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Workflow
1.0
The world leader in serving science
Revision date: 02-12-2016
Amr Osama
NGS & CE Field Application Specialist
© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of
Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
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Ion Torrent™ Technology
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Ion Technology Core Principles
Scalability
Massively scalable semiconductor technology
Simplicity
Natural biochemistry, no optical components
Speed
Runs in about two hours, not days or weeks
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Ion Technology Core Principles
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Scalability: Semiconductor Technology, the S5 series chips
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Scalability: Semiconductor Technology
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Simplicity: Natural Chemistry
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Speed: Fast Direct Detection
dNTP
H+
∆ pH
∆
Q
Sensing Layer
Sensor Plate
∆
V
Bulk Drain Source To column
Silicon Substrate receiver
Rothberg J.M. et al Nature doi:10.1038/nature10242
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Video
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(Fully automated)
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The all new Ion S5™ and Ion S5™ XL Systems.
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Ion Torrent™ Workflow
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Ion Torrent™ Workflow: Library Prep
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Ion Workflow Overview, library preparation
A DNA, or RNA
Data
Analysi
s
B Compatible
Isolate
Prepare Clonal Positive Load Chip Sequence
Library Prep Library Amplificatio Ion Sphere™
n Particles
C Template Prep
Representative
D Sequencing From complex population of DNA
DNA/RNA material fragments cloned
between 2 primers
E Compatible
Data A P B
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Library Prep Overview
P
B
Ligate adapters and nick translate
P
B
P
B
P
B
P
B
Please note: Graphic modified compared to Manual
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Ion AmpliSeq™ Target Selection Overview
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Ion Torrent™ Workflow: Template Prep
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Ion Workflow Overview
A DNA
With Ion OneTouch™ System
B Compatible
Library Prep
Isolate
Prepare Clonal Positive Load Chip Sequence
D Sequencing
Library Amplificatio Ion Sphere™
n Particles
E Compatible
Data
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Ion Workflow: Manual Template prep overview
A DNA
B
B P Insert A
Compatible
Library Prep
B’ Capture primer
C
Template Prep
D Sequencing
What Happens during amplification?
1) Amplified product binds to B’ capture primer
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Spectrum of Clonal amplification
Amplification
Low amount of
Monoclonal Product No ISP No DNA Polyclonal product product
“mixed” reads Duplicate reads
PERFECT READ
Lower signal
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Ion Workflow – Template Prep
A DNA
• At the end of the templating
reaction, ALL Ion Sphere™
Particles are collected.
B Compatible
Library Prep
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Ion Sphere™ Particle Enrichment
Post Add Magnetic Immobilize to Denature ISP
Amplification Streptavidin Bead Magnet and Wash with NaOH
Biotin Streptavidin
Monoclonal
Polyclonal
*
Empty
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Ion OneTouch™ 2 Instrument Orientation
Touchscreen
Amp Plate Handle Interface
ISP Recovery
Tube for Ion Centrifuge
OneTouch™ Oil
Tube for
Pinch valve to seal reaction
Recovery Solution during amplification
Waste Container
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Ion OneTouch™ ES Instrument Orientation
Touch pad
Mechanical Arm
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Ion Torrent™ Workflow: Sequencing
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Ion Workflow Overview
A DNA
Data
Analysi
s
B Compatible
Isolate
Prepare Clonal Positive Load Chip Sequence
Library Prep Library Amplificatio Ion Sphere™
n Particles
C Template Prep
E Compatible
Data
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Sequencing: Flows
• A “flow” is the event of exposing the chip to one particular dNTP (T,
A, C, or G), followed by a washing step
• The flow order repeats with pattern:
• ‘TACGTACGTCTGAGCATCGATCGATGTACAGC’
T A C G T A C G T C T G A G C A T C G A
… etc.
T Flows 1-4 Flows 5-8 …9-12
A
5’-Sequencing Primer-3’
C
3’-CCATCTCATCCCTGCGTGTCTCCGAC A G T C A A G C G T C C C A T G...---- Ion Sphere™
Particle
G Adaptator A Key Seq Sequence of Interrest PB
A P B
Flows 1-4
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Sequencing: Flows
• A “flow” is the event of exposing the chip to one particular dNTP (T,
A, C, or G), followed by a washing step
• The flow order repeats with pattern:
• ‘TACGTACGTCTGAGCATCGATCGATGTACAGC’
T A C G T A C G T C T G A G C A T C G A
… etc.
T Flows 1-4 Flows 5-8 …9-12
A
5’-Sequencing Primer-3’ T C
C
3’-CCATCTCATCCCTGCGTGTCTCCGAC A G T C A A G C G T C C C A T G...---- Ion Sphere™
Particle
G Adaptator A Key Seq Sequence of Interrest PB
A P B
Flows 5-8
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Sequencing: Flows
• A “flow” is the event of exposing the chip to one particular dNTP (T,
A, C, or G), followed by a washing step
• The flow order repeats with pattern:
• ‘TACGTACGTCTGAGCATCGATCGATGTACAGC’
T A C G T A C G T C T G A G C A T C G A
… etc.
