melt curve. Enter a numerical value or move the slider bar to the left
or to the right. Entering a lower percentage value or sliding the bar to the left reduces
stringency and results in less heterozygote clusters. Entering a higher percentage value or
sliding the bar to the right increases stringency and results in more heterozygote clusters.
If necessary, manually decrease sensitivity to eliminate high numbers of false positives. A high
sensitivity value generally produces more groups than a low value. For most applications, the
default 50% clustering shape sensitivity value produces acceptable results.
T
M
DIFFERENCE T HRESHOLD
T
m
difference threshold determines the lowest amount of T
m
difference between samples
which the software will call as different clusters. Choose the number of degrees Celsius by
entering a numerical value from 0.05 to 1.00. Lower values produce more homozygote
clusters.
If necessary, manually increase the T
m
difference threshold level to eliminate high numbers of
false positives. For most applications, the default Tm difference threshold of 0.15 degrees
produces acceptable results.