Post-PCR Procedures Automationin Molecular Diagnostic

part 3

Patrick MerelBiomedical Innovation Platform (PTIB), Pessac, France

pmerel@mac.com

1

Options for Post-PCR procedures• Fluorescence based detection

• Realtime PCR procedures

• New possibilities in automation

Gel Electrophoresis

Difficult

Automation through Capillary Electrophoresis (CE)

Microfluidics progress, CE chips

Sequencing

Complete automation: difficult but almost possible

Automation through CEquencing

2

Former options for Post-PCR procedures

Reverse Dot-Blot

most popular approach

favorite commercial procedure

Microtiterplate compatible

Automation of Reverse Dot-Blot

Few solutions, Roche, Abbott, …

MicroArrays, Chips automation

Generic Robotic Workstations for simple automation of reverse dot-blot protocols

3

HLA Typing by PCR/SSO on strip

REVERSE-DOT-BLOT on membrane: ‘Strip Assay’4

Automation of HLA-Typing by Reverse Slot-Blot

5

Invitrogen-Dynal, HLA RELI-SSO with the AutoRELI-48 and the RELI-Scan.

Innogenetics, HLA, HPV, CFTR with the Auto-Lipa48.

Automates Probe Hybridisation and Strip Detection

Eliminates Strip Handling and Manual Data Entry

Easy to Use Interpretation Software Combined with a comprehensive Results Management Package

Automation of reverse hybridization procedures

6

Dedicated PCR+Post-PCR workstation: the Roche Cobas Amplicor

Automation of hybridization procedures

7

Perspective for Hybridization ProceduresMicroArray-based Technologies

Liquid Arrays

Solid surfaces Arrays

Affymetrix and alike DNA Chips technology

New generation of IVD microarrays

The in-house way

What about automation?

8

Hybridization on Beads: the future of hybridization procedures?

9

❖ Luminex technology❖ Beads of various colors❖ Different probes on each color coded beads❖ Multiple colors combination detection

❖ Nanoparticules and alike❖ Quantum Dots, Nanosphere, …etc

❖ new: Digital particules

Luminex based Technologies

10

Numerous applications from various vendors and partnersAsuragenApplied Cytometry SystemsBMDBio-Rad DiagnosticsBio-Rad Life ScienceEraGen BiosciencesFisher Healthcare INOVA Diagnostics, Inc. Invitrogen MICROBIONIX GmbHMarligen Biosciences, Inc. Millipore Corporation (LINCO & Upstate) MiraiBioMultimetrix GmbHOne Lambda, Inc.PanomicsPerkinElmerQIAGENRadixTepnel LifecodesZeus Scientific, Inc.

Directly from LuminexCorp

II. Multiplex ASPE/TSPEThe PCR reaction is then subjected to a primer extension step that is specific for the allele or the infectious agent that is being analyzed. The 5’ end of the ASPE primer is attached to an xTAG universal tag sequence.

IV. Universal Array SortingThe 5’ universal tag sequence is hybridized to the complementary anti-tag sequence coupled to a particular xMAP bead set.

V. DetectionThe hybridized beads are read by the Luminex System

I. Multiplex PCR

xTag Technology

Luminex based Technologies

The Luminex High Throughput Screening System (HTS) is designed to perform hundreds of thousands of individual bioassays per day.

Supports 96 or 384 well plates; Compatible with front-end plate handling robotics; Uses the 64 bead set, not the 100 bead set; For Research use only, not for use in Diagnostic procedures

The Luminex 100/200 System are flexible analyzers based on the principles of flow cytometry.

Multiplexing: Up to 100 analytes per well.

11

The FLEXMAP 3D and MagPix are the latest multiplexing systems launched by Luminex.

Increased Multiplexing: from 50 to 500 analytes per well.

Progress in Luminex Technology Automation

12The Biorad, BioPlex 2200: A Fully-Automated, Random Access, Multiplex Platform. EIA based until now.

The following Products for the BioPlex 2200 are

Available:Syphilis IgGEpstein-Barr Virus (EBV)IgG and IgMANA MDSSCurrently in Development:Syphilis IgM*ToRC IgG*ToRC IgM*Lyme IgG/IgM*Immunity IgG*HSV 1/2*Vasculitis*Gastrointestinal (GI)*Anti-Phospholipid Syndrome*Rheumatoid Arthritis*

Nanosphere Technology

❖Ultra-sensitive detection of multiple proteins and nucleic acids simultaneously

❖Direct genomic detection without amplification or enzymes

❖5-6 orders of magnitude more sensitive than ELISA based methods for proteins

The Verigene System consists of two instruments: the Auto Processing System (APS) and the Verigene ID, which makes it easier than ever to run a variety of assays on nucleic acids and proteins with the simplicity of a sandwich assay.

The assay involves a 2-step process similar to an ELISA sandwich assay:

After isolation from a sample, DNA is hybridized to both nanoparticle probes and capture strandsSilver is catalyzed on the gold resulting in six orders of magnitude amplification of signal

13

Nanosphere Technology

14

Step 1Enter patient ID and test requisition (duration: 1 minute)Start with the Verigene ID by entering sample identification (manually or via barcode). Scan the assay barcode on the slide with the integrated wand.Intuitive Touchscreen and Software･Guides the user step-by-step through the testing process･Uses simple, icon-based instructions that minimize data entry･Tracks the samples, slides and reagent packs via barcode throughout the assay process

Step 2Pipette sample into test cartridge (duration: 1 minute)No PCR is necessary for DNA or RNA. Just pipette the prepared sample into the self-contained test cartridge.Test Cartridge･Eliminates sample contamination･Maintains consistency of assay process through microfluidics･Minimizes tech-to-tech variability by reducing manual pipetting

Step 3Insert test cartridge into Verigene APS (duration: ~90 minutes)Once the prepared sample is in the test cartridge, insert it into the Verigene APS. This automates the ClearRead nanoparticle detection method through integrated fluid processing.Verigene Auto Processing System (APS)･Operated, monitored and verified by the Verigene ID･Contains reagent pack required for processing･Simple load and run capability

Step 4Insert processed test cartridge unit into Verigene ID (duration: 3 minutes)Remove the slide from the test cartridge and insert it into the Verigene ID. Through the touch-screen, simply select and print the results.

Total duration: <95min for F2, F5, MTHFR, CFTR, HFE for Warfarin, CYP2C19

for multiplex A/B Flu+RSV

The Verigene System consists of two i n s t r umen t s : t h e A u t o Processing System (APS) and the Verigene ID, which makes it easier than ever to run a variety of assays on nucleic acids and proteins with the simplicity of a sandwich assay.

