2011 course on Molecular Diagnostic Automation - Part 3 - Detection
Patrick Merel Biomedical Innovation Platform (PTIB), Pessac, France firstname.lastname@example.org Post-PCR Procedures Automation in Molecular Diagnostic part 31
2 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
Former options for Post-PCR3 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
HLA Typing by PCR/SSO on strip 4 REVERSE-DOT-BLOT on membrane: ‘Strip Assay’
Automation of HLA-Typing by Reverse Slot-Blot 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 5
Automation of reverse hybridization procedures 6
Automation of hybridization procedures 7 Dedicated PCR+Post-PCR workstation: the Roche Cobas Amplicor
Perspective for Hybridization8 Procedures MicroArray-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?
Hybridization on Beads: the future of hybridization procedures? ❖ 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 9
Luminex based Technologies I. Multiplex PCR xTag Technology Numerous applications from II. Multiplex ASPE/TSPE various vendors and partners Asuragen The PCR reaction is then subjected to a primer extension step that is specific for Applied Cytometry Systems the allele or the infectious agent that is BMD being analyzed. The 5’ end of the ASPE Bio-Rad Diagnostics primer is attached to an xTAG universal Bio-Rad Life Science tag sequence. EraGen Biosciences Fisher Healthcare INOVA Diagnostics, Inc. Invitrogen MICROBIONIX GmbH Marligen Biosciences, Inc. IV. Universal Array Sorting Millipore Corporation (LINCO & Upstate) The 5’ universal tag sequence is MiraiBio hybridized to the complementary anti-tag Multimetrix GmbH sequence coupled to a particular xMAP One Lambda, Inc. bead set. Panomics PerkinElmer QIAGEN Radix Tepnel Lifecodes Zeus Scientific, Inc. V. Detection The hybridized beads are read by the Luminex System Directly from LuminexCorp 10
Luminex based11 Technologies The Luminex 100/200 System are flexible analyzers based on the principles of flow cytometry. Multiplexing: Up to 100 analytes per well. The Luminex High Throughput Screening System (HTS) is designed to perform hundreds of thousands of individual bioassays per day. The FLEXMAP 3D and MagPix are the Supports 96 or 384 well plates; Compatible with latest multiplexing systems launched by front-end plate handling robotics; Uses the 64 Luminex. bead set, not the 100 bead set; For Research use only, not for use in Diagnostic procedures Increased Multiplexing: from 50 to 500 analytes per well.
Progress in Luminex Technology Automation The following Products for the BioPlex 2200 are Available: Syphilis IgG Epstein-Barr Virus (EBV) IgG and IgM ANA MDSS Currently in Development: Syphilis IgM* ToRC IgG* ToRC IgM* Lyme IgG/IgM* Immunity IgG* HSV 1/2* Vasculitis* Gastrointestinal (GI)* 12 Anti-Phospholipid Syndrome* Rheumatoid Arthritis*The Biorad, BioPlex 2200: A Fully-Automated, Random Access, Multiplex Platform. EIA based until now.
Nanosphere Technology 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 13 assays on nucleic acids and proteins with the simplicity of a sandwich assay. ❖Ultra-sensitive detection of multiple proteins and nucleic acids simultaneously ❖Direct genomic detection without The assay involves a 2-step process similar to an ampliﬁcation or enzymes ELISA sandwich assay: ❖5-6 orders of magnitude more After isolation from a sample, DNA is hybridized to sensitive than ELISA based both nanoparticle probes and capture strands methods for proteins Silver is catalyzed on the gold resulting in six orders of magnitude ampliﬁcation of signal
Nanosphere Technology Step 1 Enter 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 2 Pipette 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 3 Insert 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 The Verigene System consists of ･Simple load and run capability two instruments: the Auto Processing System (APS) and the 14 Step 4 Verigene ID, which makes it easier Insert processed test cartridge unit into Verigene ID (duration: 3 than ever to run a variety of assays minutes) on nucleic acids and proteins with Remove the slide from the test cartridge and insert it into the Verigene ID. the simplicity of a sandwich assay. 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
Nanosphere latest automated platform: The Verigene SP The Verigene® SP is a bench top, free- standing instrument with a single, independent sample processing module. Random access, multifunctional test processing, including nucleic acid extraction, reverse transcription (if necessary), target ampliﬁcation (if necessary), and target 15 identiﬁcation and analysis in a Verigene® Test Cartridge.
