Automation:a key role for Molecular Diagnostic EvolutionPatrick Merel, PhDUniversity Hospital of Bordeaux, France

Biomedical Innovation Platform (PTIB)

pmerel@mac.com 1

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Molecular Diagnostic Projections

From IBC Life Sciences’ Discovery2Diagnostics conference, Oct. 08

2007 micro arrays market

$160 millions (3%)

2007 MDx market

$3 billions

credits: Harry Glorikian, managing partner of Scientia Advisors

2012 micro arrays market

$660 millions (9%)

2012 MDx market

$7 billions

Projections for 2012

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MDx Growth FactorsDemand for molecular diagnostics and gene detection products is mainly determined by the

volume of tests performed in clinical labs

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MDx Growth Factors

The need for automated and easy-to-handle techniques

Optimized sample preparation, analysis, and data evaluation

Techniques that would diagnose disease condition and medical disorders quickly for quick therapy decisions

Availability of molecular diagnostic tests for monitoring the therapeutic efficacy of expensive drugs

New diagnostics tests, primarily in the infectious disease application area

Nanobiotechnology and biochips are also expected to drive future growth

Genomics and proteomics are the major drivers of the molecular diagnostic market

Demand for molecular diagnostics and gene detection products is mainly determined by the volume of tests performed in clinical labs

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MDx Growth FactorsA major step in MDx evolution: Realtime PCR technologies

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MDx Growth Factors

Simplification of the PCR process

no more post-PCR procedures, ie. gel electrophoresis or hybridization

Single instrument based, ie. a realtime PCR thermocycler

Combination of a realtime PCR thermocycler and a Nucleic Acid (NA) extraction instrument for a potential full solution in MDx

rapid development of automated NA extraction platforms

slow progression of automated realtime PCR instruments

new area of interest, fully automated platforms including NA extraction and rtPCR

A major step in MDx evolution: Realtime PCR technologies

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NA extraction automation evolution

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NA Extraction Procedure Evolution

From single sample process to microtiterplate

by centrifugation

to generic robotic workstation

using vacuum manifolds

to magnetic beads based procedures

Biomérieux’s Boom patent and PSS Magtration technology being the major drivers

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Dedicated NA extraction platform evolution

Interstingly, this progress in NA extraction automation did’nt come from the diagnostic industry and has firstly addressed, the research and academic market.

Thus, even usefull, often poorly adapted to rout ine d iagnost ic requirements

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Dedicated NA extraction platform evolution

Interstingly, this progress in NA extraction automation did’nt come from the diagnostic industry and has firstly addressed, the research and academic market.

Thus, even usefull, often poorly adapted to rout ine d iagnost ic requirements

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Dedicated NA extraction platform evolution

The Diagnostic Industry have started to address routine MDx demands (high throughput), FDA and CE-IVD labeling more recently. With the early presence of Gen-Probe TIGRIS in blood transfusion setups.

New players are now pushing forward the ru les with fu l ly integrated platforms.

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Dedicated NA extraction platform evolution

The Diagnostic Industry have started to address routine MDx demands (high throughput), FDA and CE-IVD labeling more recently. With the early presence of Gen-Probe TIGRIS in blood transfusion setups.

New players are now pushing forward the ru les with fu l ly integrated platforms.

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Dedicated NA extraction platform evolution

The Diagnostic Industry have started to address routine MDx demands (high throughput), FDA and CE-IVD labeling more recently. With the early presence of Gen-Probe TIGRIS in blood transfusion setups.

New players are now pushing forward the ru les with fu l ly integrated platforms.

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Realtime PCR instrumentation

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Realtime PCR instrumentation

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Realtime PCR instrumentation

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Molecular Diagnostic Instrument Challenges

While it is difficult nowdays to run MDX procedures without an automated platform, some challenges remain to be fully addressed:

- from blood to buccal cells, sample type and volume are numerous. Hard to find a single and unique solution

- no clear standard for platform capacity, sample batch size, batch vs continuous flow, consolidation on core facilities vs point of care ..etc

- realtime PCR supremacy from infectious diseases to genetic testing but difficult to fully automate

- sequencing and micro-arrays based diagnostic still high cost and time demanding

- SNP detection required in many potential personalized medicine assays still looking for the ultimate gold standard for routine testing

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From 8 samples

ABI very 1st automated NA extractor

to 96 samples various platforms

from Tecan, Beckman Coulter, Hamilton to Qiagen

back to 24 batch with Biomérieux Nuclisens?

which has proved to be popular

to continuous flow for 1 to 72 samples

with Roche Cobas AmpliPrep

while Roche Magnapure 32 sample size

being of a major impact in labs

and continuous flow from 1 to 96 samples

with Qiagen QiaSymphony, QiaEnsemble?

