Ion Torrent Technology and Workflow

We Have Led Innovation in Targeted

Sequencing for 7 Years

Simple Fast Scalable Supported

Ion AmpliSeq™ Ion Chef™ System Ion Torrent™ Systems Ion Reporter™ Software
technology
 Ion GeneStudio S5 Series
Ion GeneStudio™ S5
Ion GeneStudio™ S5 Ion GeneStudio™ S5 Plus
Prime
New New

Fast. Flexible. Powerful

.
New

Ion 510™ Ion 520™ Ion 530™ Chip Ion 540™ Chip Ion 550™ Chip
Chip Chip 15–20 M reads 60–80 M reads 100–130 M reads
2–3 M reads 3–6 M reads Up to 600 bp Up to 200 bp Up to 200 bp
Up to 400 bp Up to 600 bp
For Research Use Only. Not for use in diagnostic procedures. * Throughputs based on 200bp sequencing
 Output and Turn-Around
Ion GeneStudio™ S5 Ion GeneStudio™ S5
Ion GeneStudio S5 ™
Plus Prime

Speed* 19 hrs 10 hrs 6.5 hrs

Output (max/day): 15 Gb/80 M 30 Gb/160 M 50 Gb/260 M

Chips (max/day): 1 x 540 2 x 540 or 1 x 550 2 x 550

 Ion GeneStudio S5 System and
Applications

Ion GeneStudio™ S5 System

Targeted Sequencing
RNA Applications
Applications

Microbial, Viral,
Technology and Data
Metagenomics Applications

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

Ion Torrent Workflow
4 Basic Steps
Ion Torrent™ Workflow: Library Prep
Walk-away automation with the Ion Chef™ Instrument
or
Manual library preparation and quantitation
 Ion AmpliSeq Library Preparation Overview
Amplify targets using
Amplify targets using Ion AmpliSeq RNA
Ion AmpliSeq DNA Panel
Panel
Reverse Transcriptase
RNA

Genomic DNA cDNA

Partially Digest Primer

Ligate Adapters

DNA Specific
X RNA Specific
Barcode Adapters Barcode Adapters
P1

Amplify Library
 Manual versus Automated AoC Ampliseq Library
Construction
Manual AmpliSeq Steps AmpliSeq on Chef Steps

gDNA or cDNA 8 gDNA or cDNA

Amplify targets Amplify targets

Partially digest primer sequences Partially digest primer sequences

Ligate adapters to the amplicons and purify Ligate adapters to the amplicons and purify

Option 3: Pooled Equalizer Steps

Quantify the
Option 1: Option 2: amplified library Libraries 1 to 8 @ 100pM in ~ 700µL
Equalize the Quantify the with Qubit™
library unamplified Fluorometer or
library by qPCR Agilent™ 2100
Bioanalyzer™ Only 8 hours
Instrument

Combine libraries (optional) and proceed to template

preparation

1-2 day
Ion Torrent Workflow
Clonal Amplification using Emulsion PCR

P1 Barcode + A = X

ISP = DNA Taxi

Emulsion droplet
Primer
dNTPs
Polymerase
ISP MgCl2

Isolate templated ISPs

Final Templated ISPs ready for sequencing

Cycle 1, Annealing
Cycle 1, Extension
Cycle 2, Denature
Cycle 2, Annealing
Cycle 2, Extension
Polymerase Template
Cycle 3, Denature
Cycle 3, Annealing
Cycle 3, Extension
Cycle 4, Denature
Cycle 4, Annealing
Cycle 4, Extension
ISP
And so on…

Primer

Oil Aqueous
 Actual Clonal Amplification

1*ISP + Ideal
1*Fragment

More than 1*ISP Duplicate

+ 1*Fragment Reads

Only ISP
1*ISP + More
No
than 1*Fragment
Amplification
Only fragment

Polyclonal
ISP
ISP collection and recovery
From reaction plate
Breaking solution

Centrifuge lid

To waste
Centrifuge base

Centrifuge
Cross section diagram of breaking centrifuge
Motor
 What happens next?
ES Automated Enrichment
1 2 3 4 5 6 7 8
 Robotic arm moves 8-well strip
on and off of magnet during
magnetic purification steps