16 Nanostring Molecular Barcodes The technology uses 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 simultaneously with sensitivity comparable to qPCR. +miRNA + CNV
17 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.
18 MicroArrays in Diagnostic Pathogens characterization Water-born virus, bacteria, genotyping Gene Expression monitoring Oncology Genotyping SNP detection Pharmacogenetics/Pharmacogenomics Disease predisposition, drug metabolism, drug design and development
DNA Chips : 19 from synthesis to commercial products 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
Affymetrix 20 GeneChip GeneChip Fluidics Station GeneChip Probe Array GeneChip Software GeneArray ScannerProprietary GeneChip software controls the Affymetrix ﬂuidics station and scanner functions, capturesthe ﬂuorescent image from the probe array, extracts data from the image and provides subsequentdata analysis.
Affymetrix HIV 21 GeneChipThe 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.
AmpliChip CYP450 from Roche Molecular Diagnostics a partnership IVD DNA22 with Affymetrix Array
Automated robotics for microarrays23 NanoChip® Loader 4 NanoChip ® Cartridges per Loader Nanogen’s CF ASR Single sample per six test sites Genotype for ∆F508 on initial run Screen for 24 other mutations 15 patients per cartridge 60 patients per run (4 chips)Also available as ASR: ASPA, ApoE, HFE, NanoChip ® Electronic Microarray Factor V/Prothrombin, Assay ToolBox 15 samples X 4 cartridges = 60 sample per Loader run
Multi-Processing of individual arrays: Osmetech 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. 24 eSensor CF test: 23 mutations tested for in the ACOG/ACMG-recommended cystic fibrosis carrier screening panel.
Osmetech XT-Technology § 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 capability25 CYP 450 2C9 and VKORC1 polymorphisms IVD assays New eSensor® XT-8 System
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 parts26 – Inherent ratiometric data analysis eliminates need for calibration
27 Autogenomics Technology The 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.
28 Autogenomics Menu The 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: Factor II Factor V Leiden Ashkenazi Jewish Panel Factor II-V Leiden Panel CYP450 2C19 MTHFR Warfarin Assay CYP450 3A4 FII-FV-MTHFR Panel CYP2C19 CYP450 3A5 CFTR31 MDR-1 CYP450 2C9-VKORC1 HPV Genotyping CYP450 2D6 HPV Quad UGT1A1 (Irinotecan) NAT-2 HPV Genotyping CHEK-2 HPV Quad EGFR, 5-FU (mutations) FLU A-sH1N1 MTB-DR (Drug Resistance) Respiratory Viral Panel STD Panel NTM
29 Clondiag Technology ArrayTube, 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 CLONDIAGs proprietary micro wet printing process (µWP). With the ArrayStrips, CLONDIAGS 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
Clondiag AT Procedure 30The ArrayTube Workstation ATS is designed for fast and accurate detection and analysis of all ArrayTube Tests.In combination with CLONDIAGs robust colorimetric detection method, ATS allows the analysis of the test specific interaction pattern on the AT-array carrying multiplediagnostic features wit
Clondiag Platform by Genomica 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 31 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 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. The AssayProcessor Station is designed for easy and rapid AP testing in the doctors 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. 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 33 - 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
34 Genomica clondiag-based Menu PneumoVir - Respiratory Virus Panel MetaBone HPV (17) COLLAGEN TYPE1 - SpI 35 HPV genotypes Influenza A VITAMIN D RECEPTOR – FokI y BsmI Influenza B ESTROGEN RECEPTOR - PvuII y XbalI predisposition to suffer endocrine system RSV A CALCITONIN RECEPTOR – AluI pathologies, rheumatological alterations and RSV B osteophatologies. Influenza C predisposition to suffer endocrine system pathologies, rheumatological alterations and Parainfluenza 1 osteophatologies. Rhinovirus Adenovirus Parainfluenza 2 Parainfluenza 3 Parainfluenza 4a Parainfluenza 4b Echovirus Bocavirus Coronavirus Metapneumovirus A Metapneumovirus B All products, CE-IDV