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continous flow

Continuous flow for NA extraction procedures

the answer for multiple sample batch sizes

a progress toward random access instruments?

continous innovation

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Qiagen

Gen-Probe

Gen-Probe

Roche Diagnostic

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continous flow

Continuous flow for NA extraction procedures

the answer for multiple sample batch sizes

a progress toward random access instruments?

2008 2009 2010?

continous innovation

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Qiagen

Gen-Probe

Gen-Probe

Roche Diagnostic

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What’s next with NA extraction automation

No more extraction steps?microfluidics technologies potential

No more PCR?

Fully integrated instrumentation

Integration with Clinical Chemistry Automation?

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Cepheid GeneXpert & Infinity

a precursor in microfluidics and fully automated process for MDx in a single instrument

From 1 sample to 16

on the GeneXpert

to random access

on the Infinity

Cepheid

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Cepheid GeneXpert & Infinity

a precursor in microfluidics and fully automated process for MDx in a single instrument

From 1 sample to 16

on the GeneXpert

to random access

on the Infinity

Cepheid

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Cepheid GeneXpert & Infinity

a precursor in microfluidics and fully automated process for MDx in a single instrument

From 1 sample to 16

on the GeneXpert

to random access

on the Infinity

Cepheid

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Microfluidics in MDxHandyLab Jaguar, integrated DNA extraction and realtime PCR process on Microchips

Iquum Liat Analyzer and Liat Flow Cycler, a new concept of Lab-in-a-tube for realtime PCR based assays

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Nanosphere Verigene System for FDA approved nanotechnology based MDX assays.

Direct hybridization, no PCR steps required

protein assays compatible technology

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A microfluidic cartridge for multiplexed clinical assays

Fluidic ManipulationDeliverySeparationMixingConcentration

Multiplex Molecular Analysis Genetic assay ImmunoassayElectrolyte, Ions

Universal Specimen Urine Saliva Whole blood Serum, PlasmaCulture media

Superior Advantages No PCR Minutes to result Low LOD(fM, pg/ml)Dynamic range

Revolutionizing molecular analysis

Target bindingSample delivery

New Technology for new process in MDx

Electrokinetic Concentrator

brief and rapid sample prep. Almost no DNA extraction step

BioElectronics with realtime flow impedance monitoring

<10 min total assay time

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A microfluidic cartridge for multiplexed clinical assays

Fluidic ManipulationDeliverySeparationMixingConcentration

Multiplex Molecular Analysis Genetic assay ImmunoassayElectrolyte, Ions

Universal Specimen Urine Saliva Whole blood Serum, PlasmaCulture media

Superior Advantages No PCR Minutes to result Low LOD(fM, pg/ml)Dynamic range

Revolutionizing molecular analysis

Target bindingSample delivery

New Technology for new process in MDx

Electrokinetic Concentrator

brief and rapid sample prep. Almost no DNA extraction step

BioElectronics with realtime flow impedance monitoring

<10 min total assay time

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MDx-Automation-Connectivity

Portable device

Security protection1

User authentication2

Data encryption3

Authetication4

Device LevelWireless

communication

Telecommunication

Data Transmission

SSL encryption3

Integrity control5 Digital certificates2

Transmission

Physical cable connection

Clinical data station

System Level

Security protection1

User authentication2

Data encryption6

Audit control7

1-7: Security Standards for the Protection of Electronic Protected Health Information

New concept of MDx and telecommunication

Revolutionizing molecular analysis

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Introducing clinical chemistry instrumentation parts in MDx platform

