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Automation: a key role for Molecular Diagnostic Evolution
 

Automation: a key role for Molecular Diagnostic Evolution

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from my presentation at the 4th Annual Diagnostics & Medical Devices Asia 2009 meeting in Singapore

from my presentation at the 4th Annual Diagnostics & Medical Devices Asia 2009 meeting in Singapore

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    Automation: a key role for Molecular Diagnostic Evolution Automation: a key role for Molecular Diagnostic Evolution Presentation Transcript

    • Automation: a key role for Molecular Diagnostic Evolution Patrick Merel, PhD University Hospital of Bordeaux, France Biomedical Innovation Platform (PTIB) pmerel@mac.com 1 1
    • Molecular Diagnostic Projections From IBC Life Sciences’ Projections for 2012 Discovery2Diagnostics conference, Oct. 08 2007 micro arrays market 2012 micro arrays market $660 millions (9%) $160 millions (3%) 2007 MDx market 2012 MDx market $3 billions $7 billions 2 credits: Harry Glorikian, managing partner of Scientia Advisors 2
    • MDx Growth Factors 3 Demand for molecular diagnostics and gene detection products is mainly determined by the volume of tests performed in clinical labs 3
    • MDx Growth Factors 3 Demand for molecular diagnostics and gene detection products is mainly determined by the volume of tests performed in clinical labs The need for automated and easy-to-handle techniques Optimized sample preparation, analysis, and data evaluation Techniques that would diagnose disease condition and medical disorders quickly for quick therapy decisions Availability of molecular diagnostic tests for monitoring the therapeutic efficacy of expensive drugs New diagnostics tests, primarily in the infectious disease application area Nanobiotechnology and biochips are also expected to drive future growth Genomics and proteomics are the major drivers of the molecular diagnostic market 3
    • MDx Growth Factors 4 A major step in MDx evolution: Realtime PCR technologies 4
    • MDx Growth Factors 4 A major step in MDx evolution: Realtime PCR technologies Simplification of the PCR process no more post-PCR procedures, ie. gel electrophoresis or hybridization Single instrument based, ie. a realtime PCR thermocycler Combination of a realtime PCR thermocycler and a Nucleic Acid (NA) extraction instrument for a potential full solution in MDx rapid development of automated NA extraction platforms slow progression of automated realtime PCR instruments new area of interest, fully automated platforms including NA extraction and rtPCR 4
    • NA extraction automation evolution 5 5
    • NA Extraction Procedure Evolution 6 From single sample process to microtiterplate by centrifugation to generic robotic workstation using vacuum manifolds to magnetic beads based procedures Biomérieux’s Boom patent and PSS Magtration technology being the major drivers 6
    • Dedicated NA extraction platform evolution 7 Interstingly, this progress in NA extraction automation did’nt come from the diagnostic industry and has firstly addressed, the research and academic market. Thus, even usefull, often poorly adapted to routine diagnostic requirements 7
    • Dedicated NA extraction platform evolution 7 Interstingly, this progress in NA extraction automation did’nt come from the diagnostic industry and has firstly addressed, the research and academic market. Thus, even usefull, often poorly adapted to routine diagnostic requirements 7
    • Dedicated NA extraction platform evolution 8 The Diagnostic Industry have started to address routine MDx demands (high throughput), FDA and CE-IVD labeling more recently. With the early presence of Gen- Probe TIGRIS in blood transfusion setups. New players are now pushing f o r w a rd t h e r u l e s w i t h f u l l y integrated platforms. 8
    • Dedicated NA extraction platform evolution 8 The Diagnostic Industry have started to address routine MDx demands (high throughput), FDA and CE-IVD labeling more recently. With the early presence of Gen- Probe TIGRIS in blood transfusion setups. New players are now pushing f o r w a rd t h e r u l e s w i t h f u l l y integrated platforms. 8
    • Dedicated NA extraction platform evolution 8 The Diagnostic Industry have started to address routine MDx demands (high throughput), FDA and CE-IVD labeling more recently. With the early presence of Gen- Probe TIGRIS in blood transfusion setups. New players are now pushing f o r w a rd t h e r u l e s w i t h f u l l y integrated platforms. 8
    • Realtime PCR instrumentation 9 9
    • Realtime PCR instrumentation 9 9
    • Realtime PCR instrumentation 9 9
    • Molecular Diagnostic Instrument Challenges 10 While it is difficult nowdays to run MDX procedures without an automated platform, some challenges remain to be fully addressed: - from blood to buccal cells, sample type and volume are numerous. Hard to find a single and unique solution - no clear standard for platform capacity, sample batch size, batch vs continuous flow, consolidation on core facilities vs point of care ..etc - realtime PCR supremacy from infectious diseases to genetic testing but difficult to fully automate - sequencing and micro-arrays based diagnostic still high cost and time demanding - SNP detection required in many potential personalized medicine assays still looking for the ultimate gold standard for routine testing 10