Greiner Bio-One Technology The 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 labeling35
Greiner Bio-One Procedure 1. Sample collection 2. DNA extraction 3. PCR 10 mins 30 mins 120 mins36 4. Hybridisation 15 mins at RT 5. Washing 2 mins 6. Scanning 10 mins 7. Evaluation 5 mins
CheckScanner™37 Fast and Reliable AnalysisAutomatic analysis of four HTA™Slides (or one HTA™Plate underdevelopment) with barcode identificationData analysis and reporting with CheckReport™Software
PapilloCheck®Orientation Controlfor optimised analysis OC OC OC OC OC PC PC PC PC PC PCR Control 6 6 6 6 6 45 45 45 45 45 11 11 11 11 11 51 51 51 51 51 16 16 16 16 16 52 52 52 52 52 • Type-specific 18 18 18 18 18 53 53 53 53 53 HPV DNA probes 31 31 31 31 31 56 56 56 56 56 in replicates of 5 33 33 33 33 33 58 58 58 58 58 35 35 35 35 35 59 59 59 59 59 • Print Control 39 39 39 39 39 66 66 66 66 66 • Negative Control 40 40 40 40 40 68 68 68 68 68 42 42 42 42 42 70 70 70 70 70 43 43 43 43 43 73 73 73 73 73 44 44 44 44 44 82 82 82 82 82Hybridisation Control HC HC HC HC HC SC SC SC SC SC Sample Control 38
39 Greiner Bio-One Menu ParoCheck®: 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 •PapilloCheck DNA chip comprises 12 wells, A1 biochip PapilloCheck® a total of 18 of the “high risk” – B6 defined by elevated rims. •Each well contains one PapilloCheck HPV types and 6 “low risk” HPV types can be identified. microarray with 28 probes, each in 5 replicate spots. coming: CF chip (>50 mutations) •These 140 spots are arranged in an array of 10 by 14 spots distributed over an area of about 10 mm2.
Semi Automation for Array Hybridization The TECAN HS 4800 Pro and HS 400 The systems are designed for Pro Hybridization Stations full automation, from pre- 47 represent the very latest technology hybridization and on-board for performing fully automated, denaturation up to automatic highly sensitive and reproducible slide drying with OSND™ microarray processing on slides. technology.
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. 48 Also from Advalytix.de, the ArrayBooster, SlideBooster, and PlateBooster
50 Arrays Competitor: realtime PCR Combination of NA extraction platforms + QPCR instrument most popular : Roche MagnaPure LC then 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€
Gene Expression Assays: from51 array to pcr and backthe rapid development of PCR arrays for gene expression analysis SABiosciences (acquired by Qiagen) PCRarrays Roche Applied Sciences, a development of the UPL products, RealTime Ready arrays Fluidigm Digital PCR and LifeTechnologies/BioTrove OpenArrayPathway 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 384different gene-speciﬁc products simultaneously for a single patient, or series of genes for multiple patientson a single 96 or 384 PCR plate.
Gene Expression made simple 52 with realtime PCR arraysChoose an array list fromyour provider or makeyour conﬁgure your ownarray.Extract RNAsMake cDNADispense patient’s cDNAintopre ﬁlled PCR platesQiagen PCR arrays: SybrGreenRoche PCR arrays: UPL(hydrolysis) probes withLNA
53 Gene Expression, Arrays, RT PCR Realtime PCR may have found an appropriated way to directly compete with moderate complexity arrays in MDx realtime PCR more familiar to MDx users instrumentation and automation ready in numerous MDx labs ready-to-go assays easy customization, online tools...etc PCR arrays still expensive but just starting 384 well plate compatible instrument recommended
54 And what about electrophoresis? Automation of conventional electrophoresis The CE switch CEquencing Mass-spectrometry
Automation of gel loading Robotic workstation + MT compatible gel box Biomek 2000 + OneLambda gel box 55
Automation of gel loading E-Gel 96 from Life Technologies SBS design. 56 Beckman Coulter Biomek script available
57 Automating Gel Electrophoresis With Capillary Electrophoresis
Electrophoresis Automation: Capillary Electrophoresis (CE) Fluorescence (530 nm) 200 bp 500 bp Multiplex STR Analysis after PCR 0 5 10 15 20 Electrophoresis time (minutes) CE is simply electrophoresis in a very thin capillary, under 58 high voltage. Gel and samples are automatically injected for each run.