T Flows 1-4 Flows 5-8 …9-12
C
5’-Sequencing Primer-3’ T C A G
T
3’-CCATCTCATCCCTGCGTGTCTCCGAC A G T C A A G C G T C C C A T G...---- Ion Sphere™
Particle
G Adaptator A Key Seq Sequence of Interrest PB
A P B
Flows 9+
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Sequencing: Flows
• A “flow” is the event of exposing the chip to one particular dNTP (T,
A, C, or G), followed by a washing step
• The flow order repeats with pattern:
• ‘TACGTACGTCTGAGCATCGATCGATGTACAGC’
T A C G T A C G T C T G A G C A T C G A
… etc.
T Flows 1-4 Flow 5-8 …9-12
C
5’-Sequencing Primer-3’ T C A G TT C G C A G G GT A C
G
3’-CCATCTCATCCCTGCGTGTCTCCGAC A G T C A A G C G T C C C A T G ...---- Ion Sphere™
Particle
Adaptator A Key Seq Sequence of Interrest PB
A
A P B
And so on…
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Ion TorrentTM Base calling
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Data Analysis Workflow
Ion
Torrent Browser
Raw/WELLS Unmapped BAM Reporter™
BAM
Ion S5XLTM
software
Ion S5™ End User
Sequencer Instrument Computer
Torrent Server
and Database
Plugins
Run Data Delivery
To Bases
Assessment (Unmapped
BAM)
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Data files
WELLS
Classification On Instrument
DAT files
Signal
Processing
WELLS files
Base Calling
Read Filtering
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Torrent Browser
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Torrent Browser
• Preliminary metrics
(~30 minutes after a run begins)
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Run Report
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Introduction to Ion Apps
Revision 1.0
The world leader in serving science
Revision date: 02-12-2016
Our View : Targeted Sequencing
Cost efficient, time efficient and simpler to analyze
Number of
variants 10s to 100s ~50,000 ~3,000,000
generated per run
Likely number of
1-10s 1-10s 1-10s
relevant variants
Time to analyze Hours to days Days to Weeks Weeks to Months
Total cost
$ $$ $$$
Inc. analysis
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Ion Ampliseq™ Technology for DNA analysis
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Ion AmpliSeq™ Technology, Sequence with simple, rapid NGS workflow
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AmpliSeq™ technology: Chose target on any Genome
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Ion AmpliSeq™ Technology: As Simple As PCR
+ +
12-24 576 primer pairs per tube 10 ng DNA per tube Ion AmpliSeq™ Library Kit
Ion AmpliSeq™ Custom Panels
OR
Ion AmpliSeq™ Ready-to-Use Panels
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Ion AmpliSeq™ Target Selection Overview
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Ion AmpliSeq™ technology: Inherited Disease Research Menu
Primary Immuno
Pulmonary Research Dermatology Deafness Research
Deficiency Research
Panel Research Panel Panel
Panel
Inborne Errors Of
Noonan Research Brca 1 & 2 CFTR
Metabolism Panel Research Panel Research Panel
Research Panel
Ion AmpliSeq™
Customize any of these panels on AmpliSeq.com
Exome RDY Kit
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Ion AmpliSeq™ technology, DNA Target Selection Solutions
• Community Panel
• Ion AmpliSeq™
Designer
• www.ampliseq.com
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AmpliSeq™ technology: HID-Ion Identity Panel
24h workflow
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AmpliSeq™ HID-Ion Ancestry Panel
24h workflow
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Ion AmpliSeq™ Exome RDY kit. What is the Exome?
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Ion AmpliSeq™ Exome RDY kit
* Choi M, Scholl UI, Ji W, et al. (2009) Genetic diagnosis by whole exome capture and massively parallel DNA
54 sequencing. PNAS 106(45):19096-19101.
Ion AmpliSeq™ Exome RDY - Workflow
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Ion AmpliSeq™ Exome RDY Kit
➢ Fastest, most efficient exome kit available
• Ion AmpliSeq™ Exome primer pools dried down in 96-well plate format
Plus improved AmpliSeq Library Kit formulation
• 8 exomes per plate or 4 plates of 2 exomes per plate
• 6 hour workflow & 50 mins hands-on time, with master mix only addition
steps
• Simple to automate on any X/Y robotic platform
• Simple library normalization with Ion Library Equalizer™ Kit
• No need to quantitate libraries, simple volume based pooling
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AmpliSeq™ technology: Fixed panels for bugs
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Ion 16STM - Metagenomics Kit
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Ion Total RNA-Seq Kit v2
• Single kit for RNA library construction:
Whole Transcriptome (WT) or Small RNA
• For use with Ion Sequencers
• Flexible RNA Input
Start with… RIN
• Strandedness is preserved
• Barcode compatible
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Thank you
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