We have developed software that detects and reports 5% minor variants in Sanger Sequencing traces at 95.3% sensitivity and 99.8% specificity. The software calls variants without prior knowledge of location and affords the advantages of Sanger sequencing, of robustness, low error rate, ease of use, human interpretable visual displays of the data, and low cost per sample and target. The software can confirm somatic variants found by NGS.

1. Minor Variant Finder
New Software for Detecting Somatic Mutations at Low Level in Sanger Sequencing Tracesg g q g
Edgar Schreiber, Harrison Leong, Stephan Berosik, Jeff Marks, Stephanie Schneider, Wallace George, Hardeep Sangha, Yoke Peng Lim, Evelyn Chan, Dennis Nagtalon, and Joel Colburn
Thermo Fisher Scientific Genetic Analysis Solutions, 180 Oyster Point Boulevard , South San Francisco CA 94080
Abstract Component Requirements Easy Project Set Up: Auto-Assisted or Manual Trace Organizer Report Outputs : PDF CSV VCF
We have developed software that detects and reports 5% minor variants in Sanger Sequencing traces at
95.3% sensitivity and 99.8% specificity. The software calls variants without prior knowledge of location and
affords the advantages of Sanger sequencing, of robustness, low error rate, ease of use, human
interpretable visual displays of the data, and low cost per sample and target. The software can confirm
somatic variants found by NGS.
Noise minimization and peak detection algorithms subtract the baseline noise in a control sample from a
test sample, detect candidate minor variants, and confirm them in the complementary strand.
To test the new algorithms, synthetic mixtures of minor alleles were prepared by combining DNAs containing
known mutations in known ratios. Using standard protocols for fluorescent dye terminator Sanger
Component Requirements
Computer
Windows™ computer with 2 GB hard disk space and a
minimum of 4 GB memory; 8 GB recommended.
Operating
system
Windows™ 7 SP1, 32‐bit or 64‐bit, or Windows™ 10 Pro,
64‐bit
Browser
Google™ Chrome™, Mozilla™ Firefox™, Microsoft™
Internet Explorer™ v.11, or Microsoft™ Edge
The Minor Variant Finder Software runs in a web browser
windows, but does not require connection to the internet
in order to run. Data is secure on your desktop computer.
Organize the trace files:
Trace files can be organized in two
ways:
i) manually
by setting up folders for amplicons
and test specimen and
dragging/dropping the appropriate
y j p g p p
Glossary for column “Origin”:
Common: Variant was found by MVF & NGS.
MVF: Variant was found by MVF only
sequencing, twenty‐five different amplicons and nine different genes, including TP53, KRAS, BRAF, EGFR,
FLT3, RB1, CDH1, ERBB2, and XYLT1, from cell line and FFPE DNA were sequenced on 3500, 3730, and 3130
Applied Biosystems® Genetic Analyzers.
632,452 base positions and 2334 total variant positions, spanning variants at 2.5% to 50%, were
interrogated. The software achieved 95.3% sensitivity and 99.83% specificity for automated detection of
885 variants present at the 5% level in 238,179 high quality base positions. Sensitivity was 98.8% for
variants between 7.5‐10%, 98.7% for variants between 12.5‐25%, and 100% for variants ≥ 25%. The
software displays a noise‐minimized trace view to facilitate visual inspection to confirm candidate variants.
Further, reference sequences with hg19 chromosomal locations and NGS vcf files can be imported and
aligned with the Sanger sequencing data for orthogonal verification. Hyperlinks to dbSNP for known rsSNP
The Minor Variant Finder software scans the traces of a quartet of:
• normal control forward and reverse strands
• test sample forward and reverse strands
for the presence of variants that 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. A project can encompass multiple amplicons and
reference sequences.
First steps:
Download free demo version at www.thermofisher.com/mvf
trace file pairs into these folders
or
ii) automated‐assisted
This convenient feature requires
a consistent sample naming convention
such as:
sample ID_amplicon ID_orientation
(FWD or REV)
MVF: Variant was found by MVF only.
NGS: Variant was found by NGS only.
variants are provided in the software.