MicroFluidics and CE chips, the next automated instruments for MDx? Agilent LabChip 2100 BioAnalyzer 1 PC + 1 Analytical Station Single Use Chip 90 s run/sample 12 samples / 30 min The ﬁrst available LabChip based instrument, from Agilent, in association with Caliper Life 59 Sciences
60 LabChip Pros and Cons Pros Cons Automation of electrophoresis Low throughput (actually) process Dedicated reagents Fast Still a lot hands on time Quantitative Sensibility and accuracy Cost of instrument vs conventional Standardized electrophoresis electrophoresis
LabChip automation61 from Caliper LifeSciences LabChip 90 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 3000
Bencntop Labchip instrument for High62 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. Caliper LifeSciences LabChip GX
The end of Agarose Gel: Qiagen QIAxcel 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.63 Now the QIAxcel from Qiagen
Development of automated64 sequence based diagnostic with Capillary Electrophoresis
Sequencing by CE: CEquencing The AB Prism 310 a pioneer 65
Sequencing Instrumentation Company Amersham Biosciences Beckman Coulter Licor Instrument MegaBace MegaBace MegaBace MegaBace CEQ 8000 CEQ 8800 4200 4300 500 1000 4000 750/1500 Technology capillaire capillaire capillaire capillaire capillaire capillaire Gel plat Gel plat type/lanes 48 non 96 non 384 non 8 coated 8 coated 0,25mm/ 16-32-48- 64/96 lanes nber coated coated coated capillaries capillaries 32/48/64/9 (96)/ capillaries capillaries capillaries 1x96 2x96 6 lanes 16-32-48-96 non coated samples samples Lanes length 40 cm 40 cm 40 cm 40 cm 33 cm 33 cm 41/66 cm 25/41/66 (75µm) (75µm) (75µm) (75µm) (75µm) (75µm) cm Pyrosequencing AB / Visible Applied BiosystemsMJ-Research, Biotage GeneticsBaseStation PSQ 96 PSQ 96 Long-Read Prism 310 3730 3500 3100/Avant 3730xl 100 HS/HSA MA Towergel ultra plat pyrosequencing gel Plat capillaire capillaire capillaire capillaire capillaire 75µm/100 10x 96 well 96 well Microcell 1 capillary 16 or 4 48 to 96 96 capillaries 8-24 lanes plates plate 0,5µm capillaries capillaries 16x96/384 capillaries 16 lanes 16x96/384 plates plates N.A. N.A. 14, 21, 28 47/61 cm 22/36/80 36/50 cm 36/50 cm xx20, 30,40 cm cm (50µm) cm (50µm) (50µm) (50µm)66
Low Cost multicapillary systems for sequencing based diagnostic automation 8 to 24 capillary systems made available Applied BioSystems new 3500 Genetic Analyzer Beckman Coulter CEQ 2000/8000/8800 Eight-capillary array Four-wavelength fluorescence detection/ capillary 96-well microplate format for samples 96-well microplate format for buffer Automatic gel replenishment Automatic sample denaturation and injection 67
CEquencing for HIV genotyping• HIV extraction• RT/PCR• nested PCR• Cycle sequencing reactions• Electrophoresis onto a CEquencer• Sequence Conﬁrmation• Resistance proﬁle assignment CEquencing HIV Protease gene 50min protocol 8 capillary system 4 patients/ 50min CEquencing HIV RT gene 68 1hr50 protocol 8 capillary system 4 patients/ 1hr50 4 patients RT+Prot CEquencing/2.6hours 24 patients/16hrs
69 Estimating CEquencing Cost Sequencing: 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
Sequencing based diagnostic and BioInformatics 70
Sequence conﬁrmation with CEQuence Investigator71
An Alternative To Sequencing76 Instrumentation Mass Spectrometry
77 MALDI-TOF-MS for DNA analysis• 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 courtesy of Dr Charles Cantor, Sequenom
78 MALDI-TOF-MS for SNP Genotyping Multiplex up to 40 SNPs/reaction, 150,000 genotypes/day courtesy of Dr Charles Cantor, Sequenom iPlex Gold from Sequenom