Nanosphere latest automated platform: The Verigene SP

15

The Verigene® SP is a bench top, free-

s t a n d i n g i n s t r u m e n t w i t h a s i n g l e ,

independent sample processing module.

Random access, mult i funct ional test

processing, including nucleic acid extraction,

reverse transcription (if necessary), target

amplification (if necessary), and target

identification and analysis in a Verigene® Test

Cartridge. 

Nanostring Molecular BarcodesT h e t e c h n o l o g y u s e s molecular "barcodes" and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction.

Each color-coded barcode is attached to a single target-specific probe corresponding to a gene of interest. Mixed together with controls, they form a multiplexed CodeSet.

The Reporter Probe carries the signal; the Capture Probe allows the complex to be immobilized for data collection.

Gene Expression CodeSets for the nCounter Analysis System offer a cost-effective way to analyze the expression levels of up to 800 genes s imu l taneous ly w i th sens i t i v i t y comparable to qPCR. +miRNA + CNV

16

Applied BioCode: digital arrays

Applied BioCode, Inc. (ABC) has combined photolithographic digital barcodes with immuno- and molecular chemistry to create a new, patented Barcoded Magnetic Bead (BMB) technology.

Barcoded Magnetic Bead has one of the highest multiplex capacities available (up to 1,024 targets/assay). Optically bar-coded beads are mass produced at low cost by well established semiconductor processes. These beads are functionalized with nucleic acids, proteins or other probe molecules, allowing highly multiplexed assays to be carried out in homogeneous or heterogeneous media.

The BMBs' barcode patterns give a high-contrast transmitted signal and no fluorescence background, allowing the barcode to be identified easily and accurately, with near 100% decoding accuracy.

17

MicroArrays in Diagnostic

Pathogens characterizationWater-born virus, bacteria, genotyping

Gene Expression monitoringOncology

Genotyping

SNP detection

Pharmacogenetics/PharmacogenomicsDisease predisposition, drug metabolism, drug design and development

18

Affymetrix GeneChip :For genotyping and expression monitoring

GeneChip Probe Array HIV PRT GeneChip, an array with more than 15,000 different probes

« Wafer-scale chemical synthesis » GeneChip Probe Array synthesis principle

DNA Chips :from synthesis to commercial products19

Proprietary GeneChip software controls the Affymetrix fluidics station and scanner functions, captures the fluorescent image from the probe array, extracts data from the image and provides subsequent data analysis.

GeneChip Probe Array

GeneChip Fluidics Station

GeneArray Scanner

GeneChip Software

Affymetrix GeneChip20

The GeneChip® HIV PRT assay used in conjunction with the GeneChip analysis system enables rapid, high accuracy sequence analysis on the HIV-1 protease and reverse transcriptase (codons 1242) genes.

Affymetrix HIV GeneChip21

IVD DNA Array22

AmpliChip CYP450 from Roche Molecular Diagnostics a partnership with Affymetrix

Automated robotics for microarrays

NanoChip® Loader

4 NanoChip ® Cartridges per Loader

NanoChip ® Electronic Microarray15 samples X 4 cartridges = 60 sample per Loader

run

Nanogen’s CF ASRSingle sample per six test sites

Genotype for ∆F508 on initial runScreen for 24 other mutations

15 patients per cartridge60 patients per run (4 chips)

Also available as ASR: ASPA, ApoE, HFE, Factor V/Prothrombin, Assay ToolBox

23

Multi-Processing of individual arrays: Osmetech

24

Osmetech eSensor (previously CMS division from Motorola)

At the core of the eSensor detection technology are DNA fragments attached to electrodes on the surface of a small circuit board.

This DNA microarray is the basis of detection by the eSensor System for any DNA sequence. Each electrode is electronically active and detects a different DNA sequence.

Finding the complimentary sequence in the target DNA generates a characteristic electrical signal.

Each eSensor DNA Detection System cartridge can detect several different DNA targets at once, providing a cost-effective platform for complex analysis.

eSensor CF test: 23 mutations tested for in the ACOG/ACMG-recommended cystic fibrosis carrier screening panel.

Osmetech XT-Technology

New eSensor® XT-8 System

§ Modular design–expandable from 1 to 3 towers (8 to 24 slots)

§ Improved software with touch-screen interface

§ On-chip mixing for rapid hybridization

§ Random access§ 4-label discrimination capability

CYP 450 2C9 and VKORC1 polymorphisms IVD assays25

Osmetech Key Technology Features• Homogeneous assay

– No post-PCR sample purification– No post-hybridization wash steps– Insensitive to interferences from sample matrix

• Complex samples (whole blood, tissue extracts)• Redox-active compounds (ascorbate, acetaminophen)

• Electronic detection– Simple, inexpensive instrumentation– No optics or mechanical parts– Inherent ratiometric data analysis eliminates need for calibration26

Autogenomics TechnologyThe INFINITI™ Analyzer is an automated, multiplexing, continuous flow, random access microarray platform that integrates all the discrete test processes such as sample handling, reagent management, hybridization, stringency and detection for the analyses of DNA into a totally self-contained system.

The BioFilmChip™ Microarray is a novel, film based microarray, which consists of multiple layers of hydrogel matrices about 8-10 µm thick on a polyester solid support.

27

Autogenomics MenuThe BioFilmChip microarray is based on polyester film that is coated with a proprietary

multi-layered technology for DNA and protein analysis.

Arrays will have densities of less than 100 spots/chip and will be primarily assay specific.

Analysis can be performed utilizing:

Hybridization assay, Primer Extension assay, In situ hybridzation, Immunoassay (Competitive / Sandwich)

Available RUO Menu: Available CE Menu: Available IVD Menu:

Ashkenazi Jewish PanelCYP450 2C19CYP450 3A4CYP450 3A5MDR-1HPV GenotypingHPV QuadNAT-2CHEK-2EGFR, 5-FU (mutations)MTB-DR (Drug Resistance)Respiratory Viral PanelSTD PanelNTM

28

Factor IIFactor V LeidenFactor II-V Leiden PanelWarfarin AssayCYP2C19

MTHFRFII-FV-MTHFR Panel CFTR31 CYP450 2C9-VKORC1CYP450 2D6UGT1A1 (Irinotecan)HPV GenotypingHPV Quad FLU A-sH1N1

Clondiag TechnologyArrayTube, ArrayStrip, Assay Processor

Unique feature of the AT platform is the combination of probe array chip and micro reaction vial into one single platform, allowing easy and reliable array handling with conventional lab equipment.

The probe arrays are made up of a transparent glass chip supporting a filter membrane like coating with the embedded probe molecules.