We have demonstrated that Minor Variant Finder calls 5% somatic variants with 95.3% sensitivity and 99.8%
specificity in Sanger sequencing traces.
Introduction
A minor variant is a heterozygous allele in which the proportion of the secondary allele is less than a
classical Mendelian germ line heterozygous variant of 50:50. Detecting minor variants has become
1) Create a project
2) Import ab1 trace files
into project
Immediate QC Check of Imported Trace Files
For minor variant detection, it is essential to
use sequencing trace files of high quality
Standardizing the sample name format
greatly facilitates the set up of complex
projects with multiple amplicons.
Clear Presentation of Results
h l h f h
5% Limit of Detection with 95.3% Sensitivity 99.8% Specificity
MVF Algorithm Performance
g yg g
essential to combating, managing, and advancing our understanding of cancer, inherited and
infectious diseases, and the impact of heteroplasmy on mitochondrial diseases.
We have developed Minor Variant Finder (MVF) desktop software for the de novo discovery of minor
variants, as well as next generation sequencing confirmation (NGC) of minor variants.
Key software features include:
 Novel algorithms for calling minor variants. Testing demonstrates a 5% limit of detection with
95.3% sensitivity and 99.8% specificity.
 Easy‐to‐use workflow.
use sequencing trace files of high quality.
Guidance for achieving high quality traces is
provided in companion protocols.
Quality threshold settings can be customized to
The Results Overview shows a map of the
amplicon(s), genome or amplicon
coordinates, DNA sequence and the
position of variant candidates for both
NGS and Sanger data. Clicking on a variant
will open a comprehensive, detailed
Results Table.
The Results Table lists the
Algorithm Performance was tested on a large
data set. Synthetic mixtures of minor alleles
were prepared by combining DNAs containing
known variants in known ratios, in which DNA
was obtained from cell line DNA or FFPE cell
line DNA. DNA was also extracted from lung
tumor FFPE samples, where the percent minor
variant was determined by sequencing on Ion
Torrent™ Personal Genome Machine® (PGM™)
next generation sequencing (NGS). 25
amplicons across 9 genes, TP53, KRAS, BRAF,
EGFR, FLT3, RB1, CDH1, ERBB2, and XYLT1, were
sequenced using BigDye® Terminator v3 1 or
 Electropherograms and results workflow to enable users to easily review and accept variant
calls.
 Functionality to import the human hg19 chromosome reference and next‐generation
sequencing variants file for the NGS confirmation workflow, if the computer is web‐enabled.
 Ability to align and compare results between Sanger sequencing and Next Generation
Sequencing (NGS) for orthogonal verification.
 Hyperlinks to dbSNP for known rsSNP variants are provided in the software.
 Trace file Quality Check, with preset thresholds for quick quality check review.
 Capability to export results in a variety of formats from pdf reports, comma‐separated files, to
industry standard formats like vcf.
P j bli d h i
Q y g
meet individual experimental needs. Proven
default settings are provided.
The QC module in MVF contains all the trace
viewing functionality of the popular, free Applied
Bi t S S ft
High trace score value (>40) and signal to noise ratio
(>180) are recommended for reliable minor variant
detection.
details for all variant
candidates and their Review
status. A colored “Review
Indicator” indicates the
goodness for the variant call:
Green means the algorithm is
confident; Yellow and Red
mean increasing levels of
uncertainty requiring extra
scrutiny by the user.
Hyperlink to
dbSNP if variant
is a known SNV.
sequenced using BigDye Terminator v3.1 or
BigDye® Direct Sequencing kits on POP‐7™
polymer on 3500, 3500xL, 3730xL, or 3130xL
Applied Biosystems® Genetic Analyzers.
Characteristics of Test Data Set
632,452 Total Number of basecalls
2,334 total number of variant positions
Test Data
% Minor
Variant
# of Expected
Variants
# of Found
Variants
# of
Quartets
# of Basecalls in
Used Range
Sensitivity Specificity
2.50% 399 138 399 143899 34.6% 99.76%
5% 885 843 754 238179 95.3% 99.83%
7.5‐10% 578 571 491 170214 98.8% 99.80%
12 5 25% 302 298 200 49657 98 7% 99 79%
Summary for sensitivity (in %) for detection of rare (minor)
variants at known % level (x‐axis).
