Whole exome or panel sequencing projects performed by next generation sequencing (NGS) technologies typically reveal a large number of variants which may require verification by an orthogonal method. To that end, automated fluorescent Sanger sequencing is the method of choice since it is accurate, affordable and easy to perform. To facilitate the re-sequencing of any exon in the human genome we have recently made available to the scientific community a free to use tool called Primer Designer™ . The tool provides the designs for over 350.000 PCR primer pairs that cover 99% of all exons in the human genome. Amplicons generated with these primers can be readily sequenced using the BigDye®Direct, BigDye® Terminator v1.1 and v3.1 sequencing chemistries on the 3500xl Genetic Analyzer capillary electrophoresis platform. For variant identification the sequencing trace files are analyzed with Applied Biosystems Variant Reporter® Software which requires the import of a text file with a reference sequence for sequence alignment and comparison. Here we show the utility and workflow of a new on-line tool called VR Toolkit™ that generates a reference file from Primer Designer –derived PCR amplicons that contains the coordinates of the chromosomal location. Use of this annotated reference file in Variant Reporter® Software allows the generation of an output file that can be compared and matched to a variant call file (vcf) from an NGS instrument. The VR Toolkit enables the connection and synchronization between NGS data and traditional Sanger sequencing data analyzed with Variant Reporter® Software and should be of benefit for all researchers seeking to validate NGS data by Sanger sequencing.

1. Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • lifetechnologies.com
From NGS back to Sanger Sequencing: Connecting and Synchronizing
NGS and CE Variant Files with the VR Toolkit™
P16.70-M
Edgar Schreiber, Stephan Berosik, Samsani Sivakumar, Sylvia Chang, Stephen Sharp
Thermo Fisher Scientific Life Sciences Solutions
180 Oyster Point Boulevard, South San Francisco, CA 94080 USA
Abstract
Whole exome or panel sequencing projects performed by next generation sequencing
(NGS) technologies typically reveal a large number of variants which may require verification
by an orthogonal method. To that end, automated fluorescent Sanger sequencing is the
method of choice since it is accurate, affordable and easy to perform. To facilitate the re-
sequencing of any exon in the human genome we have recently made available to the
scientific community a free to use tool called Primer Designer™ . The tool provides the
designs for over 350.000 PCR primer pairs that cover 99% of all exons in the human
genome. Amplicons generated with these primers can be readily sequenced using the
BigDye® Direct, BigDye® Terminator v1.1 and v3.1 sequencing chemistries on the 3500xl
Genetic Analyzer capillary electrophoresis platform. For variant identification the
sequencing trace files are analyzed with Applied Biosystems Variant Reporter® Software
which requires the import of a text file with a reference sequence for sequence alignment
and comparison. Here we show the utility and workflow of a new on-line tool called VR
Toolkit™ that generates a reference file from Primer Designer – derived PCR amplicons that
contains the coordinates of the chromosomal location. Use of this annotated reference file
in Variant Reporter® Software allows the generation of an output file that can be compared
and matched to a variant call file (vcf) from an NGS instrument. The VR Toolkit enables the
connection and synchronization between NGS data and traditional Sanger sequencing data
analyzed with Variant Reporter® Software and should be of benefit for all researchers
seeking to validate NGS data by Sanger sequencing.
This is my NGS vcf file; the NGS run
detected a lot of variants
I ‘d like to verify this particular variant
with Sanger Sequencing on my Applied
Biosystems CE Genetic Analyzer.
• How can I do this quickly,
inexpensively and efficiently ?
• How can I match the NGS data with
CE Sanger data ?
www.lifetechnologies.comYour laboratory
Primer Designer ToolFind and order suitable PCR
primers for re-sequencing locus/
allele of interest
Use primers to PCR and sequence
e.g. with BigDye Direct followed
by CE: get .ab1 sequence files
Obtain annotated reference file
(vrr) from VR Toolkit for primer
assay used
Primer Designer Tool
VR Toolkit
custom-synthesis of primers
delivered to your bench
Analyze .ab1 sequencing traces
with Variant Reporter Software;
export result file (.txt)
vrr
CE
variant
file
NGS
variant
file (vcf)
Need to confirm
something with
Sanger sequencing
Send vcf file and CE variant file
to VR Toolkit for matching
Primer Designer Tool
VR Toolkit
NGS
variant
file (vcf)
CE
variant
file
vrr+ +
Matched report with variants found in
NGS and CE and genome position
Workflow matching NGS vcf file
with Sanger Sequencing Variant s
go to
go to
http://www.lifetechnologies.com/primerdesigner
Step 1:
Find Suitable PCR/Sequencing primers using Primer Designer™ Tool
Enter genome
position for variant of
interest from vcf file
Pre-designed primer pairs (assays) are easy to
find with the Primer Designer Tool.
For this particular NGS variant we can use the
position information from the vcf file.
Pick and order a primer pair for this amplicon ; I am adding M13-tails for
use with BigDye® Direct Sequencing chemistry (un-tailed is also available)
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)
Step 3: Obtain a Variant Reporter (VR) Reference File with Genome Positions
Primer Designer Tool
The VR ToolKit will create a specific reference file (.vrr) with genome coordinates for
your assay for subsequent CE data analysis using Variant Reporter® Software.
ReferenceFile_2014.04.28-18_56_25-UTC.vrr
Annotated
reference file
contains amplicon
and primer
sequences and
hg19 positions
Open VR Toolkit from
button in Primer
Designer tool
(free; no charge)
Step 4:
Analyze Sanger CE Data using Variant Reporter® Software and VR Tool Reference File
Here is the
reference file
from VR Toolkit
imported into
Variant Reporter®
Software
Identification
of the variant
allele using
BigDye® Direct
Sequencing
traces and
Variant
Reporter®
Software.
Alignment with
the reference
file.
Export variant report result file
VRtool_XPC_227180_Project
Variant Export.txt
Now match it with the NGS vcf file
Step 5:
Upload NGS vcf file and exported CE Variant File (.txt) into VR Toolkit and Match
VR Toolkit matches NGS vcf result with Sanger sequencing variant (VR )result
Export report as csv file.
Connecting the Dots between NGS Variant
file (.vcf) and CE Variant File (.txt)
• Need to confirm NGS-detected variants in human exome with
Sanger Sequencing ?
• Order ready to go pre-designed PCR/Sequencing primer pairs
(= assays) for the human exome from Primer Designer portal
(www.lifetechnologies.com/primerdesigner)
• Perform rapid and robust PCR & Sanger sequencing with stream-
lined Big Dye Direct or traditional BigDye chemistry
• Generate a reference file with genome annotation for the ordered
assay with the VR Toolkit (free)
• Analyze .ab1 Sanger sequencing file with this reference file in
Variant Reporter™ software (output is a CE variant file .txt )
• Match CE variant file (.txt) with NGS variant file (.vcf) in VR Toolkit
(free)
• For Research Use Only – Not for use in diagnostic procedures
www.lifetechnologies.com/primerdesigner
Primer Designer™ Tool and integrated VR Toolkit™ software
• Find and order primers
for nearly any exon in
the human exome
• Alignment of Sanger
sequencing traces with
human genome
positions (hg19)
• Matching variant(s)
from NGS vcf file with
Sanger sequence variant