Probe deposition is performed by applying state-of-the-art spotting techniques (for the fabrication of oligonucleotide, polynucleotide and protein arrays) and by oligonucleotide in-situ synthesis using CLONDIAG's proprietary micro wet printing process (µWP).

With the ArrayStrips, CLONDIAG'S reliable ArrayTube platform is now available in a 96 well microplate compatible format.

The AP cartridge is integrating target amplification, hybridization and signal detection in a single device.

part of Inverness Medical Innovation

29

Clondiag AT Procedure

The ArrayTube Workstation ATS is designed for fast and accurate detection and analysis of all ArrayTube Tests.

In combination with CLONDIAG's robust colorimetric detection method, ATS allows the analysis of the test specific interaction pattern on the AT-array carrying multiple diagnostic features wit

30

Clondiag Platform by Genomica

31

1-This microarray (3x3 mm) includes 120 cDNA spots immobilised on a polymer-coated slide which hybridise with specific DNA sequences from the sample.

2-Amplified DNA is marked with biotin and added to the Array Tube

3-These labelled products recognize the specific probes on the microarray during hybridisation, and are immobilised

4-The microarray is incubated with a streptavidin-peroxidase conjugate which binds to the amplified products via a reaction between the streptavidin and the biotin label

5-In the presence of tetramethylbenzidine (TMB), the peroxidase activity of the conjugate induces the appearance of an insoluble product which precipitates at the hybridisation sites on the microarray.

6-Final image. Dark spots indicate hybidization.

Genomica middle high throughput platform

With the CAP (Clinical Array Processor) and the CAR (Clinical Array Reader) Up to 12 ArrayStrips can be inserted into one microplate frame allowing the parallel testing of up

to 96 samples.

In contrast to common microplate test formats, each well containing a microarray enables the analysis of one sample for multiple parameters in parallel.

32

Clondiag Assay Processor

The AssayProcessor Station is designed for easy and rapid AP testing in the doctor's office or in the routine lab. After sample injection, the AP is placed into the Station, and the test is started via simple touch screen command. All assay steps run automatically controlled without any further user interaction.

The size of a test strip the AP cartridge is integrating target amplification, hybridization and signal detection in a single device. Central part of the reaction cartridge is a high quality micro probe array enabling the parallel analysis of multiple test parameters in a single step reaction.

APS Features

- fully automated AP test processing for both nucleic acid and serological AP assay formats

- robust fluorescence detection concept for qualitative and quantitative measurements- integrated data matrix reader for automated identification of test and test parameters- integrated PC and built-in touch screen, interface for external data transfer and printing- ease of operation- complies with current IVD guide

APS control unit with 4 processing modules in parallel

33

MetaBone

COLLAGEN TYPE1 - SpIVITAMIN D RECEPTOR – FokI y BsmIESTROGEN RECEPTOR - PvuII y XbalICALCITONIN RECEPTOR – AluI

predisposition to suffer endocrine system pathologies, rheumatological alterations and osteophatologies.

HPV

35 HPV genotypes

predisposition to suffer endocrine system pathologies, rheumatological alterations and osteophatologies.

Genomica clondiag-based MenuPneumoVir - Respiratory Virus Panel (17)

Influenza A Influenza B RSV A RSV B Influenza C Parainfluenza 1 Rhinovirus AdenovirusParainfluenza 2 Parainfluenza 3 Parainfluenza 4a Parainfluenza 4b Echovirus Bocavirus Coronavirus Metapneumovirus AMetapneumovirus B

All products, CE-IDV

34

Greiner Bio-One TechnologyThe HTA™Slide is a standard 25 x 75 mm glass slide partitioned into 12 flat compartments, each with a printable surface of 6 x 6 mm. Therefore 12 samples can be processed simultaneously.

Greiner Bio-One’s HTA™Plate is an innovative and reliable platform for diagnostic applications of microarrays.

•96 x 36 mm2 printable area •4 individual sections with 24 wells each •Removable wash collars •Low autofluorescence •Barcode labeling

35

Greiner Bio-One Procedure

1. Sample collection 10 mins

2. DNA extraction 30 mins

3. PCR120 mins

4. Hybridisation 15 mins at RT

5. Washing 2 mins

6. Scanning 10 mins

7. Evaluation 5 mins

36

37CheckScanner™Fast and Reliable Analysis

Automatic analysis of four HTA™Slides (or one HTA™Plate under development) with barcode identification

Data analysis and reporting with CheckReport™Software

38

PapilloCheck®

OC OC OC OC OC PC PC PC PC PC

HC HC HC HC HC SC SC SC SC SC

6 45 45 45 45 456 6 6 6

11 51 51 51 51 5111 11 11 11

16 52 52 52 52 5216 16 16 16

18 53 53 53 53 5318 18 18 18

31 56 56 56 56 5631 31 31 31

33 58 58 58 58 5833 33 33 33

35 59 59 59 59 5935 35 35 35

39 66 66 66 66 6639 39 39 39

40 68 68 68 68 6840 40 40 40

42 70 70 70 70 7042 42 42 42

43 73 73 73 73 7343 43 43 43

44 82 82 82 82 8244 44 44 44

Orientation Control for optimised analysis PCR Control

Hybridisation Control Sample Control

• Type-specific HPV DNA probes in replicates of 5

• Print Control• Negative Control

Greiner Bio-One MenuParoCheck®: Inflammation of the gums and the paradontium are among the most frequent human diseases. ParoCheck® is the first biochip approved as an “In-Vitro-Diagnostic” (IVD), and with it up to 20 different characteristic indicative pathogens can be detected.

CarnoCheck®: With this biochip 8 different animal species can be unequivocally identified in foods or other products.

PapilloCheck®: Early recognition of human papillomaviruses (HPVs). With this newly developed biochip PapilloCheck® a total of 18 of the “high risk” HPV types and 6 “low risk” HPV types can be identified.

coming: CF chip (>50 mutations)

•PapilloCheck DNA chip comprises 12 wells, A1 – B6 defined by elevated rims.•Each well contains one PapilloCheck microarray with 28 probes, each in 5 replicate spots.•These 140 spots are arranged in an array of 10 by 14 spots distributed over an area of about 10 mm2.