 Biotinylated templated ISPs are

1) ISP sample
purified from the rest of the sample.
2) MyOne™ Beads
3) Wash Solution
4) Wash Solution
5) Wash Solution
6) Empty
7) Melt-Off Solution
8) Empty
 Ion Sphere™ Particle Enrichment
Post Add Magnetic Immobilize to Remove 2nd strand Enriched
Amplification Streptavidin Bead Magnet and Wash with NaOH ISP

S
S S A
A A

S S
A A

“no template”
“no biotin” “no capture” “no yield”
Ion Sphere™ Particle Enrichment (mixed reads)

Post Add Magnetic Immobilize to Melting off 2nd strand

Amplification Streptavidin Bead Magnet and Wash with NaOH

S
S A S
A A

*This mixed read species can be minimized through proper dilution.

Proper DNA to Ion Sphere™ Particle ratio is critical!
 Ion Torrent Workflow

Ion GeneStudio S5 Series

System
 Fully Automated Template Preparation

Initialize Start Sequencing Run

Ion S5/S5 XL or • Remove chips from Ion Chef
Ion GeneStudio S5 Series • Remove adapter
Instrument • Load chip onto sequencer

Run time: ~2.5 hours

Data analysis times:

Ion S5 & Ion GeneStudio S5 ~16.5 hours
Ion S5 XL ~5 hours
Ion GeneStudio S5 Plus ~7.5 hours
Ion GeneStudio S5 Prime ~4 hours

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

 Plug and Play Reagents

Temp Controlled Chip

Large HD Touchscreen Clamp

Sequencing Reagent Wash Solution

Cartridge & Automated
Waste Cartridge Cleaning Solution
(behind W2 Solution)
 Easy to use pre-made, RFID tagged
consumables
Cartridge W2 Solution

RFID
• Cartridge/W2 bottles - Tracking

single use

• Cleaning bottle -
multiple use (4x)

• Waste container – RFID

Tracking
reusable

• RFID tracking –
traceable
How Does Sequencing Works?

Hydrogen ions released

under non-buffering
conditions
Sequencing Detection by pH Change
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

Ion Torrent leverages on semiconductor technology

Direct detection of hydrogen ions and conversion to an electrical signal

read by a transistor substrate
 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 ...---Ion Sphere™
3’-CCATCTCATCCCTGCGTGTCTCCGAC AGTCAAGCGTCCCATG
Particle
G Adaptator A Key Seq Sequence of Interest P1 seq

Flows 1-4
 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 Interest P1 seq

Flows 5-8
 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 AG
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 Interest P1 seq

Flows 9+
 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 T T 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
A Adaptator A Key Seq Sequence of Interest P1 seq

And so on…
 Sequencing: Ionograms
• An “ionogram” is the output of the signals in flow space
• Must be read “up-and-down” along with “left-to-right”
• Height of bar indicates how many nucleotides incorporated during flow
• “Negative” or “zero” flows indicate no nucleotide incorporation
– These observations are omitted when converting to nucleotide space

TTT
Key Sequence
AA TT AA
TC
TCAG C TG

Sequence: AATCTTCTG…
Torrent Suite Software
Plan, monitor, and track your
runs all within a web interface
while reviewing the quality and
accuracy of your sequencing
run, in addition to variant
calling, detecting SNPs and
indels.
Ion Reporter Software
Call and annotate SNPs/InDels,
CNVs, and fusions. Interpret
your DNA and RNA variants
faster and more easily from
semiconductor sequencing data
using a web-enabled solution
Torrent Suite Software
Ion Reporter Software
TRÂN
TRÂNTRỌNG
TRỌNGCẢM
CẢMƠN!
ƠN!