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SAMPLE–TO–ANSWER SOLUTIONS
Development and demonstration of the
Apollo 200 breadboard, a four sample
rapid human identification system
Stevan Jovanovich, Ph.D.
President/CEO
Bill Nielsen
VP Systems Integration
IntegenX Inc.
Pleasanton, CA
www.integenx.com
1 | IntegenX 10/4/2010
Outline
• Company introduction
• Development timeline
• Development of a four channel rapid DNA analysis
system
2 | IntegenX 10/4/2010
IntegenX Background
IntegenX is automating workflows for
DNA sequencing and human identification.
• Founded in 2003 to automate and miniaturize sample
preparation
• Initial funding - $15 MM in grants and contracts: developed
core technologies and applications
 DoD, NIH, DHS, Strategic Investor, forensics partners, Northrop Grumman
 $12.3 MM contract under negotiation with DoD/DTRA for biodefense
• Equity funding - $20.7 MM in A & B rounds: Product
development
• Licensed 18 patent families and filed 12 patent families
• 50 employees, multidisciplinary, engineering, science,
microfabrication, manufacturing, and commercial
3 | IntegenX 10/4/2010
Fully Integrated and Automated Runs
Representative full profile: buccal swab to STR profile fully integrated
and automated run using Promega® PowerPlex chemistry.
4 | IntegenX 10/4/2010
Key IntegenX Technology: Microscale On-chip Valves (MOVe™)
Closed
Open
wafer
wafer
Fluid Channel
MOVe™ valves can:
•Pump 10 nL to 100 L
•Mix fluids`
Air Chamber
•Process beads
•Enable programmable fluidics
•Integrate multiple processes
Fabricated in glass and plastics
Dye Mixing - WATCH THIS VIDEO
5 | IntegenX 10/4/2010
Fluid Channel
PDMS film
wafer
plastic wafer
•Split or combine fluids
MBI-040 - WATCH THIS VIDEO
PDMS film
Apply
pressure
Apply
vacuum
Product Portfolio
Sample
In
Preprocess
Extraction, Purification,
Concentration
DNA Sequencing
Human ID Testing
6 | IntegenX 10/4/2010
Molecular Biology
Repair, Ligate, Amplify, Dilute
Sanger Sequencing
Apollo 100/100xl
Launched Q2 2010
Analysis and
Detection
Answer
Out
Apollo 100: Automated DNA Sequencing Sample Preparation
•
Input: 96 PCR or plasmid
prep samples per run
•
Process:
- Robot transfers reagents
and samples to chips
•
•
On-chip cycle sequencing
On-chip SPRI bead clean-up
- Robot unloads finished
samples into 96 well plate
•
7 | IntegenX 10/4/2010
Output: Six 96-well
plates/day, Ready-to-Inject™
Product Portfolio
Sample
In
Preprocess
Extraction, Purification,
Concentration
DNA Sequencing
Molecular Biology
Repair, Ligate, Amplify, Dilute
Sanger Sequencing
Apollo 100/100xl
Launched Q2 2010
Next Gen Sequencing:
Apollo 324
Launch Q4 2010
Next Gen Sequencing
Apollo 396
Launch Q3 2011
Integrated Sample-to-Sequence System
for biodefense and molecular diagnostics
Launch 2014
Human ID Testing
8 | IntegenX 10/4/2010
Analysis and
Detection
Answer
Out
Integrated Sample-to-Sequence System
• Goal: detect genetically engineered bioagents and
emerging pathogens
• Requirements
• Input, down to 10 bacteria
• Output, DNA sequence
• Push button operation
• Integrates
• Upstream cell lysis and DNA purification
• Rolling circle amplification
• Next Generation Sequencer
• TRL 4 in 2012
9 | IntegenX 10/4/2010
Product Portfolio
Sample
In
Preprocess
Extraction, Purification,
Concentration
DNA Sequencing