39

Asian Market DevelopmentHybribio, HongKong

Flow-through biochip [HPV (21), HBV (rtPCR), HPV (rtPCR)]Dr Chip, Taiwan

Polymer Biochip [enterovirus, RVP (10), milk pathogens (7), food pathogens (7)]Guilin Immunetech Co, Ltd, GIMT, China

rtPCR, ELISA biochip [CMV]BioCore, Seoul, Korea

Slide Chip, gelPCR [HPV (32), HCV(10) - CT, NG, MP, CMV, EBV, HSV, MT]LGLife Sciences, Seoul, Korea

Slide Chip, rtPCR [HPV (32)- HPV, TBSeegene, Seoul, Korea

CEPCR [Sepsis, RVP (18), HPV (18), TB, HBVgeno]GelPCR [JAK2, FLT3, BCR-ABL, PML, AML, MTHFR, CYP2C9, ApoE, Food]

40

European Market DevelopmentAttomol, Germany

DNA-LINA [VZV, HSV, B19, bacteria]

AID, Germany

CEPCR [STD(6), HPV (5)], StripPCR [ HLA, F2-F5-MTHFR, Hfe, ApoE-B, Osteop,CYP2C9, HFI]

Greiner Bio-One, Germany

CE-IVD BioChips [HPV (24), CF (49), ParoCheck, CarnoCheck, MycoDtect]

Vircell, Spain

Speed-Oligo (stripPCR) [Mycoplasma-Legionella-Chlamydia Pnemonia]

Progenika, Spain

LIPOchip, BLOODchip, PHARMAchip

41

European Market Development

Innogenetics, Belgium4-Mat technology, 3D porous microarray

Eppendorf, GermanyBioChip System, SilverQuant detection, DualChip GMO assay, Gene Expression on-demand assays

42

Italian Market DevelopmentAb Analitica, Padova, Italy

PCR [B19, CMV, EBV, HCV, HCVgeno strip, HHV6, HSV, HBV, HPV strip (32)]

rtPCR [CMV, EBV, HSV, HHV6, HHV8, HPV (8)]

Ce-Gel-PCR [col.cancer, CPIG clonality, KRAS, BCR-ABL, AZF, DDK, APoE, COL1A1, VDR, STR, VNTR]

strip-PCR [F2-F5-MTHFR, Hfe]

BCS Biotech, Cagliari, Italy

rtPCR [Scrapie], ProDect Chip [ceivd HPV (25), RVP(8), Pan-Entero, L-C-M pneumoniae, Milk(8), Food(10)]

ELISA-PCR [HPV(19), HCV, HCVgeno, HBV, HBVgeno, HAV, HGV, HDV, HSV, CMV, VZV, HHV6, HHV8, EBV, Rub, JC/BK, Entero, B19, HIV, HTLV, SARS, MT, MBhsp65, BA, HP, MP, MG, TC, CP, BP, BB, ToxoB1, ToxoP30, SMA, F2, F5, MTHFR, F8, Hfe, BCR-ABL, K-Ras, GMO]

EuroClone, Siziano, Italy

CE-IVD stripPCR [F2-F5-MTHFR, Infertility, Hfe]

43

Choose your probes (oligos, cDNA)

Choose the way for spotting them

Hybridization experiments

Scanner Detection

Biocomputing Analysis

Making Your Own Array Tests44

Array Construction

45

Array Hybridization

46

Semi Automation for Array Hybridization

The TECAN HS 4800 Pro and HS 400 Pro Hybridization Stations represent the very latest technology for performing fully automated, highly sensitive and reproducible microarray processing on slides.

The systems are designed for full automation, from pre-hybridization and on-board denaturation up to automatic slide drying with OSND™ technology.47

Semi Automation for Array Hybridization

BioMicro’s MAUIs are Systems for automating the fluid processing of microarray samples in low-to-high-throughput laboratories.

The MAUI System integrates and optimizes the sample processing performed on microscope slide arrays via its patented instrument and MAUI Mixer hybridization chambers.

Also from Advalytix.de, the ArrayBooster, SlideBooster, and PlateBooster

48

AffymetrixAgilent TechnologiesAlpha InnotechApplied BiosystemsApplied PrecisionBiomedical Photometrics Inc.Bio-RadGenetixIlluminaInvitrogenMolecular DevicesPerkinElmerTecanTelechem-ArrayItVIDAR Sys. Corp.etc...

Array Detection

49

Arrays Competitor: realtime PCRCombination of NA extraction platforms + QPCR instrument

most popular : Roche MagnaPure LCthen Qiagen EasyOne, Biomerieux EasyMag

In France, 30% Roche LightCycler, 30% AB-Prism, 30% Cepheid (IL) SmartCycler

QPCR average cost in France 12€ (in house - including NA extraction @4-5€) or 18-45€ (CE-IVD)

Clondiag, Greiner, Innogenetics like arrays: 40-65€

50

Gene Expression Assays: from array to pcr and back

the rapid development of PCR arrays for gene expression analysisSABiosciences (acquired by Qiagen) PCRarrays

Roche Applied Sciences, a development of the UPL products, RealTime Ready arraysFluidigm Digital PCR and LifeTechnologies/BioTrove OpenArray

Pathway Focused: Profile the expression of a panel of genes relevant to a pathway or disease state.

Simple and Accurate: Simple real-time PCR method provides high sensitivity and wider dynamic range. Requires as little as 1 ng total RNA.

Pre-aliquoted primer set plates and Master mix formulation enable the PCR Array to amplify 96 or 384 different gene-specific products simultaneously for a single patient, or series of genes for multiple patients on a single 96 or 384 PCR plate.

51

Gene Expression made simple with realtime PCR arrays

Choose an array list from your provider or make your configure your own array.

Extract RNAs

Make cDNA

Dispense patient’s cDNA into

pre filled PCR plates

Qiagen PCR arrays: Sybr Green

Roche PCR arrays: UPL (hydrolysis) probes with LNA

52

Gene Expression, Arrays, RT PCRRealtime PCR may have found an appropriated way to directly compete with moderate complexity arrays in MDx

realtime PCR more familiar to MDx usersinstrumentation and automation ready in numerous MDx labsready-to-go assayseasy customization, online tools...etc

PCR arrays still expensive but just starting

384 well plate compatible instrument recommended

53

And what about electrophoresis?Automation of conventional electrophoresis

The CE switch

CEquencing

Mass-spectrometry

54

Robotic workstation+

MT compatible gel box

Biomek 2000 + OneLambda gel box

Automation of gel loading

55

Automation of gel loading

E-Gel 96 from Life Technologies

SBS design.Beckman Coulter Biomek script available56

Automating Gel Electrophoresis

With Capillary Electrophoresis

57

Fluo

resc

ence

(530

nm

)

0 5 10 15 20

Electrophoresis time (minutes)

200 bp

500 bp

Multiplex STR Analysisafter PCR

CE is simply electrophoresis in a very thin capillary, under high voltage. Gel and samples are automatically injected for each run.

Electrophoresis Automation: Capillary Electrophoresis (CE)

58

MicroFluidics and CE chips, the next automated instruments for MDx?

