High data quality and accuracy are recognized characteristics of Sanger re-sequencing projects and are primary reasons that next generation sequencing projects compliment their results by capillary electrophoresis data validation. We have developed an on-line tool called Primer Designer™ to streamline the NGS-to-Sanger sequencing workflow by taking the laborious task of PCR primer design out of the hands of the researcher by providing pre-designed assays for the human exome. The primer design tool has been created to enable scientists using next generation sequencing to quickly confirm variants discovered in their work by providing the means to quickly search, order and receive suitable pre-designed PCR primers for Sanger sequencing. Using the Primer Designer™ tool to design M13-tailed and non-tailed PCR primers for Sanger sequencing we will demonstrate validation of 28-variants across 24-amplicons and 19-genes using the BDD, BDTv1.1 and BDTv3.1 sequencing chemistries on the 3500xl Genetic Analyzer capillary electrophoresis platform.

1. Michael Wenz, Stephan Berosik, Achim Karger, Pius Brzoska, Fangqi Hu, Xiaoqing You.
Thermo Fisher Scientific, 180 Oyster Point Blvd., South San Francisco, CA 94080
RESULTS
A New PCR Primer Design Tool for Sanger Sequencing Confirmation
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • lifetechnologies.com
ABSTRACT
High data quality and accuracy are recognized characteristics of Sanger re-
sequencing projects and are primary reasons that next generation sequencing
projects compliment their results by capillary electrophoresis data validation. We
have developed an on-line tool called Primer Designer™ to streamline the NGS-to-
Sanger sequencing workflow by taking the laborious task of PCR primer design out
of the hands of the researcher by providing pre-designed assays for the human
exome. The primer design tool has been created to enable scientists using next
generation sequencing to quickly confirm variants discovered in their work by
providing the means to quickly search, order and receive suitable pre-designed
PCR primers for Sanger sequencing.
Using the Primer Designer™ tool to design M13-tailed and non-tailed PCR primers
for Sanger sequencing we will demonstrate validation of 28-variants across 24-
amplicons and 19-genes using the BDD, BDTv1.1 and BDTv3.1 sequencing
chemistries on the 3500xl Genetic Analyzer capillary electrophoresis platform.
INTRODUCTION
The Applied Biosystems® Primer Designer™ tool enables NGS scientists to quickly
and accurately validate variants by Sanger sequencing. An essential part of
successful Sanger sequencing is the quality of the design, synthesis and
purification of the gene specific PCR primers. By navigating to
www.lifetechnologies.com and entering the search terms “Sanger Primers” or
“Primer Designer™ Tool” researchers can now save valuable time by choosing
from a collection of >300,000 human exome amplicon targets and view their
associated primer pair sequences for free. The pre-designed primers for PCR and
Sanger sequencing can be ordered directly from Life Technologies with or without
M13 tails, HPLC or desalt-purification only. With the Primer Designer™ Tool it is
possible to confirm novel SNPs by complimentary Sanger sequencing within days.
MATERIALS AND METHODS
The Primer Designer™ Tool was used to identify primers for 192-gene targets or
amplicons from the “Top 60” genes covered in the Ion TargetSeq™ Exome Kit. Of
these 192-targets, 24-amplicon targets were selected for discussion here based on
28-SNPs variants found using NGS that could be subject to confirmation by Sanger
sequencing.
Four sets of 192-amplicon target primers each were ordered through Life
Technologies including M13-tailed/desalted, M13-tailed HPLC purification, non-
tailed/desalted and non-tailed/HPLC purified. The Primer Designer™ Tool primer
design is such that each primer pair, regardless of location within the human
exome, has a target Tm of 63°C +/- 3°C to support universal PCR conditions which
was used for all 192-amplicons. All data were generated on the Applied
Biosystems® 3500xl Genetic Analyzer and Veriti® thermal cycler.
The Primer Designer™ tool primers generated amplicons of no greater than 600-bp
using starting input such as target information (Gene, SNP ID, RefSeq, or FASTA
sequence), chromosome position, or a .vcf file from NGS. The following will
demonstrate how NGS SNP validation was achieved.
Materials PN
AmpliTaq Gold® 360 PCR Mix 4398881
BigDye® Direct PCR/Sequencing Kit 4458688
BigDye® Terminator v1.1 Sequencing Kit 4337451
BigDye® Terminator v3.1 Sequencing Kit 4337456
BigDye XTerminator® Purification Kit 4376484
UP DN/Rnase Free Water 10977-023
DNA Buffer (10mM Tris/0.1mM EDTA, pH 8.0) T0223
Primers ordered through the Life Technologies web site were delivered dried down at
ambient temperature and were re-suspended to a 100 µM working solution in a DNA re-
suspension buffer; primers were then pooled to an optimal concentration based upon
PCR needs; 2.4 µM each for common PCR reactions or 0.8 µM primer for BigDye®
Direct PCR. BigDye® Terminator v1.1, v3.1 and Direct sequencing chemistries were all
performed across a subset of the 192-designed primers and across all of the 24-
amplicon/28-SNP NGS confirmation targets.
The Primer
Designer™ search is
started by entering
either
specific target
information,
a chromosome region
of interest or by
pointing to a NGS .vcf
file.
A search of the
>300,000 PCR pre-
designed primer pairs
will return a list of
primer pair options to
choose from. By
clicking on the “View
Primer on Map” option
nearest to the primer
set of interest a map of
the target exome within
the gene of interest can
be displayed.
Click the “View
Details” option beneath
the primer pair of
interest to reveal
additional information
including the primer
chromosome location,
Gene, Cosmic, and
SNP detail related to
the designed PCR
primer set, view both
forward and reverse
primer sequences for
download or for
placement of an oligo
synthesis order.
