Patented Anti-Correlation Technology
for Capillary (Sanger) Sequencing
Analysis
Mutation Surveyor® software
features patented physical comparison
“anti-correlation” (U.S. Patent 8,086,410)
technology and a unique multi-step
alignment algorithm to rapidly and
accurately detect DNA variants from
Sanger Sequencing traces, including
single nucleotide polymorphisms (SNPs),
insertions and deletions (indels),
duplications, and low frequency variants
associated with mosaicism or heteroplasmy.
Compatible with outputs from all major
sequencing platforms, Mutation Surveyor
software provides an unattended operation
that is capable of detecting variants
in 2000 sequence traces in less than
15 minutes. Accuracy is greater than
99% using Phred 20 bi-directional
sequence traces, and sensitivity greater
than 5% of the primary peak, or 1
part diseased in 17 parts normal cells.
The software's Windows® based operation
features a biologist-friendly interface,
automatic download of GenBank reference
files for complete annotation, contig
assembly, and custom reporting that
is superior in nature to those features
found in similar programs 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.
Anti-Correlation
Technology Provides Variant Accuracy
>99%

Figure 1:
Two Single Nucleotide Polymorphisms
detected in a sample trace. The anti-correlation
technology of Mutation Surveyor software
displays peaks in the “Mutation Electropherogram”
(also known as Mutation Trace) where
the sample peak profile differs from
the reference peak profile.
Accuracy of the software
in the bi-directional analysis mode
is over 99% with Phred-20 sequence
traces, and our collaborators have
demonstrated an accuracy of 95% when
processing single-directional sequence
traces. The software's patented anti-correlation
technology compares the sample trace
to the reference trace at each nucleotide
position, using physical changes in
nucleotide peak profiles as a basis
for SNP detection to provide excellent
accuracy and sensitivity. Changes
in sample traces relative to the reference
are determined through three physical
characteristics of the sample trace:
dropping factor, overlapping factor,
and signal-to-noise ratio. Peak differences
are displayed in the “Mutation Electropherogram”
in the middle of the screen, with
larger peaks indicating greater discordance
between the sample and reference position.
Each mutation is given a Phred-like
confidence score calculated from the
physical differences of the sample
and reference, providing the user
with a confidence value for each mutation
call.
Robust Alignment
Algorithm Accurately Detects Homozygous
and Heterozygous Insertions, Deletions,
and Duplications

Figure
2: A homozygous deletion
(TTT) and a SNP detected in a sample
trace. Mutation Surveyor software’s
unique multi-step alignment algorithm
gaps the sample trace at the position
of the deletion (red bar) and continues
sample alignment to the reference
trace.
Mutation Surveyor software
also features a unique multi-step
alignment algorithm for alignment
of traces and insertion and deletion
(indel) detection. While other software
packages rely on base calls to determine
indels, Mutation Surveyor software
remains insensitive to base call errors
by monitoring DNA sample migration
time during sample trace alignment
to the reference trace. Sample migration
time is then contracted (insertion)
or expanded (deletion) to accurately
align sample traces to the reference
to detect and display indel events.
Sensitivity
Down to 5% Primary Peak
Sensitivity is greater
than 5% of the primary peak, which
allows detection of variants buried
in the baseline of both the forward
and reverse traces, alerting researchers
to the presence of possible low frequency
mutations.

Figure
3: Somatic mutation G>GA
detected in TP53. The sensitivity
of Mutation Surveyor software can
detect minor alleles that are present
within the baseline of bi-directionally
aligned peaks. Sensitivity is greater
than 5% of the primary peak.
BasePatch Function
Corrects Sequencing Errors
BasePatch is a function
unique to Mutation Surveyor software
and is not present in any other software
package. This function helps to reduce
baseline noise, sharpen peaks, and
correct for mobility shift problems
in sequence traces. When used in conjunction
with our anti-correlation algorithm,
it will resolve "N" calls
made by the original basecall software,
reduce false positives due to baseline
noise, and align the ends of traces
more efficiently for an extended region
of mutation detection.

Figure
4: This illustration shows
two analyses with the same parameters
except that the right image has BasePatch
activated while the left image does
not. You can see that the trace on
the right has less noise in the Mutation
Electropherogram than the image on
the left. Also, the lane quality score
and Phred-like score increased for
the analysis with BasePatch on. BasePatch
is intended to increase the overall
quality of the trace for better alignment
and mutation detection.
Webinars
Mutation Surveyor Software Introductory
Overview (View
on )
Working With Your Mutation Surveyor
Project in the Graphical Analysis
Display Part I (view
on )
Working With Your Mutation Surveyor
Project in the Graphical Analysis
Display Part II (view
on )
Working With Your Mutation Surveyor
Project in the Graphical Analysis
Display Part III (view
on )
Optimizing Analysis Settings with
Mutation Surveyor- Part 1 (view
on )
Optimizing Analysis Settings with
Mutation Surveyor- Part 2 (view
on )
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