Agilent LabChip 2100 BioAnalyzer

1 PC + 1 Analytical StationSingle Use Chip90 s run/sample

12 samples / 30 min

59The first available LabChip based

instrument, from Agilent, in association with Caliper Life Sciences

LabChip Pros and ConsPros

Automation of electrophoresis process

Fast

Quantitative

Sensibility and accuracy

Standardized electrophoresis

Cons

Low throughput (actually)

Dedicated reagents

Still a lot hands on time

Cost of instrument vs conventional electrophoresis

60

61

LabChip 3000

The LabChip 3000 is dedicated to drug-discovery and immunoassays: Serine/Threonine Kinases;Tyrosine Kinases; Phosphatases; Proteases; Lipid-modifying Enzymes; and G-protein coupled receptors (GPCRs).

LabChip 90

LabChip automation from Caliper LifeSciences

Bencntop Labchip instrument for High Throughput DNA and RNA Analysis

Caliper LabChip GX/GXII instruments with LabChip GxP Software are computerized systems designed to automate the analysis of DNA, RNA or proteins using Caliper Sipper Chip technologies.

With sample acquisition time less than a minute the instrument can thoroughly analyze 96 samples in less than an hour.

62

Caliper LifeSciences LabChip GX

The end of Agarose Gel: Qiagen QIAxcel

63

The QIAxcel System is based on a proprietary multiplexed fluorescence detection with inexpensive solid-state light sources and micro-optical collector.

It uses a multiple usage and disposable multi-capillary-gel cartridge.

High resolution separation is provided for 12 samples, every 5-7min.

Resolution is 3-5 bp in DNA fragment sizes between 15-5000 bp.

Sensitivity is 0.1 ng/µl of diluted PCR product solution.

Now the QIAxcel from Qiagen

Development of automated sequence based diagnostic

with Capillary Electrophoresis

64

Sequencing by CE: CEquencing

The AB Prism 310 a pioneer

65

Sequencing Instrumentation

36/50 cm (50µm)

22/36/80 cm (50µm)

47/61 cm (50µm)

14, 21, 28 cm

N.A.N.A.20, 30,40 cm

48 to 96 capillaries

16x96/384 plates

16 or 4 capillaries

1 capillaryMicrocell 0,5µm

16 lanes

96 well plate

10x 96 well plates

75µm/100 lanes

capillairecapillairecapillairegel Platpyrosequencinggel ultra plat

37303100/AvantPrism 310Long-Read Tower

PSQ 96MA

PSQ 96HS/HSA

BaseStation 100

Applied BiosystemsVisible Genetics

Pyrosequencing AB / BiotageMJ-Research,

36/50 cm (50µm)

96 capillaries16x96/384

plates

capillaire

3730xl

8-24 capillaries

66

40 cm (75µm)

16-32-48-(96)/

16-32-48-96 non coated

capillaire

MegaBace 750/1500

33 cm (75µm)

8 coated capillaries

1x96 samples

capillaire

CEQ 8000

Beckman Coulter

33 cm (75µm)

8 coated capillaries

2x96 samples

capillaire

CEQ 8800

41/66 cm

0,25mm/32/48/64/9

6 lanes

Gel plat

4200

Licor

25/41/66 cm

40 cm (75µm)

40 cm (75µm)

40 cm (75µm)

Lanes length

64/96 lanes384 non coated

capillaries

96 non coated

capillaries

48 non coated

capillaries

type/lanes nber

Gel platcapillairecapillairecapillaireTechnology

4300MegaBace 4000

MegaBace 1000

MegaBace 500

Instrument

Amersham BiosciencesCompany

capillaire

3500

xx

Eight-capillary arrayFour-wavelength fluorescence detection/capillary96-well microplate format for samples96-well microplate format for bufferAutomatic gel replenishmentAutomatic sample denaturation and injection

Low Cost multicapillary systems for sequencing based diagnostic automation

67

8 to 24 capillary systems made availableApplied BioSystems new 3500 Genetic AnalyzerBeckman Coulter CEQ 2000/8000/8800

CEquencing for HIV genotyping

CEquencing HIV Protease gene50min protocol

8 capillary system4 patients/ 50min

CEquencing HIV RT gene1hr50 protocol

8 capillary system4 patients/ 1hr50

4 patients RT+Prot CEquencing/2.6hours

24 patients/16hrs

• HIV extraction

• RT/PCR

• nested PCR

• Cycle sequencing reactions

• Electrophoresis onto a CEquencer

• Sequence Confirmation

• Resistance profile assignment

68

69Sequencing: 3+1+(0.4+4.5+0.4 €)x2=$19 per 700b sequence

CEQuencing with 8 capillaries: 33.000b per 24h (48x2x700b)

CEquencing cost for ds33.000 bases: $892

CEquencing cost for ds1Mb: $27.000

BioIT, sequence confirmation: 5min/1000b ; 7hrs/33.000b

Estimating CEquencing Cost

Sequencing based diagnostic and BioInformatics

70

Sequence confirmation with CEQuence Investigator

71

Major and Minor HIV mutations

Protéase Reverse Transcriptase1 31 61 1 31 61 91 121 Q151M Y181C 211 2412 V32i 62 2 32 A62V 92 122 152 182 212 2423 L33 63 3 33 63 93 123 153 183 213 2434 34 64 4 34 64 94 124 154 M184V 214 2445 35 65 5 35 K65R 95 125 155 185 T215Y/F 2456 M36i 66 6 36 66 96 126 156 186 216 2467 37 67 7 37 D67N/E/S 97 127 157 187 217 2478 38 68 8 38 68 98 128 158 Y188C/H/L 218 2489 39 69 9 39 T69D/N 99 129 159 189 K219Q/E 249

L10i 40 70 10 40 K70R L100i 130 160 G190A/S 220 25011 41 A71V/T 11 M41L 71 K101E 131 161 191 221 25112 42 72 12 42 72 102 132 162 192 222 25213 43 G73S 13 43 73 K103N 133 163 193 223 25314 44 74 14 E44A/D L74V 104 134 164 194 224 25415 45 75 15 45 V75M/S/A/T 105 135 165 195 P225H 25516 M46i/L 76 16 46 76 V106A 136 166 196 22617 i47V V77i 17 47 F77L 107 137 167 197 22718 G48V 78 18 48 78 V108i 138 168 198 22819 49 79 19 49 79 109 139 169 199 229

K20R i50V 80 20 50 80 110 140 170 200 23021 51 81 21 51 81 111 141 171 201 23122 52 V82A/F/S/T 22 52 82 112 142 172 202 23223 53 83 23 53 83 113 143 173 203 233