Molecular Biology
Repair, Ligate, Amplify, Dilute
Sanger Sequencing
Apollo 100/100xl
Launched Q2 2010
Next Gen Sequencing:
Apollo 324
Launch Q4 2010
Next Gen Sequencing
Apollo 396
Launch Q3 2011
Integrated Sample-to-Sequence System
for biodefense and molecular diagnostics
Launch 2014
Human ID Testing
Rapid DNA Human Identity Testing
Apollo 200
Early Access Q3 2011
10 | IntegenX 10/4/2010
Analysis and
Detection
Answer
Out
SAMPLE–TO–ANSWER SOLUTIONS
Apollo 200: sample-to-answer rapid
DNA identification system
11 | IntegenX 10/4/2010
Rapid DNA-based Human Identification
• Law enforcement real-time
screening of arrestees
• Forensic identification
• Missing person identification
•
•
•
•
Field-based antiterrorism
Linking IEDs to bomb makers
Border control
Immigration for kinship
validation and asylum
• Disaster recovery/mass
casualty situations
• Applied and diagnostic
markets
12 | IntegenX 10/4/2010
Current DNA Human Identity Analysis
DNA analysis today is fragmented using 8 to 10 different technologies
embedded in 6 or 7 different manufacturer’s instrumentation.
Cell Lysis
Extraction
1hr 45 min
Qiagen EZ1
Quantify
2 hr 20 min
ABI 7000
RT-PCR
PCR
Amplify
Dilute
15 min
Tecan
Genesis
3 hr 30 min
ABI 9700
8 to 10 hours
13 | IntegenX 10/4/2010
Detect
Multimek
Analyze
2 hr
ABI 3130
1 hr
ABI
Gene Profiler
Apollo 200 DNA Analysis
IntegenX’s process uses a single fully integrated and automated
system with a faster process.
Cell Lysis
Extraction
PCR
Amplify
Dilute
Detect
Process
Data
CODIS
Output
2 hours
• Full automation
 Load reagents and samples
 Push go
 CODIS data in 2 h
• Four channel system operational
today
14 | IntegenX 10/4/2010
Development of an Integrated Transportable Human ID System
2006
2007
2008
Module development
15 | IntegenX 10/4/2010
2009
2010
2011
Development of an Integrated Transportable Human ID System
2006
2007
2008
2009
2010
2011
Single channel
system development
SingleChannel
Breadboard
2.5 x 2 x 1.5 ft
March 2008
16 | IntegenX 10/4/2010
Development of an Integrated Transportable Human ID System
2006
2007
2008
2009
2010
2011
Single channel
system development
SingleChannel
Breadboard
2.5 x 2 x 1.5 ft
STR data from a buccal swab processed on fully integrated and
automated single-channel system using MiniFiler™ reagents.
17 | IntegenX 10/4/2010
Development of an Integrated Transportable Human ID System
2006
2007
2008
2009
2010
Four channel
system development
Four Channel
Breadboard
2 x 2 x 2 ft
September
2009
18 | IntegenX 10/4/2010
2011
SAMPLE–TO–ANSWER SOLUTIONS
Four channel rapid identification system
19 | IntegenX 10/4/2010
Four Channel Integrated Breadboard
Capillary Separation & Detection
Subsystem
Polymer Gel Fill
Device
Post -Amplification
Subsystem
Reagent
Distribution Device
Waste
Nucleic Acid
Purification
Subsystem
Reagent Reservoirs
STR Reaction
Subsystem
System Footprint = 2ft x 2 ft x 1.25 ft
20 | IntegenX 10/4/2010
System Process Flow and Reagent Inputs
21 | IntegenX 10/4/2010
Disposable Cartridge
MOVe™
Chip
DNA Profiler Cartridge
Precious Reagent
Cartridge
DNA Profiler
Cartridge Installed
On-System
22 | IntegenX 10/4/2010
Process
1. Load disposable MOVe sample cartridge
2. Install reagent cartridge, reagent bottles, premix
fill device and separation polymer syringe
3. Load samples into cartridge and identify samples
