Sanger sequencing using fluorescent BigDye® terminator chemistry and semi‐automated capillary electrophoresis (CE) has long been considered the gold standard for identifying sequence variations such as disease‐causing mutations. The robustness, low error rate, ease of use, human interpretable visual displays of the signals generated by the instruments, and low cost per sample and target have all contributed to this reputation. Homozygous and heterozygous germ line mutations are reliably detected and reported using established DNA sequencing analysis software such as the Applied Biosystems Variant Reporter™ software. However, somatic variants with an allelic proportion of 25% or less are often undetected (i.e. not "called") by the software and thus escape awareness if not detected by careful visual inspection of the electropherograms. With the rapid adoption of next generation sequencing technology (NGS) and its use for characterization of specific and discrete mutations in tumor samples, an urgent need has emerged to establish an orthogonal technology for reliable and sensitive detection of somatic mutations which may occur at proportions of 10% or lower compared to the normal allele. To this end, we have developed an innovative algorithm, software, and a protocol that specialize in the detection and reporting of minor mutations by Sanger sequencing. Moreover the algorithm preserves the ability to generate the familiar displays of the data to facilitate human review. Using panels of prepared mixtures of minor alleles in the range of 2.5%, 5%, 10% and 20%, we have achieved 94.6% sensitivity and 99.8% specificity for automated detection of mutations present at the 5% level with high quality data. In conclusion, we have demonstrated that standard protocols for fluorescent dye terminator Sanger sequencing in conjunction with the new algorithm delivered in Variant Finder software may enable the identification of de novo somatic mutations to a level of 5%. This technology will also be useful for the confirmation of minor variants identified by NGS platforms.

1. Improved Detection of Low Level Sequence
Variants by Sanger Sequencing using a New
Noise Reduction Algorithm
ABSTRACT
Sanger sequencing using fluorescent BigDye® terminator chemistry and semi‐automated capillary
electrophoresis (CE) has long been considered the gold standard for identifying sequence variations such
as disease‐causing mutations. The robustness, low error rate, ease of use, human interpretable visual
displays of the signals generated by the instruments, and low cost per sample and target have all
contributed to this reputation. Homozygous and heterozygous germ line mutations are reliably detected
and reported using established DNA sequencing analysis software such as the Applied Biosystems Variant
Reporter™ software. However, somatic variants with an allelic proportion of 25% or less are often
undetected (i.e. not "called") by the software and thus escape awareness if not detected by careful visual
Introduction
Detecting and Distinguishing Minor
Variants from Background Noise
Minor variants are single nucleotide
polymorphisms (SNPs) which present
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inspection of the electropherograms. With the rapid adoption of next generation sequencing technology
(NGS) and its use for characterization of specific and discrete mutations in tumor samples, an urgent
need has emerged to establish an orthogonal technology for reliable and sensitive detection of somatic
mutations which may occur at proportions of 10% or lower compared to the normal allele.
To this end, we have developed an innovative algorithm, software, and a protocol that specialize in the
detection and reporting of minor mutations by Sanger sequencing. Moreover the algorithm preserves
the ability to generate the familiar displays of the data to facilitate human review. Using panels of
prepared mixtures of minor alleles in the range of 2.5%, 5%, 10% and 20%, we have achieved 94.6%
sensitivity and 99.8% specificity for automated detection of mutations present at the 5% level with high
quality data.
In conclusion, we have demonstrated that standard protocols for fluorescent dye terminator Sanger
sequencing in conjunction with the new algorithm delivered in Variant Finder software may enable the
identification of de novo somatic mutations to a level of 5%. This technology will also be useful for the
confirmation of minor variants identified by NGS platforms.
For Research Use only – Not for use in diagnostic procedures.
p y p ( ) p
as a minor component i.e. with a
contribution of less than 25% at a
given allele.
Minor variants may occur
spontaneously or evolve during
tumorigenesis (somatic mutation) or
in viral, bacterial or mitochondrial
mixed populations.
Minor variants are difficult to detect
by conventional fluorescent Sanger
sequencing since the reduced peak
trace of the minor variant may be
hidden in the “background noise.”
“Background noise” at the baseline of a typical
fluorescent Sanger sequencing trace. The
arrows point to two genuine minor variants.
Could you tell?
