Low-frequency
DNA Variant discovery from Sanger
Sequencing Traces
Mutation Surveyor software's patented
“anti-correlation” technology provides
the enhanced sensitivity needed
for the detection of low frequency
alleles in Sanger sequencing traces,
including low-frequency variants
associated with somatic mutations,
heteroplasmy, and mosaicism. Many
software packages, such as Applied
Biosystems SeqScape®, Variant Reporter®,
and Minor Variant Finder; DNAStar's
SeqMan Pro™; Gene Code's Sequencher®;
or JSI Medical Systems' SEQUENCE
PILOT, rely on basecall differences
and have been optimized for variant
detection with alleles having heterozygous
mixtures of 50%. However, today's
sequencing projects demand software
with an analytic sensitivity capable
of detecting low frequency mutations
that are often buried in background
noise. Mutation Surveyor software
provides a solution to these issues
by offering a unique trace-to-trace
comparison of sample sequence traces
to a reference trace, calculating
and displaying the physical differences
between sample and reference peak
in a mutation electropherogram.
This technology has demonstrated
accuracy in the bi-directional analysis
mode over 99%, with sensitivity
to greater than 5% of the primary
peak. The sensitivity of the anti-correlation
method has proven to be beneficial
in the direct comparison of normal
to cancer cells for the detection
of somatic mutations in Sanger sequencing
traces from all major capillary
electrophoresis outputs.
Mutation
Surveyor Software Detects Minor
Alleles Buried in the Baseline

Figure
1: Low frequency G>GA
mutation detected in Mutation Surveyor
software. A green bar in the Mutation
Electropherogram indicates the presence
of a possible low frequency variant.
Mutation
Surveyor software’s trace-to-trace
anti-correlation technology evaluates
physical characteristics of the
sample trace relative to the reference,
including: signal-to-noise ratio,
overlapping factor, and dropping
factor. Often somatic/low-frequency
alleles do not result in major changes
to the primary peak. However, Mutation
Surveyor software’s sensitivity
is capable of detecting these variants
by monitoring minor mutation peak
intensity calculated in the Mutation
Electropherogram. When minor alleles
of the same color are detected in
both the paired forward and reverse
directional trace files, Mutation
Surveyor software places a green
bar in the Mutation Electropherogram
of the Graphical Analysis Display
(GAD) at the location of event.
The above image represents a low-frequency
peak detected in exon 5 of the TP53
gene.
Mutation
Quantification Tool Provides Major
and Minor Peak Contribution

Figure
2: The Mutation Quantifier
report displays the contribution
from each allele. The low-frequency
variant in this example has a minor/mutant
peak contribution near 6% in both
the forward and reverse trace.
The
Mutation Quantification tool of
the software automatically calculates
the major and minor allele contribution
through two methods. The Standardized
Allele Ratio method provides the
percent decrease in normal intensity
and the percent increase in mutant
intensity, and the Simplified Allele
Ratio method uses Relative Fluorescence
Units (RFU) to provide the relative
percent contribution of the major
and minor allele. The Simplified
Allele Ratio report indicates that
the low-frequency variants in this
sample were detected with only 6%
contribution of the primary peak.
Mutation
Quantifier Tool Displays Major and
Minor Allele in Electropherogram
Window

Figure
3: Electropherogram window
displaying the major and minor allele
of the forward and reverse trace.
This window can be accessed from
the Mutation Quantifier report,
Simplified Allele Ratio method.
Flexible
Reporting Options for Low-Frequency
Variants

Figure
4: The Custom Report lists
possible low-frequency mutations
with a teal background. Custom color
schemes can be added, and mutations
can be edited with the click of
a mouse.
The interactive reporting and display
options of Mutation Surveyor software
provide the flexibility needed for
the user to make edits and to add,
delete, or confirm low-frequency
mutations in a number of ways. Rapid
customization of reporting options
also provides useful ways to identify
low-frequency mutations, including
background and text color-coding.
Users can easily add mutations to
the GAD at locations of possible
somatic mutations and the Mutation
Report will populate with the changes.
Additional details can be found
on the Mutation
Quantification page.
Download application note
Automated
Somatic Mutation Detection in DNA
Sequence Traces
Webinars
Optimizing Analysis Settings with
Mutation Surveyor- Part 1 (View
on )
Low Frequency Variant Detection in
Mutation Surveyor (view
on )
Trademarks property of their respective owner
|
|