L24i i54V/L/T i84V 24 54 84 114 144 174 204 23425 55 85 25 55 85 115 145 175 205 23526 56 86 26 56 86 F116Y 146 176 206 P236L27 57 87 27 57 87 117 147 177 207 23728 58 N88D/S/T 28 58 88 118 148 178 208 23829 59 89 29 59 89 119 149 179 209 239

D30N 60 L90M 30 60 90 120 150 180 L210W 240

mutations primaires

mutations secondaires

mut majeure ANRS : pas JAMA

mut mineure JAMA : pas ANRS

72

LIMS Solutions for Genomic Applications

SCC soft computer, Soft Lab, SoftMolecular, SoftCytogenetics, SoftHLA

Geospiza, GeneSifter

SoftGenetics, Nextgene

CLC bio, DNA Workbench, Genomics Workbench

GenomeQuest, web based platform

BiotiqueSystems, BLIS

Genomatix, GGA, ChipInspector, ..etc

InteRNA, Intess

Genologics,

Biorepositories, BioChronicles, Geneus, Omix...etc

73

Molecular Diagnostic ITSequence DatabasesMutation Databases

SNP Databases

Journal References

74

© 200675

Inte

grat

ed a

naly

sis

Act

iona

ble

Expert board

Sequence profilesReference Database

Algorithms

Intranet Internet

Interface (XML, HL7)

Data management

Public databases

IDNSTM

Lab-

Dat

a

Labo

rato

ryComplex Data

§ Microbial identification: bacteria, mycobacteria

§ Fungal identification§ Veterinary and food pathogens § HIV drug-resistance: genotyping

and resistance assessment§ HCV genotyping and HBV resistance

testing§ Epidemiological genotyping of

bacteria and viruses: MLST for meningococci, Campylobacter, Influenza, others

§ HLA typing

IDNS modules for research, clinical diagnostics, epidemiology

An Alternative To Sequencing Instrumentation

Mass Spectrometry

76

• DNA consists of a four-letter alphabet: A, C, G, T and each letter has defined molecular mass:

• dAMP = 313.2 Da - dCMP = 289.2 Da - dGMP = 329.2 Da - dTMP = 304.2 Da

• Matrix-assisted laser desorption ionization time-of flight mass spectrometry (MALDI-TOF-MS) has been mainly commercialized by Sequenom Inc. (San Diego, CA) and has emerged as a strong contender in the high throughput genotyping field. In this technology, the PCR products serve as templates for an optimized primer extension reaction, generating allele-specific oligonucleotide products.

• High resolution resolves non-specific background from signal => the very high sensitivity

• Large mass window allows multiplexing many analyses into a single spectrum

• Absolute concentration can be measured with higher precision than other methods

• Full automation and data interpretation means no mass spec expertise required

77 MALDI-TOF-MS for DNA analysis

courtesy of Dr Charles Cantor, Sequenom

MALDI-TOF-MS for SNP Genotyping78

Multiplex up to 40 SNPs/reaction, 150,000 genotypes/day

iPlex Gold from Sequenomcourtesy of Dr Charles Cantor, Sequenom

MALDI-TOF MS sensivity for CNV analysis

Relative SNP Allele Ratio

Copy Number Variation

Requires Well-characterized SNPs for CNV region and Heterozygote samples

Provides Relative copy number and Minimal quantitative informative data

Useful for Post-array validation, Fine mapping of specific CNV regions, Potential CNV discovery from genotyping experiments

Flexibilit Doesn’t rely upon a SNP, Can use heterozygote or homozygote

Provides Absolute copy number, Highly quantitative, informative data

Useful for Fine mapping of specific CNV regions, Copy number association studies, Post-array validation of uncharacterized regions

Analysis of Copy Number Variation using Quantitative Interspecies Competitive PCR Nigel M. Williams, Hywel Williams, Elisa Majounie Nadine Norton, Beate Glaser, Huw R. Morris, Michael J. Owen and Michael C. O’Donovan. Nucleic Acids Research, 2008.

courtesy of Dr Charles Cantor, Sequenom

79

Numerous applications for MALDI-TOF MS

Detection of low pourcentage mutations (>20% by sequencing, 1-3% by MS)

Detection of low abundance transcriptsie, Sequenom SEQureDX T21 test

DNA methylation measurements

Resequencing

Signature sequence identificationbacteria typing, HCV quasispecies analysis, respiratory virus surveillance...etc

80

The major players in MDx

Ibis Biosciences,

T5000 Biosensor System

(an Abbott Company)

81

Sequenom, Inc., MassArray

Sequence based diagnosticIs getting highly automated

With microtiterplate compatible operation and multicapillary throughput

New area to take care with: bioinformatics

Lot of time required for analysis

Informatics skilled technicians needed

Collection of data

Integration with LIS

megaBase, gigaBase and full genome sequencing may be the next molecular tool revolution after realtime PCR

MALDI-TOF MS a serious alternative to sequencing instrumentation

82

From Sequencing to Genotyping: SNPs

Interest in SNPs is increasing in the diagnostic field

pharmacogenomics, pharmacogenetics, personalized medicine

Disease risk prediction

Disease predisposition

Many options to analyze SNPs with a high throughput

Sequencing, PyroSequencing, Real-time PCR, Invader, MassArray, OLA, BeadArray, SBE…etc

83

15,000,0000

2 haploid genomes differ at 1 nucleotide every 1331 bp

1 0,S N P

84

5,000,000SNPs have been identi"ed so far

85

99%SNPs have no biological effect.

Over 60.000, however, are within genes, and some are associated with disease.

86

SNP Genotyping Methods

According Ivo Gut, Human Mutation 17:475-492 (2001)

87

SNP Detection in MDxImplementing such technologies still requires a lot of effort.

•the easiest way: realtime PCR approach for a limited number SNP to analyze per patient•while SNPs number increase reasonably, switch to sequencing approach•when SNPs to analyze start to be numerous, switch to microarray based assays

•the complete picture: whole genome sequencing?

88

•<10

•>10 <96•>96

Real-time PCR for SNP genotyping89

The TaqMan assay for SNPs analysis

Real-time PCR for SNP genotyping90

Real-time PCR for SNP genotyping91

The Amplifluor assay for SNPs analysis

Sequencing for SNP genotyping

92

Sequencing for SNP genotyping

93With AB-Prism 3700

Sequencing for SNP genotyping

94

With Beckman Coulter CEQ 8000/8800

Chaque incorporation de dNTP libère un pyrophosphate PPI (relation équimolaire)

Chaque PPI est converti en ATP en présence d’Adénosine 5’ phosphosulfate APS

Grâce à l’ATP, la luciférine est transformée en oxyluciférine. Cette réaction s’accompagne d’une émission de lumière (relation proportionnelle aux nbres de molécules d’ATP utilisées)

PyroSequencing for SNP genotyping95

PyroSequencing for SNP genotyping

The PSQ HS 96A

96(Pyrosequencing) Biotage, a Qiagen company

Major SNP Technology Providers97

Illumina SNP Technologies

The Golden Gate Assay on the Sentrix Universal-16 BeadChip or

Universal-96 Array Matrix multi-sample

array formats.