Three step process to confirm NGS results by Sanger Sequencing using the
VR Toolkit ( ww.apps.lifetechnologies.com/vrtoolkit )
1) Build a reference
sequence based on the
designed primers
2) Run Variant Reporter®
Software locally and load
the reference file created
in step #1
3) Use the “Variant
Confirmation” application
in the VR Tool kit.
Step 1:Input the Primer ID that
corresponds to the PCR primer
pair ID that was generated by
the Primer Designer™ Tool. A
reference file (.vrr) is generated
by the VR Toolkit that can be
imported by Variant Reporter®
Software.
Step 2: Use Variant Reporter
software to identifying SNPs
contained within the Sanger
sequencing data. The
reference file generated in step
#1 is used for analysis and
SNP identification.
The example shows data
generated by the Primer
Designer™ Tool for the XPC
gene; a control gDNA (bottom
two traces) does not produce
the SNP while the specimen of
interest does. The variant
report is exported from Variant
Reporter® for step #3 below.
Step 3: Generate a Variant
Confirmation Table by
selecting:
• the NGS .vcf file,
• reference file (step #1) and
• Variant Reporter® result
(step #2)
and clicking on “Generate
Report” in the VR Toolkit. NGS
confirmation is indicated when
the NGS(.vcf) and VR (.txt)
results match.
Sanger sequencing results using BigDye® Direct and BigDye® Terminator sequencing
chemistries confirm 27/28-SNPs identified using NGS. In addition to the BigDye®
chemistries, each primer set was synthesized with and without M13-tails as well as with
standard desalting and HPLC purification methods where each version generated the
same Sanger sequencing result. Note that in some instances, high-lighted in orange, a
definitive sequencing answer is found in only one sequencing direction due to additional
sequence content such as homopolymers or heterozygous insertion/deletions obscuring
the sequencing results of the opposite strand.
In choosing the sequencing chemistry it is important to consider the proximity of the
SNP of interest to the PCR priming sites as the BigDye® Terminator v3.1 and POP-7™
combination may miss variants adjacent to the 5’-region due to a 25-30-bp
compression. Tailed PCR and the use of M13 fwd/rev sequencing primers affords
sequencing through the PCR specific priming region as the sequencing priming site is
M13 sequence specific; this is beneficial if a SNP of interest is near the 5’-gene specific
(PCR) priming region where there is a risk of missing the SNP if 5’-sequencing
resolution is sub-par for the first 5-10-bp. The BigDye® Direct PCR/Sequencing kit is
only compatible with M13-tailed PCR primers. The BigDye® Direct/POP-7™ and
BigDye® Terminator v1.1/POP-6™ chemistry and 3500xl Genetic Analyzer run module
combinations provide comparable 5’ sequencing resolution although the 3500xl
POP-6™ sequencing run module used to capture at least 600-bp is significantly longer
than the POP-7™ module.
CE Verification Results for 24-amplicons/28-SNPs
CONCLUSIONS
We demonstrated the successful use of the Primer Designer™ Tool and a next generation
sequencing data confirmation by Sanger sequencing analysis workflow using 24-genes
containing 28 SNPs. The Primer Designer™ Tool generated PCR primer designs for 192-
amplicon gene targets that performed well using universal PCR conditions as well as with
the BigDye® Terminator sequencing chemistries. In addition to the Primer-Designer™ Tool
a data analysis workflow for the comparison the NGS data to the Sanger sequencing
results using the VR Tool Kit was demonstrated. The combination of these free tools
enables efficient and effective confirmation of NGS variants.
TRADEMARKS/LICENSING
For Research Use Only. Not for use in diagnostic procedures.
AmpliTaq Gold is a registered trademark of Roche Molecular Systems, Inc.
The authors declare a conflict of interest.
BigDye Direct BigDye Terminator v1.1 BigDye Terminatorv3.1
CE Genotype CE Genotype CE Genotype
NGS/CE
Confirmed
NOTCH2 247286 A A/T A/T A/T A/T YES
PDE4DIP 247326 C C/T C/T C/T C/T YES
PDE4DIP 247326 G G/A G/A G/A G/A YES
EXT2 328600 T T/C T/C T/C T/C YES
CCND2 115094 C C/G C/G C/G C/G YES
ARID2 336029 T T/C T/C T/C T/C YES
ALK 192315 A A/T A/T A/T A/T YES
MSH6 359011 A A/G A/G A/G A/G YES
MSH6 359024 T C C C C YES
FN1 205929 C C/T C/T C/T C/T YES
BCR 362193 G G/A G/A-fwd G/A-fwd G/A-fwd YES
BCR 362194 C C/T C/T C/T C/T YES
BCR 362197 G G/A G/A G/A G/A YES
MN1 221970 A A/G A/A A/A A/A NO
CYP2D6 225801 G G>T G>T G>T G>T YES
CYP2D6 225801 G G>A G>A G>A G>A YES
CYP2D6 225801 T T>G T>G T>G T>G YES
CYP2D6 225801 G G>C G>C G>C G>C YES
FANCD2 363573 G G/T G/T G/T G/T YES
XPC 227180 G G/A G/A G/A G/A YES
FOXP1 235541 A A/T A/T A/T A/T YES
EPHB1 240070 G G/A G/A G/A G/A YES
FBXW7 254918 G G/A G/A G/A G/A YES
MTRR 257547 A A/G A/G-rev* A/G-rev* A/G-rev* YES
TNFAIP3 281646 C C/T C-f/indel-r C-f/indel-r C-f/indel-r YES
TNFAIP3 281648 A A/C A/C A/C A/C YES
SYNE1 282848 A A/G A/G A/G A/G YES
SYNE1 282961 T T/C T/C T/C T/C YES
Gene
Primer#/
Assay Reference
NGS
Genotype