Introduction to Ion Torrent™ Technology &

Workflow

1.0
The world leader in serving science
Revision date: 02-12-2016
 Amr Osama
NGS & CE Field Application Specialist

For Research Use Only. Not for use in diagnostic procedures.

© 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

200bp 200bp 200bp

Sequencing Analysis Analysis
Output Reads Read Length
Time Time* Time*
S5/S5 XL S5 XL S5

Ion 520™ Chip 2.5 hr ~1 hr ~5 hr 1-2 Gb 3-5 M Up to 400bp

Ion 530™ Chip 2.5 hr ~2.5 hr ~8 hr 3-5 Gb 15-20 M Up to 400bp

Ion 540™ Chip 2.5 hr ~5 hr ~16.5 hr 10-15 Gb 60-80 M Up to 200bp

• Analysis time to BAM file

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Scalability: Semiconductor Technology

Wafer Chip Chip Cross

Section

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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

DNA → Ions → Sequence

• Nucleotides flow sequentially over Ion semiconductor chip
• One sensor per well per sequencing reaction
• Direct detection of natural DNA extension
• Millions of sequencing reactions per chip
• Fast cycle time, real time detection

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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.

• Same fluidics, same sequencing performances

• Different computational performances, the Ion S5™ XL System is
faster in data processing

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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

QC of library: Quality & Quantity

qPCR, Bioanalyzer™, Qubit™
P instruments
B

P
B
Please note: Graphic modified compared to Manual

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Ion AmpliSeq™ Target Selection Overview

• As little as 10 ng of input DNA,

enabling FFPE analysis
• Rapid results with 3-5-hour library
P
B construction
P
B • Library construction automation on
P B
the Ion ChefTM instrument
P B
• www.ampliseq.com
Please note: Graphic modified compared to Manual

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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

C Template Prep Data

Analysi
s

Isolate
Prepare Clonal Positive Load Chip Sequence
D Sequencing
Library Amplificatio Ion Sphere™
n Particles

E Compatible
Data

With Ion Chef ™ System

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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

E 2) Polymerase extends from B’

Compatible
Data
3) Complementary sequence is extended

4) Amplified product disassociates from bead

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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

• This includes both templated and

C
non- templated ISPs
Template Prep • Monoclonal ISPs
• Polyclonal ISPs
D Sequencing • Empty Spheres

E • Empty spheres will take up wells

Compatible
Data on the chip and therefore will need
to be removed.

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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

*This species can be minimized through proper dilution.

Proper DNA to Ion Sphere™ Particle ratio is critical!

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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

Minimal Site-Prep, Simple Installation

• Plug in electrical outlet
• Update firmware via USB drive or ethernet connection
• Installation includes one-time instrument initialization

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Ion OneTouch™ ES Instrument Orientation

Touch pad

Mechanical Arm

Pipet Tip Loader

8-well strip tube

(flanked by magnets)

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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

Massive parallel of each loaded Ion

D SphereTM particle on the Ion ProtonTM
Sequencing
Instrument

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 Suite™ Software

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)

Single run analysis Multiple run analysis

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Data files

DAT Processing DAT

WELLS
Classification On Instrument
DAT files

Signal
Processing
WELLS files

Base Calling

Read Filtering

Unaligned BAM files On (Virtual)

Server
WELLS
Alignment BAM
Aligned BAM files

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 Torrent Browser

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Torrent Browser

• A web accessible data portal used to view

preliminary Ion Torrent™ data.