 Project management enabling data sharing.
Novel classification and signal processing algorithms were developed to identify minor variants. The
algorithms take advantage of the reproducibility of the peak pattern in the Sanger sequencing traces,
when the same locus of interest is sequenced in different specimens. The baseline noise is also
typically very similar between different traces, provided the different DNAs are sequenced
i lt l i th t i l t i t t t Th l d l d
The How: Detecting Minor Variants out of “Reproducible Noise”
Biosystems Sequence Scanner software.
Multiple Options for Reference Import - Import NGS vcf file
Creating a reference sequence in text
format is optional; by default the forward
t l i d
The algorithm also requires confirmation of the minor variant in both forward and reverse strands to
minimize false positives. The candidate variants the algorithm finds can easily be reviewed and
accepted in the six electropherogram view. The Control electropherograms are shown in the lowest
panel. The Test electropherograms are shown in the middle panel. The algorithm‐generated Test trace
with the baseline noise minimized and the candidate variant revealed (marked by the red arrow) is
Reviewing and Confirming Candidate Variants
12.5‐25% 302 298 200 49657 98.7% 99.79%
>25% 170 170 170 30503 100.0% 99.77%
Summary
• The Minor Variant Finder software detects and calls minor variants as low as 5% from
Sanger sequencing traces (.ab1 files) at high sensitivity (95.3%) and specificity (99.8%).
• The algorithm neutralizes background noise signal by comparison of test sample(s) and a
normal control sample.
Th l ith t t t l t h ith th b k d i t li dsimultaneously, using the same reagents, primer lots, instruments, etc. The newly developed
algorithms subtract the baseline noise in the control sequence from the baseline in the test sequence
to reveal and thereby identify minor variants. The algorithms generate a noise minimized trace,
labeled ‘NSS Algorithm Generated’ trace in the software, to enable easy reviewing by the user.
control sequence is used.
Import of a reference sequence with
chromosomal positions (GRCh37; hg19)
enables a link to the dbSNP database if a
known rsSNP is detected. It is required if a
NGS vcf file is imported for a verification
project. Multiple reference sequences
(each up to 10 kb length) can be set up.
with the baseline noise minimized and the candidate variant revealed (marked by the red arrow) is
displayed in the top panel. In addition, the algorithm calculates the peak height of the minor variant as
a percent of the primary plus secondary peak heights for both the forward and reverse traces (circled
in red.) Highly divergent peak heights between the forward and the reverse traces suggest the
candidate is a false positive. Note: Peak heights in Sanger sequencing are not quantitative, and hence
the ratio between primary and secondary peak heights does not precisely reflect allele frequency.
• The algorithm generates test electropherograms with the background noise neutralized.
• The algorithm calculates the ratio of the minor peak height relative to the peak heights of
the primary plus secondary peak heights, in both the forward and reverse strands.
• The minor variant is detected on both the forward and reverse strands.
• The minor variant candidates are presented for review and confirmation
(accept/reject/comment) by the user in a high resolution viewer tool that enables deep
scrutiny of traces.
• Reviewed and user confirmed results are reported in pdf, csv and vcf file formats.
• The software aids in verification of minor variant findings obtained by NGS by reading the
vcf file and aligning Sanger results with the NGS results.
• A hyperlink to dbSNP is provided for known variants (if reference is available with
chromosomal positions)
Reproducible peak pattern in the primary sequence is
shown between the Test sample (middle) and the Control
sample (lower).
Baseline noise peak pattern is also reproducible between
Test (middle) and Control (lower). The minor variant in
the test sample is revealed (top panel) when the baseline
noise in Control is subtracted from baseline noise in Test.
A vcf file can be imported into the
project for verification of NGS results.
The MVF software automatically
filters out all AF=0 listings in the vcf
file.
chromosomal positions).
• Free demo version is available for download at www.thermofisher.com/mvf.
• For Research Use Only – Not for use in diagnostic procedures.
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