The Illumina GoldenGate Genotyping Assay is a flexible, pre-optimized assay that uses a discriminatory DNA polymerase and ligase to interrogate 96, or from 384 to 1,536, SNP loci simultaneously.

98

Illumina SNP Technologiesa novel approach to microarraysIllumina’s BeadArray Technology is based on 3-micron silica beads that self assemble in microwells on either of two substrates: fiber optic bundles or planar silica slides.

Each bead is covered with hundreds of thousands of copies of a specific oligonucleotide that act as the capture sequences.

The BeadXpress Reader is a high-throughput, dual-color laser detection system that enables scanning of a broad range of multiplexed assays developed using the VeraCode digital microbead technology.

99

Beckman Coulter SNPstream Technology

100

1.  Primer Design

2.  Multiplex PCR

3.  PCR Cleanup

4.  Single Base Extension

reaction

5.  Tag-Array hybridization

6.  Image Scanning

7.  Automated Genotype Calling

Autoprimer.com

SNPware® Reagent Kits

SNPstream Software Suite

Final Step: Interpretating the results

250 SNP/patient for 3000 patientsDNA extraction campaign = 10 daysPCR setup = 10 daysDNA PCRs = 11 nightsPrimer extension reactions and readings = 12 days Project duration ≈ 32 days (1 month)

101

Pre-PCR Setup (1)✓ Beckman Coulter, Inc. (BCI) Biomek Nx sp8 for DNA

extraction, PCR setup and PCR plate loading

✓ Roche LightCycler 480 for robotic PCR

102

Post-PCR Setup (2)✓ BCI Biomek Nx sp8 for primer extension reaction setup, hybridization

setup, plate washes, PCR product purif., sequencing reaction setup, sequence reaction purif. ...etc

✓ BCI SNPstream scanner

✓ BCI CEQ 8000

103

High Throughput SNP detection: portable to routine MDx?

Screening for a large set of SNP is still high end and technical demanding

Large set of SNP studies more related to research and clinical studies rather than routine MDx

However, more and more SNP are related with disease-associations. So still a high potential for MDx developments.

How these technologies will face the $100 full genome sequencing era? Technologies for 1m SNPs vs Full genome sequence with >5m SNPs?

104

Basics 1 kilobase 1kb 1 000 bases

Virus: 3500 to 8 x 105 bases

Bacteria >1Mb(Escherichia coli =

4,7 Mb)

1 megabase1Mb 1 000 000 bases1 million

1 gigabase1 Gb1000 Mb1 billion

Eucaryotes 10-3.105 Mb

yeast1,3 Mb

drosophila165 Mb

Homo sapiens

3400 Mb

3Gb

20 000-25 000 genes

Transcriptome

2% Genome105

2nd Generation Sequencing:

routine MDX tools?

NextGen Sequencers - NextGen Sequencing - NGS

Whole Genome Sequencer - Whole Genome Sequencing - WGS

AB & BCICE Sequencing

AB 96 capillaries:2,8 Mb/24h400b/read

BCI 8 capillaires:45KB/24h700b/read

AB Solid, PGM, Illumina HiSeq, Roche FLX, Jr

PacBio

Roche:0,5 GB/10hrs

Illumina:200 GB/8days

AB/LT:210 GB/7days

106

Sanger vs NextGen

107

Other Players

George Church Lab. + Danaher Motion: Polonator G.007

Helicos BioSciences Corp.: HeliScope SMS

Upcoming 3rd generation sequencers

The Polonator G.007 is the first

"open source" gene sequencing

instrument to hit the lab market

in which the instrument's

software (Web ware) and

specifications are freely

available to the public.

At $150,000, the Polonator is the

cheapest instrument on the

marketThe HeliScope™

Single Molecule

Sequencer is the first

genetic analyzer to

harness the power of

direct DNA

measurement, enabled

by Helicos True Single

Molecule Sequencing

(tSMS)™ technology.

108

Roche Applied-Science (454)

GS-20, GS-FLX, GS-FLXti

GS-junior

109

Roche GS-FLX technology

110

Roche New Instrument

The GS Junior Launch Nov. 2009

111

GS FLX ti GS junior

Throughput 0.7 GB / day 35 MB / day

Read Length 4-500b 4-500b

Reads per run > 1million 70-100,000

Instrument price ~$648,000 ~$100,000

Potential applications shown:

HIV drug resistance testing, 8 patients @ 1500X

HLA class 1+2 full sequencing

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)

112

NextGen Sequencers add-

ons- Nebulizers + nitrogen tankNebulization is required to shear fragments for DNA >70-800bp

- emPCR Breaking KitThis device is required for the preparation of consistently sized reactors for emulsion PCR.

- Magnetic Concentrator IVGN +€5000- MT plate centrifuge BCI +€15.000

- Multisizer™ 3 COULTER counter +€15.000The most versatile and accurate particle sizing and counting analyzer available today. Using The Coulter Principle, also known as ESZ (Electrical Sensing Zone Method), the Multisizer 3 COULTER COUNTER provides number, volume, mass and surface area size distributions in one measurement, with an overall sizing range of 0.4 µm to 1,200

- Agilent BioAnalyzer +€20.000

- Titanium cluster station +€29.000

Roche GS-FLX:

not included

113

Automating the process

The REM e System, is a liquid handler accessory designed to fully automate the emPCR enrichment and sequence primer annealing s t ep s i n t he Genome Sequencer FLX System workflow.

114

The Fluidigm Access Array System to automate preparation of 48 samples with up to 48 amplicons in one run — then sequence all 2,304 unique amplicons in a single Roche GS Junior System run.

Still required•MT plate centrifuge BCI +€15.000•M u l t i s i z e r 3 B C I counter +€15.000•Agilent BioAnalyzer +€20.000

illuminaGenome Analyzer

(Solexa)

115

Illumina-Solexa TechnologyThe approach relies on attachment of randomly fragmented genomic DNA to a planar, optically transparent surface and solid phase amplification to create an ultra-high density sequencing flow cell with >10 million clusters, each containing ~1,000 copies of template per sq. cm. These templates are sequenced using a very robust four-color DNA sequencing-by-synthesis technology that employs reversible terminators with removable fluorescence.

