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Post-PCR Procedures Automationin Molecular Diagnostic
part 3
Patrick MerelBiomedical Innovation Platform (PTIB), Pessac, France
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