• Hosted on Torrent Server with remote

access from any PC (with granted access)

• Enables assessment of run quality and

preliminary alignment results

• Preliminary metrics
(~30 minutes after a run begins)

• Loading density and enrichment efficiency

• Easy navigation via filtering options

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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

Targeted Sequencing Whole Exome Whole Genome

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

Your Ion Torrent Ion

Sample AmpliSeq™ Suite™ Reporter™
Designer Software Software
> > >

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AmpliSeq™ technology: Chose target on any Genome

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Ion AmpliSeq™ Technology: As Simple As PCR

Single-tube, ultra-high multiplex for targeted resequencing

+ +

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

• As little as 10 ng of input DNA,

enabling FFPE analysis
• Rapid results with 3-5-hour library
P
B construction
P
B • Library construction automation on
P B
the Ion ChefTM instrument
P B
• www.ampliseq.com
Please note: Graphic modified compared to Manual

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Ion AmpliSeq™ technology: Inherited Disease Research Menu

Cardiovascular Hematology Neurological Ophthalmic

Research Panel Research Panel Research Panel Research Panel

Primary Immuno
Pulmonary Research Dermatology Deafness Research
Deficiency Research
Panel Research Panel Panel
Panel

Endocrinal Dysmorphia-dysplas Gastrointestinal Renal Research

Research Panel ia Research Panel Research 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

Mendeliome panel New panel

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Ion AmpliSeq™ technology, DNA Target Selection Solutions

• Ready to use Panel

• DNA & RNA

• Exome / Transcriptome Panel

• Community Panel

• Ion AmpliSeq™
Designer
• www.ampliseq.com

Ion AmpliSeq™ Library Kit 2.0

Core reagent kit for library construction

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AmpliSeq™ technology: HID-Ion Identity Panel

• The HID-Ion AmpliSeq™ Identity Panel is a ready-to-use panel that

includes 124 autosomal markers, and provides discrimination of
individuals similar to STR genotype match probabilities used by forensic
analysts

24h workflow

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AmpliSeq™ HID-Ion Ancestry Panel

• The HID-Ion AmpliSeq™ Ancestry Panel is a ready-to-use panel that

includes 165 autosomal markers. Used with multiplex PCR and
next-generation sequencing technologies, it can provide biogeographic
ancestry information to help guide your investigation process.

24h workflow

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Ion AmpliSeq™ Exome RDY kit. What is the Exome?

• The exome is comprised of exons

• Exons are short sequences of DNA representing the protein coding
regions in the genome
• It is estimated that there are about 180,000 exons arranged in 22,000
genes in the human genome
• The protein-coding exons comprise about 35-40Mb or ~1.3-1.5% of the
human genome

Image Credit: https://www.my46.org/intro/whole-genome-and-exome-sequencing

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Ion AmpliSeq™ Exome RDY kit

•~294,000 primer pairs across 12 primer pools

• >24,500-plex PCR!
•Total DNA input as low as 50 ng
•Covers >97% of CCDS (Release 12)
• >19,000 coding genes, >198,000 coding exons, no
UTRs, miRNAs, or ncRNAs

•Amplicon size range 225-275 bp

* 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

Amplify Digest Ligate Amplify

target primers adapters library

• Simplest, fastest exome enrichment workflow

• <50 minutes hands on time
• 6 hours for exome library
• qPCR is recommended method of quantitation for
best barcode balancing
• BioAnalyzerTM instrument and QubitTM Fluorometer
compatible
• Ion Equalizer™ Kit compatible

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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

• Ion AmpliSeq™ TB Research Panel

• The panel assesses 8 genes related to antimicrobial resistance (embB, eis,
gyrA, inhA, katG, pncA, rpoB, rpsL).

• Ion AmpliSeq™ Ebola Research Panel

• The panel was developed as a quick, accurate and cost effective method to
identify the present of Ebola virus (EBOV).

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Ion 16STM - Metagenomics Kit

Primer Sets Master Mix Buffers Control DNA

• Set V2-4-8 • Environmenta • DNA Dilution • E. coli control
• Set V3-6,7-9 l Master Mix Buffer DNA
v2.0 • Negative
Control Water

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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

5-100 ng miRNA in total RNA

1-100 ng enriched small RNA >6

1-500 ng poly(A) RNA

100 ng total RNA
(use Dynabeads® mRNA DIRECT™ Micro Kit for polyA >7
selection)

25-500 ng rRNA-depleted total RNA

• Strandedness is preserved
• Barcode compatible

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 Thank you

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