From Clinical Chemistry Environment to MDx

The Vidiera NSP

Primary tubes on racks

NA extraction by filtration

Qiagen chemistry

Quantification by spectrometry

Normalization

PCR setup

Archieving

Beckman Coulter

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Connectivity in High Throughput MDx settings

Abbott Molecular

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Siemens

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Connectivity in High Throughput MDx settings 20

Roche Diagnostic

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Connectivity in High Throughput MDx settings 20

Roche Diagnostic

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MDx new fields of application

Personalized medicine has brought attention onto SNP analysis procedures in MDx

Oncology developments have made gene expression analysis a growing field for MDx

Emerging infectious diseases, drug resistance monitoring, NextGeneration Sequencing instruments have renewed the interest for sequence based MDx

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MDx outside realtime PCR

SNP and Gene Expression assays are missing consensus technology in routine diagnostic, except for low complexity assays that are making use of realtime PCR.

Sequencing technologies mature, but processing is long and missing automation.

The promise of NextGen sequencing technologies.

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SNP analysis MicroArrays SequencingMonoplex to Low Multiplexing Realtime PCR

Custom Multiplex FlexibilityABI-SNPlexSequenom-iPlex-MassArrayBeckman Coulter-SNPStream

Low to None Flexibility but very high MultiplexingiLLumina-Golden Gate iLLumina-InfiniumAffymetrix-GeneChip

Affymetrix-Roche CYP450very 1st FDA available kit

Low-Cost solutions (manual)Greiner BioOne CE-IVD chipsEppendorf BioChipLGLife SciencesDrChip

Low-Cost solutions (semi-automated)ClonDiag-Genomica CE-IVD chipsAutogenomics IVD, RUOOsmetech IVDHybribioBioCore

Newest alternatives (fully automated)Nanosphere CE-IVD GeneFluidics RUO

Capillary sequencing drivenstill technical demandingnot fully automated

Core facility oriented task

Whole Genome Sequencing- NextGeneration Sequencing technologieslot of interestfuture of modern medicine, personalized medicine

Still work to do on:cost/patientprocess automationBioIT process

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Increasing interest for automated micro-array processing

Greiner BioOne ClonDiag Genomica

Autogenomics

Nanosphere

Osmetech

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Roche GS-FLXti0.4 Gb/run

1m reads @ 400b

$7768/run 0.4Gb$19.41/Mb$648k/inst.

Illumina GA25-10 Gb/run

60m reads @ 50b

$8250/run 5Gb$0,33/Mb$460k/inst.

AB Solid 3.010-20 Gb/run

100m reads @ 50b

$6873/run 5+5Gb$0,69/Mb

$599k/inst.

Illumina GA2:Setup time: 2-3 d

6-11 Gb/runRun time: 3-6 dimages: 900 GB

Primary Analysis: 350 GBPA CPU time: 100 hrsFinal File Size: 75 GB

Roche GS-FLXti:Setup time: 3-4 d

0.4Gb/runRun time: 10 hrsimages: 27 GB

Primary Analysis: 15 GBPA CPU time: 220 hrs

Final file size: 4 GB

NextGen Sequencing

the future of MDx?

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AB Solid 3.0:Setup time: 3-5 d

5-12.5 Gb/run/slideRun time: 3.5-10 d

images: 2.5 TBPrimary Analysis: 750 GBPA CPU time: in run time

Final file size: 140 GB

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Roche GS-FLXti0.4 Gb/run

1m reads @ 400b

$7768/run 0.4Gb$19.41/Mb$648k/inst.

Illumina GA25-10 Gb/run

60m reads @ 50b

$8250/run 5Gb$0,33/Mb$460k/inst.

AB Solid 3.010-20 Gb/run

100m reads @ 50b

$6873/run 5+5Gb$0,69/Mb

$599k/inst.

Illumina GA2:Setup time: 2-3 d

6-11 Gb/runRun time: 3-6 dimages: 900 GB

Primary Analysis: 350 GBPA CPU time: 100 hrsFinal File Size: 75 GB

Roche GS-FLXti:Setup time: 3-4 d

0.4Gb/runRun time: 10 hrsimages: 27 GB

Primary Analysis: 15 GBPA CPU time: 220 hrs

Final file size: 4 GB

NextGen Sequencing

the future of MDx?

