Automated fluorescent dye-terminator DNA Sequencing using capillary electrophoresis (also known as CE or Sanger sequencing) has been instrumental in the detailed characterization of the human genome and is now widely used as gold standard method for verification of mutation findings, notably in tumor samples. The primary information of the DNA sequencing process is the identification of the nucleotides and of possible sequence variants. A largely unexplored feature of fluorescent Sanger sequencing traces is the quantitative information embedded therein. With the growing need for quantifying somatic mutations in tumor tissue it is desirable to exploit the potential of the quantitative information obtained from sequencing traces. Materials and Methods To this end, we have developed a software tool that converts a Sanger sequencing trace file into a .comma separated value (.csv) file containing numerical data of peak data characteristics that can be explored and analyzed using conventional spreadsheet software. The web-based tool can be accessed at: http://apps.lifetechnologies.com/ab1peakreporter . The output file contains the peak height and quality values for each nucleotide and peak height ratios for all 4 bases at any given locus allowing the detection and assessment of subtle changes at any given allele. Results and Discussion We demonstrate the utility of this tool by analyzing mixed DNA samples with known amounts of spiked in variant alleles from the human TP53 gene ranging from 2.5%, 5%, 7.5%, 10%, 15% and 25% and show that the minor alleles could be readily detected below the 10% level. Conclusion Enabling high sensitivity detection of minor alleles with a widely available and simple to use technology like Sanger sequencing will be useful for verification of results obtained from next generation sequencing (NGS) platforms.

1. Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • lifetechnologies.com
Detecting and Quantifying Low Level Variants in Sanger Sequencing Traces
Edgar Schreiber, Stephan Berosik, Michael Wenz
Thermo Fisher Scientific Life Sciences Solutions
180 Oyster Point Boulevard, South San Francisco, CA 94080 USA
Introduction
Automated fluorescent dye-terminator DNA Sequencing using capillary electrophoresis (also known as
CE or Sanger sequencing) has been instrumental in the detailed characterization of the human genome
and is now widely used as gold standard method for verification of mutation findings, notably in tumor
samples. The primary information of the DNA sequencing process is the identification of the nucleotides
and of possible sequence variants. A largely unexplored feature of fluorescent Sanger sequencing traces
is the quantitative information embedded therein. With the growing need for quantifying somatic
mutations in tumor tissue it is desirable to exploit the potential of the quantitative information obtained
from sequencing traces.
Materials and Methods
To this end, we have developed a software tool that converts a Sanger sequencing trace file into a
.comma separated value (.csv) file containing numerical data of peak data characteristics that can be
explored and analyzed using conventional spreadsheet software. The web-based tool can be accessed at
http://apps.lifetechnologies.com/ab1peakreporter .
The output file contains the peak height and quality values for each nucleotide and peak height ratios
for all 4 bases at any given locus allowing the detection and assessment of subtle changes at any given
allele.
Results and Discussion
We demonstrate the utility of this tool by analyzing mixed DNA samples with known amounts of spiked
in variant alleles from the human TP53 gene ranging from 2.5%, 5%, 7.5%, 10%, 15% and 25% and show
that the minor alleles could be readily detected below the 10% level.
Conclusion
Enabling high sensitivity detection of minor alleles with a widely available and simple to use technology
like Sanger sequencing will be useful for verification of results obtained from next generation
sequencing (NGS) platforms.
http://www.lifetechnologies.com/primerdesigner
How To Re-Sequence Any Exon of Any Human Gene of Interest (e.g. TP53)
•Step 1: Find Suitable PCR/Sequencing primers using Primer Designer™ Tool
FREE
Enter “TP53” here
Click here
23 PCR Sanger Sequencing primers
are available for the p53 gene
Here we are
sequencing exons
8-9 of the P53
gene and look for
minor variants in
mixtures of 2
genomic DNAs at
various
compositions
Step 2: Get Sanger Sequencing Data Fast and Easy
Stream-lined PCR and Sanger Sequencing with the BigDye® Direct Sequencing Kit
Set up PCR ( 10 min )
primer pair Primer Designer
gDNA
water
2x PCR Master Mix
(included in kit)
PCR on Veriti® Fast
Thermal Cycler (60 min)
Add Sequencing Mix (5min )
on top of PCR reaction
(no plate transfer needed !)