MALDI-TOF MS sensivity for CNV analysis 79 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. Relative SNP Allele Copy Number Variation RatioRequires Well-characterized SNPs for CNV Flexibilit Doesn’t rely upon a SNP, Can use heterozygote or homozygoteregion and Heterozygote samples Provides Absolute copy number, Highly quantitative, informative dataProvides Relative copy number and Minimal quantitative informative data Useful for Fine mapping of specific CNV regions, Copy numberUseful for Post-array validation, Fine mapping of specific CNV regions, Potential association studies, Post-array validation of uncharacterized regionsCNV discovery from genotyping experiments courtesy of Dr Charles Cantor, Sequenom
80 Numerous applications for MALDI-TOF MS Detection of low pourcentage mutations (>20% by sequencing, 1-3% by MS) Detection of low abundance transcripts ie, Sequenom SEQureDX T21 test DNA methylation measurements Resequencing Signature sequence identiﬁcation bacteria typing, HCV quasispecies analysis, respiratory virus surveillance...etc
81 The major players in MDx Ibis Biosciences, T5000 Biosensor System (an Abbott Company) Sequenom, Inc., MassArray
82 Sequence based diagnostic Is 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
83 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
1, 0 0 15,000,000 SNP2 haploid genomes diﬀer at 1 nucleotide every 1331 bp 84
99% SNPs have no biological eﬀect.Over 60.000, however, are within genes, and some are associated with disease. 86
87 SNP Genotyping Methods According Ivo Gut, Human Mutation 17:475-492 (2001)
88 SNP Detection in MDx Implementing such technologies still requires a lot of effort.•<10 •the easiest way: realtime PCR approach for a limited number SNP to analyze per patient•>10 <96 •while SNPs number increase reasonably, switch to sequencing approach•>96 •when SNPs to analyze start to be numerous, switch to microarray based assays •the complete picture: whole genome sequencing?
89 Real-time PCR for SNP genotypingThe TaqMan assay for SNPs analysis
Sequencing for SNP genotyping With AB-Prism 3700 93
Sequencing for SNP genotyping With Beckman Coulter CEQ 8000/8800 94
95 PyroSequencing for SNP genotypingChaque incorporation de dNTP libère un pyrophosphatePPI (relation équimolaire)Chaque PPI est converti en ATP en présenced’Adénosine 5’ phosphosulfate APSGrâce à l’ATP, la luciférine est transformée enoxyluciférine. Cette réaction s’accompagne d’uneémission de lumière (relation proportionnelle aux nbresde molécules d’ATP utilisées)
PyroSequencing for SNP genotyping The PSQ HS 96A 96 (Pyrosequencing) Biotage, a Qiagen company
Illumina SNP Technologies The Golden Gate Assay on the Sentrix Universal-16 BeadChip orUniversal-96 ArrayMatrix multi-sample array formats.The IlluminaGoldenGateGenotyping Assayis a flexible, pre-optimized assaythat uses adiscriminatoryDNA polymeraseand ligase tointerrogate 96, orfrom 384 to1,536, SNP locisimultaneously. 98
Illumina SNP Technologies a novel approach to microarrays Illumina’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 1. Primer Design Autoprimer.com 2. Multiplex PCR 3. PCR Cleanup 4. Single Base Extension SNPware® Reagent Kits reaction 5. Tag-Array hybridization 6. Image Scanning 7. Automated Genotype SNPstream Software Suite Calling100
Final Step: Interpretating the results250 SNP/patient for 3000 patientsDNA extraction campaign = 10 daysPCR setup = 10 daysDNA PCRs = 11 nightsPrimer extension reactions and readings = 12 daysProject 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
High Throughput SNP detection:104 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?