in software
4. Hit run
5. CODIS .xml files ready in two hours
23 | IntegenX 10/4/2010
BeadStorm™ Fluidic Cartridge
Sample / Lysis
Chambers
Connect to
Post-Amp
• Sample introduced to Sample /
Lysis Chamber
• Lysis buffer metered to chamber
• Heat assisted lysis
• Beads metered to Bead Capture /
Wash Chamber
Bead Capture /
Wash Chambers
24 | IntegenX 10/4/2010
MOVe™ Fluidic
device
•
•
•
•
•
Lysis solution moved to chamber
Mix and capture beads
Meter wash solution to chamber
Mix and capture beads
Push bead / wash solution to
cycling chamber with metering by
Post Amp module
BeadStorm™ Yield for Bead Dilutions
• Bead concentration
optimized to yield >1 ng
which produces the
best STR reaction
products
• The beads normalize
the reaction input DNA,
alleviating the need for
further quantification
• Multiple bead
chemistries have been
implemented
25 | IntegenX 10/4/2010
Thermocycler Device
Patent pending
26 | IntegenX 10/4/2010
STR Premix Loading and Reactions
•Magnetic beads with genomic DNA
in wash solution are transferred
from BeadStorm™ to PostAmp
•Beads are captured in tubing as
solution flows by
•Premix is loaded into tubing by
syringe device
•Premix is brought to place by MOVe
fluidics in the PostAmp
•STR thermocycling reactions are
performed by resistive heater and
fan
27 | IntegenX 10/4/2010
Fast Runs
PowerPlex chemistry 36 mins
IdentiFiler
28 | IntegenX 10/4/2010
Post Amplification Sub-System
• Meters sample during capture
of DNA/bead solution prior to
thermocycling
• Positions STR reagents in the
thermocycler
• Performs dilution of amplified
STR products prior to CE
injection
• Delivers products to cathode
for CE injection
• Delivers buffer to cathode for
CE pre-run and run
Patent pending
29 | IntegenX 10/4/2010
Reagent Cartridge
Capillary Separation and Detection Subsystem
Capillaries provide proven performance, low fabrication cost and 100+ run stability
Access to the fully developed technologies associated with capillary array
electrophoresis minimizes risk
• Capillaries are thermally controlled by contact to aluminum plate
• Interface with polymer fill, anode and cathode is facilitated by the capillary
electrophoresis module
• 200 µm OD x 75 µm ID fused silica capillaries
• Self coating separation polymer
30 | IntegenX 10/4/2010
TrEKI (Transient Electro-Kinetic Injection)
• Capillary coupled with tube that
is interfaced to MOVe fluidics
• Each channel has
capillary/tubing/electrode
assembly
•Dual electrodes
Patent pending
31 | IntegenX 10/4/2010
TrEKI Sample Injection and Run
Patent pending
32 | IntegenX 10/4/2010
Diode Array Detector
Four Channel Detector
Optical Path
Single Channel Detector
Patent pending
33 | IntegenX 10/4/2010
Separation Performance
Allelic ladder run on four channel breadboard system
34 | IntegenX 10/4/2010
Separation Performance
120
140
425
35 | IntegenX 10/4/2010
475
Separation Performance
36 | IntegenX 10/4/2010
Separation Performance
37 | IntegenX 10/4/2010
DevLink Integration Software
Generic Instrument
Control Panel
(Other Interfaces)
TCP/IP
TCP/IP
User Layer
(Normal Login Account)
Service Layer
(High-priority/privileged runtime
environment)
Generic Instrument
Driver
State
Machine
Instrument
Abstractions
USB
DLL
Shell API
Robot
DAQ
External Analysis
XML Machine
