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Heterozygous
Insertion and Deletion (Indel) Detection
and Deconvolution in Sanger Sequencing
Traces
Mutation Surveyor software employs
a multi-step alignment algorithm
to detect homozygous and heterozygous
deletions, insertions, and duplications
(indels) from Sanger Sequencing
traces. Included in the software
is a unique and interactive Heterozygous
Indel Detection tool not found in
other sequencing software 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. This tool automatically
detects and deconvolutes heterozygous
indels into wild-type and novel
alleles, eliminating the need for
tedious and error-prone manual deconvolution.
Mutation Surveyor software is able
to detect heterozygous indels with
sensitivity at 20% contribution
from the minor allele by monitoring
the migration time changes between
the sample and reference trace.
After alignment of the sample trace
to the reference trace the novel/mutant
sequence is deduced by subtracting
the reference from the sample sequence.
The mutant trace is automatically
shifted to realign with the reference
sequence, providing the location
of the variant in an interactive
display window.
Automatic
Deconvolution of Heterozygous Indels
in Heterozygous Indel Detection
Tool
Figure
1: A 4bp heterozygous deletion
of TTTA is detected in the Heterozygous
Indel Detection tool. Trace A is
the reference and trace B is the
sample trace containing a mixture
of alleles. The sample trace is
automatically deconvoluted to display
the normal/wild-type allele in trace
C and the novel/mutant allele in
trace D. The mutant allele is then
shifted to realign to the reference
in trace E and the location depicted
in trace F. The mutant percentage,
displayed above each allele, is
calculated by taking the ratio of
the mutant intensity over the mutant
plus normal intensity (total).
When
a heterozygous indel is detected
during the alignment process, Mutation
Surveyor software will automatically
deconvolute the mixture to determine
the location of the event. For further
review, the Heterozygous Indel Detection
tool can be opened to examine and
edit the indel. Trace A is the reference
trace and trace B is the sample
trace containing the mixture of
two alleles. The sample trace is
automatically deconvoluted into
wild-type allele in trace C and
novel/mutant allele in trace D.
Finally, the tool shifts the mutant
trace to align to the reference
in part E, providing a clear location
of the event in trace F. The tool
is highly interactive, allowing
users to zoom in and out of the
display window, and print images
at high resolution.
Mutation Surveyor software’s alignment
algorithm compares the mobility
of the sample trace to the reference
trace to detect insertion, duplication,
and deletion events. When a heterozygous
indel is detected, a horizontal,
brown bar is placed at the location
of the event in the Mutation Electropherogram.
The event can then be viewed in
the Heterozygous Indel Detection
tool by double-clicking on the mutation
call in the Mutation Table, or by
opening the tool from the icon at
the top of the screen. The software
is able to regularly and accurately
detect various sizes of indels,
with recent studies showing accurate
detection of 3 to 30 base pair indels.
Graphical
Analysis Display Indicates Start
Site of Heterozygous Indel Event
with Brown Horizontal Bar in Mutation
Electropherogram
Figure
2: A heterozygous indel
displayed in the Mutation Surveyor
Graphical Analysis Display (GAD).
The software places a horizontal
brown bar at the start site location
of the event for easy identification.
Download application note
Detection
of Insertions and Deletions, Including
Deconvolution of Het-Indels in Sanger
Resequencing Projects using Mutation
Surveyor® Software
Webinar
Het_Indel Detection in Mutation Surveyor
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