Operator`s manual

18
LightCycler® 480 Gene Scanning Software
Gene Scanning with the LightCycler® 480 System
Components of the LightCycler® 480 System Gene Scanning Assay
Data Analysis using the LightCycler2.2
®
480 Gene
Scanning Software
LightCycler® 480 Gene Scanning Software analyzes the High Resolution Melting curve
data to identify changes in the shape of the curve, which indicate the presence of sequence
variations in the PCR product.
Correct interpretation of the data depends to a large extent on the software algorithms
used. LightCycler® 480 Gene Scanning Software has been developed specifically to pro-
vide the most accurate analysis of High Resolution Melting curves.
The standard workflow followed by the LightCycler® 480 Gene Scanning Software has
four basic steps:
Detecting negatives:1. LightCycler® 480 Gene Scanning Software automatically uses a
negative filter to detect negative samples, i.e., samples with low fluorescence signals
that lack a prominent melting curve. The software also allows you, if you wish, to
identify the negative samples in the run manually.
Normalizing:2. The second step in the analysis is to normalize the raw melting curve
data by setting the pre-melt (initial fluorescence) and post-melt (final fluorescence)
signals of all samples to uniform values. Pre-melt signals are uniformly set to a relative
value of 100%, while post-melt signals are set to a relative value of 0%. Normalizing
the initial and final fluorescence in all samples aids interpretation and analysis of the
data. In some cases, samples with homozygous SNPs may be distinguished from the
wild type by the displacement of their melting curves, which is easier to see in the
normalized data.
unnormalized melting curves normalized melting curves