116

Illumina new instruments 117

HiSeq 2000Genome Analyzer IIx

cBot Cluster System Paired-End ModuleInstrumentation Accessories

HiSeq 2000 HiSeq 2000 GA IIx

Throughput 200 Gb / 8 days 100 Gb / 8 days 85 Gb / 14 days

Capacity 25 Gb / day 12,5 Gb / day 85 Gb / 14 days

Read Length 2 x 100bp 2 x 100bp 2 x 150bp

Genomes 2/run @ $10,000 1/run 1/run

Instrument price ? 828 000 $ ? 600 000 $ ? 564 000 $

HiSeq 1000

Redefining NGS workflow 118

NextGen Sequencers add-

ons

Total: €126.000

Illumina GA2:

not included - Cluster Station +$50.000The Cluster Station is a standalone, software-controlled system for the automated generation of clonal clusters from single molecule fragments on Illumina Genome Analyzer flow cells.

- Paired-End Module +$45.000The Paired-End Module provides fully automated template preparation for the second round of sequencing in a paired-end sequencing run.

- IPAR +$60.000IPAR is a bundled hardware and software solution that provides real-time quality control and integrated online processing of primary data during sequencing runs

- Agilent BioAnalyzer +€20.000

119

Life Technologies

Applied BioSystemsSOLID systems

120

AB SOLID high throughput

How it Works

Start with a fragment or mate-paired library depending on the application and information you need.

Prepare clonal bead populations in microreactors containing template, PCR reaction components, beads and primers.

After PCR, you denature the templates and perform a bead enrichment step to separate beads with extended templates from undesired beads.

Then deposit the 3’ modified beads onto a glass slide. Once loaded onto the Analyzer, primers hybridize to the adapter

sequence.

A set of four color dye-labeled probes compete for ligation to the sequencing primer. Specificity of probe ligation is achieved by interrogating every 4th and 5th base during

the ligation series. Five to seven rounds of ligation, detection and cleavage record the color at every 5th

position with the number of rounds determined by the type of library used. With mate-paired sequencing, this process

is repeated for the second tag.

121

Illumina new instruments 122

5500xl 5500

Throughput 200 Gb / 7 days 100 Gb / 7 days

Capacity 30 Gb / day 15 Gb / day

Read Length 75 +35 75 +35

Genomes 2/run @ $3,000 1/run

Instrument price 595,000 $ 349,000 $

New Microfluidic FlowChip

Redefining NGS workflow 123

The SOLiD EZ Bead System automates the SOLiD System work flow from emulsion PCR (ePCR) to templated bead

Fragment library preparation workflow with the steps performed on

the Tecan Freedom EVO 75 or others

Ion Torrent/Life Technologies

$50k benchtop inst.

<$500 10Mb run

or

Pacific Biosciences

Single Molecule Sequencer

30Gb/day >1000b frag.

The Challengers?

124

125 2nd Generation Sequencers and MDx

1.Cost of instrumentation

2.Cost of reagents

3.Procedure time

4.BioIT requirements

1.Massive amount of useful data

2.Genome, Transcriptome, Microbiome applications

3.Reagent cost decreasing

4.$3-10K full genome access

126The $100 Full Genome sequence and its impact on the Mdx market

The new era of personalized medicine (P4 medicine)

The DTC-Genomics market

The 3rd generation sequencing technology breakthrough

What 3rd gen. sequencers will provide and how it will change the MDx market?

The DTC-Genomics Players

> 40 players and growing

127

‣ Illumina (everygenome)➡ full genome @ $48k -> $19k->14k

‣ Knome➡ full genome @ $100k->$68k->$25k?

‣ Complete Genomics➡ full genome @ $12k - 7k

-23AndMe $1000->$499->$99 (111)-Athleticode-deCODEme-DNAancestry $99 (mtDNA)-DNAdirect-DNAVision -EthnoAncestry $129-299 (Y/mtDNA)-Genebase $448 (mtDNA+91)-GeneEssence $1195 (84)-Genelex-GeneLink-GenePartner-GeneTonix -GeneWize-MatrixGenomics-Medomics-MyredhairGene-Navigenics $2500->$999 (28)-Pathway Genomics (USA) $249 (77)-ScientificMatch-TruGenetics

‣ Life Technologies➡ full genome @ $3k

‣ illumina➡ full genome @ $10k

socialcompare.com DTC-Genomics Players http://t.co/60acprd

Upcoming 3rd generation sequencing technology breakthrough

> 20 upcoming players

128-Complete Genomics-Pacific BioSciences-Life Technologies -ZS Genetics-Ion Torrents-Oxford Nanopore -BioNanometrix-Lightspeed Genomics-IBM-Reveo et ReVase -Genome Corp.-GenoVoxx-Halcyon Molecular-Base4Innovation-Intelligent BioSystems-LaserGen

-Mobious Biosystems-NaBsys-Netbio-Population Genetics-Seirad-U.S. Genomics...and growing

Upcoming 3rd generation sequencers: the promise129

Instrument target price $10k

$500k in 2010 then down to $10k by 2015

compared to >$600k today

Full genome target price $100 before end of 2012?

$10k end 2009, $3000 end of 2010, $1000 by 2011?

compared to $50k in 2009 (or $20-10k reagents)

1 day assay for full genome sequence at 40X

compared to 10-15 days today

Upcoming 3rd generation sequencing revolution?

If (1 NA extraction + 1 3rd gen. test) = full genome at 40X in > 8hrs

then MDx for genetic marker, mutation, deletion, SNP, VNTR, STR = in silico diagnostic?

if 3rd gen. seq. capable of full transcriptome and epigenome analysis

then what about array based diagnostics, what about gene expression technologies?

if 3rd gen. seq. capable of >100X sequencing for the microbiome

then what about PCR based assays for infectious diseases?

if 3rd gen. seq. deliver the promise

then shall we enter into the new era of P4 medicine?

(personalized, predictive, preventive and participative)

130

What is the Future of Molecular Diagnostic Procedures?More automation needed

For sample preparation (higher throughput, larger vol.)

For Fast-PCR (in 96 well format)

Links to be improved

Between sample preparation and PCR

Between molecular diagnostic and clinical chemistry

Between molecular diagnostic and pre-analytical steps

Full Integration

131

What is the Future of Molecular Diagnostic Procedures?Evolution through RealTime PCR

entering the ‘array’ gene expression business

New Genotyping and Pharmacogenetics era

Technologies development in the SNP field

Evolution of Sequence Based Diagnostics

The $100 full genome sequence: the upcoming revolution

The MDx future: in silico diagnostic?

132

P. Merel

Any Question?

133