Descriptors (datadriven behavior
models)
External World
(Everything else connected to the computer)
38 | IntegenX 10/4/2010
DevLink System Control Software
39 | IntegenX 10/4/2010
Early Four Channel Data
11/10/2009 - Fully-Integrated Run (System #4). Run time ~3hrs.
Channel 1 - Sample 18M
16 alleles
Channel 2 - Sample 1F
16 alleles
Channel 3
Sample
24M
Channel
3 - Sample 24M
16 alleles
Channel 4 - Sample 18M
16 alleles
40 | IntegenX 10/4/2010
Integration of GeneMapper ID-X
Data automatically inserted to GeneMapper ID-X
over TCP/IP from IntegenX TraceAnalyzer.
GeneMapperID Connector
Allele Report, delivered
to requesting computer
via TCP/IP
41 | IntegenX 10/4/2010
GeneMapperID-X automatically
runs with a DevLink service
implementing TCP/IP interface.
System Development
System tuning
Total possible loci
Not called
Off ladder
Correct calls
Incorrect calls
16 loci
736
22
3.0%
22
3.0%
663
90.1%
28
3.8%
13 loci+amelogenin
644
1
0.2%
11
1.7%
624
96.9%
8
1.2%
Final system test
Total possible loci
Not called
Off ladder
Correct calls
Incorrect calls
42 | IntegenX 10/4/2010
16 loci
608
16
0
591
1
2.6%
0.0%
97.2%
0.2%
13 loci+amelogenin
532
0
0%
0
0%
532
100%
0
0%
Four Channel Breadboard System Test Summary
Demonstration and test of unit at IntegenX
Total of 40 samples
%
Complete call (16 Loci)
37
93%
> 13 Loci
38
95%
< 13 Loci
0
0%
Mechanical Failure
2
5%
Other Failures
0
0%
Subject matter experts
representing the
customer observed
and ran four channel
system at IntegenX
Pleasanton.
Installation and test at customer site
Total of 53 samples
%
Complete call (16 Loci)
22
41%
> 13 Loci
39
74%
< 13 Loci
6
11%
Mechanical Failure
7
13%
Other Failures
1
1.9%
43 | IntegenX 10/4/2010
IntegenX installed at
customer site, training
and hand-off to
customer for testing.
Sample 24M, All Calls Correct
44 | IntegenX 10/4/2010
Sample 18M, All Calls Correct
45 | IntegenX 10/4/2010
Liquid Blood Samples
•
•
•
47 | IntegenX 10/4/2010
Full profile using PowerPlex chemistry and
DNA IQ from 15 µL fresh blood
System run time = 2 hrs
Proteinase K in lysis buffer
Eight Channel Development Program
• Field prototypes ready in Q3 2011
• Sample Cartridge
 2 x 4 channel
 On cartridge reagent storage
 Simple installation
• Separation and Detection System
 Reproducible field replacement
 Simple connections at the cathode and anode
 High sensitivity detection
Patent pending
48 | IntegenX 10/4/2010
Summary
• Sample-to-answer results in two hours
• Four channel system has made correct calls on
hundreds of samples
• The system has been placed externally and used
by minimally trained outside experts
• Eight channel product in development for release
in Q3 2011
49 | IntegenX 10/4/2010
The Team

Helen Franklin - Project Manager

Bill Nielsen (VP Systems Integration) - Systems Design & Architecture

Stefanie Pagano - Systems Development and Integration

Greg Bogdan - Systems Hardware Engineer

Rich Belcinski - System Software Control & Data Processing

Joe Carrow - Engineering Design

Omar El-Sissi - Systems & Process Integration

Jacklyn Buscaino – Reagents and Process Integration

Jim Knittle - Injection, Separation & Detection

Boris Kobrin - Microfabrication

Debi Frost - Operations and Manufacturing

Howie Goldstein - Commercial

John Storella - Legal
50 | IntegenX 10/4/2010