Integrated and seamless PCR
primer removal with cycle
sequencing
PCR on Veriti® Fast Thermal
Cycler (70 min)
Add Bead Mix on top of
PCR/SEQ reaction (5min )
(no plate transfer needed !)
Mount on Vortexer
(20 min)
Set up CE run (5min )
Put plate into Genetic Analyzer
(no plate transfer needed !)
Capillary Electrophoresis
(60 min)
The Data Analysis Challenge:
Minor Variants (< 20%) Are Often Undetected or Overlooked
• True heterozygous mutations are
reliably called (green circle) in
current analysis software but
minor variant detection is limited
to ~20-30% of major peak height.
• Analysis software does not provide
quantitative trace data.
• Not sensitive enough for somatic
mutation detection.
50 % G
40 % G
30 % G
20 % G
10 % G
Minor variants < 20% are often
visible as humps but not “called” by
analysis software (red circles)
• Need for a new tool that reveals and quantifies minor variants: ab1 Peak Reporter !
The ab1 Peak Reporter Tool Provides a Solution for Detection of Minor
Variants in Sanger Sequencing Traces
• The tool extracts numerical information from .ab1 files and returns as .csv files.
• The extracted data can be analyzed manually by downstream spreadsheet software,
such as, Microsoft Excel® for studying base peak characteristics (e.g. allele proportions).
• Aids in detection and quantitation of minor variants
• Aids in quantifying methylation events in bi-sulfite converted DNA
• Access tool after login into your personal Life Technologies user account
• https://apps.lifetechnologies.com/ab1peakreporter
• Detailed instructions for use and further information can be found in the application
note
• http://tools.lifetechnologies.com/content/sfs/brochures/seq-quantification-app-note.pdf
FREE
Extract zip file to
individual .csv files
Upload .ab1 files to
cloud-based tool
ab1 Peak Reporter
(96 files limit)
Receive output file (zip)
back to your computer
ab1 Peak Reporter Workflow
approx. 1 min
Open and analyze
trace data file with
Excel
ab1 Peak Reporter Shows the Numbers Behind the Peaks
10 % mutant admixture (“A”-trace) viewed in signal intensity line plot shows distinct peak for minor allele
Signal (rfu)
G A T CScan # Base
call
The tool calculates peak height ratios (PHR) for all 4 bases in
a broader 7-scan window (rfu peak “N” /rfu max of major
peak) and a narrow 1-scan window (rfu peak “N” /rfu major
peak at base call scan). For detection and quantitation of
true minor variants the values of these two ratios should be
similar.
Other output includes: signal (rfu) G,A,T,C, peak height ratios,
quality values, base calls 1st and 2nd peak, kb basecaller
amplitudes, sequence finder and other run information logs.
TP53 Gene Exon 11 Sequencing of gDNA with
0, 2.5, 5, 7.5, 10, 15 and 25% Admixture of Mutant Allele
Peak Height Ratio Values of Minor Variant
PHR
7-scan
PHR
1-scan window
ab1 Peak reporter data
analysis of ab1 Sanger
sequencing files: shown
are the peak height ratios
(PHR) in 7-scan and 1-scan
mode between mutant
peak (a “C” and major
peak. Note the
remarkable concordance
of the ratio values with
the actual amount of
mutant allele present
(column “sample”).
TP53 Gene Exon 8-9 Sequencing of gDNA with
2.5, 5, 7.5, 10 % Admixture of Mutant Allele
Peak Height Ratio Values of Minor Variant
ab1 Peak reporter data analysis of ab1 Sanger sequencing files for a different
segment of the TP53 gene (exon 8-9) : shown are the peak height ratios (PHR) in 1-
scan mode between minor mutant peak (a “T” and major peak C in the forward
sequencing reaction (fwd) and an “A” and major peak G in the reverse sequencing
reaction (rev) . Here the PHR values from the forward sequencing reaction
appeared to be close to the actual amount ; whereas in the reverse sequencing
reaction the minor allele presented more prominent.
A
T
Summary
• Sanger sequencing is the method of choice for verification
of uncertain NGS results since it is easy to perform
(BigDye® Direct chemistry), fast, economical and rich in
information (long sequence reads)
• The Primer Designer tool is a free resource for finding
suitable PCR/sequencing primers for almost any exon in
the human genome.
• Minor allele variations can be investigated by analysis of
Sanger sequencing traces with the free ab1 Peak reporter
tool which provides quantitative data of peak traces.
• For Research Use Only – Not for use in diagnostic
procedures