The Polonator G.007 is the first "open source" gene sequencing instrument to hit the lab market in which the instruments Other Players software (Web ware) and specifications are freely available to the public. At $150,000, the Polonator is the cheapest instrument on the The HeliScope™ market Single Molecule Sequencer is the first genetic analyzer to harness the power of direct DNAGeorge Church Lab. + Danaher measurement, enabledMotion: Polonator G.007 by Helicos True Single Molecule Sequencing (tSMS)™ technology.Helicos BioSciences Corp.:HeliScope SMSUpcoming 3rd generationsequencers 108
Roche New Instrument 111 The GS Junior Launch Nov. 2009 GS FLX ti GS juniorThroughput 0.7 GB / day 35 MB / dayRead 4-500b 4-500bLengthReads per > 1million 70-100,000runInstrument ~$648,000 ~$100,000price Potential applications shown: HIV drug resistance testing, 8 patients @ 1500X HLA class 1+2 full sequencing
NextGen Sequencers Roche GS-FLX: Workﬂow 112 Workﬂow 3-4 days (setup) + 1 day (run) IT steps: 1. Generation of a single-stranded template DNA library (~8-16 hours) 2. Emulsion-based clonal ampliﬁcation of the library (~8 hours) GS-FLX Software 3. Data generation via sequencing-by-synthesis (9 hours) 4. Image and Base calling analysis (~8 hours) ▪GS Reference Mapper 5. Data analysis using different bioinformatics tools•Long Single Reads / Standard Shotgun (required input = 3–5μg,5μg recommended) ▪GS De Novo Assembler~1,000,000 single reads with an average read length of 400 bases ▪GS Amplicon Variant Analyzer•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 Third Party Softwarea 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-basedenrichment process.•Amplicon Sequencing (1-5ng or 10-50ng)
NextGen Roche GS-FLX:Sequencers add- onsnot included - Nebulizers + nitrogen tank Nebulization is required to shear fragments for DNA >70-800bp - emPCR Breaking Kit This 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 +€15.000 The 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 +€29.000 113
The REM e System, is a liquid handler accessory designed to fully automate the emPCR enrichment and sequence primer annealingAutomating the steps in the Genome Sequencer FLX System workﬂow. process The Fluidigm Access Still required Array System to automate preparation of 48 samples •MT plate centrifuge BCI + €15.000 with up to 48 amplicons in one run — then sequence •M u l t i s i z e r 3 B C I counter +€15.000 all 2,304 unique amplicons in a single Roche GS Junior •Agilent BioAnalyzer + €20.000 System run. 114
Illumina-Solexa Technology 116The approach relies on attachment of randomly fragmented genomic DNA to a planar, optically transparent surfaceand solid phase amplification to create an ultra-high density sequencing flow cell with >10 million clusters, eachcontaining ~1,000 copies of template per sq. cm. These templates are sequenced using a very robust four-colorDNA sequencing-by-synthesis technology that employs reversible terminators with removable fluorescence.
Illumina new instruments 117 Genome Analyzer IIx HiSeq 2000 HiSeq 1000 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 150bpcBot Cluster System Paired-End Module Genomes 2/run @ $10,000 1/run 1/run Instrumentation Accessories Instrument price ? 828 000 $ ? 600 000 $ ? 564 000 $
NextGen Illumina GA2:Sequencers add- onsnot included - Cluster Station +$50.000 The 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 +$45.000 The Paired-End Module provides fully automated template preparation for the second round of sequencing in a paired-end sequencing run. - IPAR +$60.000 IPAR 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 +€20.000Total: €126.000 119
120 Life TechnologiesApplied BioSystems SOLID systems
AB SOLID high throughput 121 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.
Illumina new instruments 122 New Microfluidic FlowChip 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 $
Redeﬁning NGS workﬂow 123 Fragment library preparation workﬂow with the steps performed on the Tecan Freedom EVO 75 or others The SOLiD EZ Bead System automates the SOLiD System work ﬂow from emulsion PCR (ePCR) to templated bead
124Ion Torrent/Life Technologies $50k benchtop inst. <$500 10Mb run or Paciﬁc Biosciences Single Molecule Sequencer 30Gb/day >1000b frag.The Challengers?
125 2nd Generation Sequencers and MDx 1.Cost of instrumentation 1.Massive amount of useful data 2.Cost of reagents 2.Genome, Transcriptome, Microbiome applications 3.Procedure time 3.Reagent cost decreasing 4.BioIT requirements 4.$3-10K full genome access
The $100 Full Genome sequence and126 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?
Upcoming 3rd generation sequencers:129 the promise 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 sequencing130 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)
What is the Future of Molecular131 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
What is the Future of Molecular132 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?