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AB Solid 3.0:Setup time: 3-5 d

5-12.5 Gb/run/slideRun time: 3.5-10 d

images: 2.5 TBPrimary Analysis: 750 GBPA CPU time: in run time

Final file size: 140 GB

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Numerous fields of applications

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HIV Ultra Deep Sequencing for drug resistance monitoring

4000X coverage

minority populations detection

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NextGen Sequencers Workflow

Workflow 3-4 days (setup) + 1 day (run)

1. Generation of a single-stranded template DNA library (~8-16 hours)

2. Emulsion-based clonal amplification of the library (~8 hours)3. Data generation via sequencing-by-synthesis (9 hours)4. Image and Base calling analysis (~8 hours)5. Data analysis using different bioinformatics tools

+IT steps:

GS-FLX Software▪GS Reference Mapper▪GS De Novo Assembler▪GS Amplicon Variant Analyzer Third Party Software

Roche GS-FLX:

•Long Single Reads / Standard Shotgun (required input = 3–5μg,5μg recommended)~1,000,000 single reads with an average read length of 400 bases

•Paired End Reads (required input = 5μg @25 ng/μl or above, in TE; >10kb)◦3K Long-Tag Paired End Reads. Sequence 100 bases from each end of a 3,000 base span on a single sequence read (Figure). Co-assemble GS FLX Titanium shotgun reads with 3K Long-Tag Paired Ends reads from Standard series runs.

•Sequence Capture (required input = 3–5μg)◦Roche NimbleGen Sequence Capture using a single microarray hybridization-based enrichment process.

•Amplicon Sequencing (1-5ng or 10-50ng)◦The DNA-sample preparation for Amplicon Sequencing with the GS FLX System consists of a simple PCR amplification reaction with special Fusion Primers. The Fusion Primer consists of a 20-25 bp target-specific sequence (3' end) and a 19 bp fixed sequence (Primer A or Primer B on the 5' end).

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NextGen Sequencers Workflow

Workflow 3-4 days (setup) + 1 day (run)

1. Generation of a single-stranded template DNA library (~8-16 hours)

2. Emulsion-based clonal amplification of the library (~8 hours)3. Data generation via sequencing-by-synthesis (9 hours)4. Image and Base calling analysis (~8 hours)5. Data analysis using different bioinformatics tools

+IT steps:

GS-FLX Software▪GS Reference Mapper▪GS De Novo Assembler▪GS Amplicon Variant Analyzer Third Party Software

Roche GS-FLX:

•Long Single Reads / Standard Shotgun (required input = 3–5μg,5μg recommended)~1,000,000 single reads with an average read length of 400 bases

•Paired End Reads (required input = 5μg @25 ng/μl or above, in TE; >10kb)◦3K Long-Tag Paired End Reads. Sequence 100 bases from each end of a 3,000 base span on a single sequence read (Figure). Co-assemble GS FLX Titanium shotgun reads with 3K Long-Tag Paired Ends reads from Standard series runs.

•Sequence Capture (required input = 3–5μg)◦Roche NimbleGen Sequence Capture using a single microarray hybridization-based enrichment process.

•Amplicon Sequencing (1-5ng or 10-50ng)◦The DNA-sample preparation for Amplicon Sequencing with the GS FLX System consists of a simple PCR amplification reaction with special Fusion Primers. The Fusion Primer consists of a 20-25 bp target-specific sequence (3' end) and a 19 bp fixed sequence (Primer A or Primer B on the 5' end).

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Conclusion

Realtime PCR has been a revolution for MDx

New procedures and lab organization

A 2 step process: NA extraction and PCR/detection

Bringing a new capacity for simple automation

Huge development and options for NA extraction

mag. beads procedures as a standard

Integration of realtime PCR step through full automated instrument

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ConclusionDevelopment of fully integrated instrument for MDx is bringing new concept and technologies

Microfluidics, bioelectronics, nanotechnologies

Telecommunication integration vs clinical chemistry settings integration

The needed evolution of SNP procedures

Finally low cost micro-array platforms with options for semi and full automation

The next upcoming revolution in MDx: making use of NextGeneration instrumentation in routine clinical diagnostic settings

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