Detecting Minor Variants out of “Reproducible Noise”
Experienced users of fluorescent Sanger sequencing systems may have noticed the remarkable
consistency and reproducibility of the primary peak pattern in the sequence trace profiles when the
same locus of interest is sequenced in different specimens. This phenomenon is due to the sequence
context dependent nucleotide incorporation efficiencies during the polymerization process (Carr et al.
2008). The assumption is that the same principle applies not only to the primary predominant base but
also to the other three bases that generate a characteristic pattern that is commonly referred to as the
“background noise.” The feature of “reproducible noise” can be exploited to algorithmically reveal the
signal indicative of a potential minor variant.
To this end the dye traces of a bi directional (forward and reverse strand) sequencing from a test
The Variant Finder tool scans the
traces of a quartet of:
• normal control forward strand
• normal control reverse
• test sample forward
• test sample reverse
for the presence of variants that
User interface of the Variant
Finder Detector tool:
sample files (control and test
samples) are transferred into
the Analysis fields for
forward reverse traces. An
To this end, the dye traces of a bi‐directional (forward and reverse strand) sequencing from a test
sample where a minor variant is potentially present is compared to the dye traces of a normal control
sample where it is known that the variant is bona fide absent.
How is the Signal of the Minor Variant Distinguished from Background Noise? The main idea is that
the background noise of the control sample can be used to remove background noise in the test
sample if the noise between the two is similar enough. If the primary sequence backbone for the two
samples is the same and the samples were processed in a similar fashion, there is a good chance that
the background noise will be sufficiently similar. With this noise removed, variants in the test sample
are revealed because the variants are not associated with the common primary sequence backbone.
To complete the variant detection decision, pattern recognition is used to distinguish bona fide variant
signals from any noise remaining in the traces.
occur in matching (i.e. sequence
complementary) positions on both
strands.
At least one normal control pair
(fwd/rev) must be present.
Many test sample pairs for the
same amplicon can be analyzed in
the same session.
output directory is selected
and variant finding is
started by “ANALYZE.”
When analysis is completed change
to Variant Finder Viewer for review
Candidate(s) for minor variant are presented In the VIEWER window for visual review
and manual call for “Accept” or “Reject”
Full view of trace
quartet
Black bars indicate variant
candidates
Noise‐purified trace
view for variant
candidate
Verifying Very Low Level Minor Variants: A 5% variant in the human TP53 Gene
Using a slider in
the center of the
viewer, peaks can be
scrutinized at highest
detail. Colors for base‐
specific traces can be
switched on or off.
Correlating Minor Variant Findings by Ion Torrent™ PGM™ and Sanger CE Variant Finder
Original trace view
for variant candidate
in test sample
Original trace view
for normal control
sample
switched on or off.
This allows a thorough
assessment whether a
peak represents a
genuine variant or a
nonspecific noise
signal.
Sequence Scanner View Variant Finder Viewer
Sensitivity & Specificity Statistics Summary
Conclusions
• The Variant Finder tool facilitates detection and calling of minor
variants as low as 5% from Sanger sequencing traces (.ab1 files).
• The algorithm neutralizes background noise signal by
comparison of test sample(s) and a normal control sample.
• The minor variant is detected on forward and reverse strands.
• The minor variant candidates are presented for review in a
convenient viewer tool and reported in a csv output file.
• The tool aids in verification of minor variant findings by NGS.
Sample a3 Variant c35G>A at 5% (NGS)
is barely visible in traditional Sequence
Scanner viewer.
Sample a3 Variant c35G>A at 5% (NGS)
is clearly detected on both strands in the new
Variant Finder viewer (note: T‐trace only is shown).
Summary for sensitivity (in %) for
detection of rare (minor) variants at
known % level (x‐axis)
375 file sets generated on Applied Biosystems 3730 or 3500
Genetic Analyzers were analyzed:
Overall Specificity was 99.8%
Sensitivity at 5% rare is 94.6%
ROC curve plotting Sensitivity vs
Specificity for the 5% variants (n=75)
at different algorithm settings:
The performance of the current algorithm is indicated by
the red circle.
The tool aids in verification of minor variant findings by NGS.
• For Research Use only – Not for use in diagnostic procedures.
Acknowledgements: Geoff Bien and David Chi Life Technologies Services Lab West Sacramento CA
and Manjula Aliminati for providing the KRAS NGS and Sanger data, Nakul Natarad and Kara Norman
for early access to Acrometrix® MegaMix™ technology.
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