    • From 8 samples to 96 samples various platforms ABI very 1st automated NA from Tecan, Beckman extractor Coulter, Hamilton to Qiagen while Roche back to 24 batch Magnapure 32 with Biomérieux sample size Nuclisens? being of a major which has proved to impact in labs be popular to continuous flow and continuous for 1 to 72 flow from 1 to 96 samples samples with Qiagen with Roche Cobas QiaSymphony, AmpliPrep QiaEnsemble? 11 11
    • G Continuous flow for NA extraction procedures Gen-Probe the answer for multiple sample batch sizes a progress toward random access instruments? Roche Diagnostic 12 continous flow continous innovation Qiagen 12
    • G Continuous flow for NA extraction procedures Gen-Probe the answer for multiple sample batch sizes a progress toward random access instruments? Roche Diagnostic 12 continous flow continous innovation Qiagen 2009 2008 2010? 12
    • What’s next with NA extraction automation No more extraction steps? microfluidics technologies potential No more PCR? Fully integrated instrumentation Integration with Clinical Chemistry Automation? 13 13
    • Cepheid GeneXpert & Infinity a precursor in microfluidics and fully automated process for MDx in a single instrument From 1 sample to 16 on the GeneXpert to random access on the Infinity Cepheid 14 14
    • Cepheid GeneXpert & Infinity a precursor in microfluidics and fully automated process for MDx in a single instrument From 1 sample to 16 on the GeneXpert to random access on the Infinity Cepheid 14 14
    • Cepheid GeneXpert & Infinity a precursor in microfluidics and fully automated process for MDx in a single instrument From 1 sample to 16 on the GeneXpert to random access on the Infinity Cepheid 14 14
    • Microfluidics in MDx 15 HandyLab Jaguar, integrated DNA extraction and realtime PCR process on Microchips Iquum Liat Analyzer and Liat Flow Cycler, a new concept of Lab-in-a- tube for realtime PCR based assays Nanosphere Verigene System for FDA approved nanotechnology based MDX assays. protein assays compatible technology Direct hybridization, no PCR steps required 15
    • New Technology for new process in MDx Revolutionizing molecular analysis 16 A microfluidic cartridge for multiplexed clinical assays Superior Universal Fluidic Multiplex Advantages Specimen Manipulation Molecular Urine Analysis No PCR Delivery Saliva Minutes to result Separation Whole blood Genetic assay Low LOD(fM, pg/ml) Mixing Serum, Plasma Immunoassay Dynamic range Concentration Culture media Electrolyte, Ions Electrokinetic Concentrator Target binding Sample brief and rapid sample delivery prep. Almost no DNA extraction step BioElectronics with realtime flow impedance monitoring <10 min total assay time 16
    • New Technology for new process in MDx Revolutionizing molecular analysis 16 A microfluidic cartridge for multiplexed clinical assays Superior Universal Fluidic Multiplex Advantages Specimen Manipulation Molecular Urine Analysis No PCR Delivery Saliva Minutes to result Separation Whole blood Genetic assay Low LOD(fM, pg/ml) Mixing Serum, Plasma Immunoassay Dynamic range Concentration Culture media Electrolyte, Ions Electrokinetic Concentrator Target binding Sample brief and rapid sample delivery prep. Almost no DNA extraction step BioElectronics with realtime flow impedance monitoring <10 min total assay time 16
    • Revolutionizing molecular MDx-Automation-Connectivity analysis 17 New concept of MDx and telecommunication Device Level Data Transmission System Level Wireless communication Physical cable connection Telecommunication Portable device Transmission Clinical data station  Security protection1  Security protection1  SSL encryption3  User authentication2  User authentication2  Integrity control5  Data encryption3  Data encryption6  Digital certificates2  Authetication4  Audit control7 1-7: Security Standards for the Protection of Electronic Protected Health Information 17
    • From Clinical Chemistry Environment to MDx 18 Introducing clinical chemistry instrumentation parts in MDx platform The Vidiera NSP Primary tubes on racks NA extraction by filtration Qiagen chemistry Quantification by spectrometry Normalization PCR setup Archieving Beckman Coulter 18
    • Connectivity in High Throughput MDx settings 19 Abbott Molecular Siemens 19
    • Connectivity in High Throughput MDx settings 20 Roche Diagnostic 20
    • Connectivity in High Throughput MDx settings 20 Roche Diagnostic 20
    • MDx new fields of application 21 Personalized medicine has brought attention onto SNP analysis procedures in MDx Oncology developments have made gene expression analysis a growing field for MDx Emerging infectious diseases, drug resistance monitoring, NextGeneration Sequencing instruments have renewed the interest for sequence based MDx 21
    • MDx outside realtime PCR 22 SNP and Gene Expression assays are missing consensus technology in routine diagnostic, except for low complexity assays that are making use of realtime PCR. Sequencing technologies mature, but processing is long and missing automation. The promise of NextGen sequencing technologies. 22
    • SNP analysis MicroArrays Sequencing Monoplex to Low Multiplexing Affymetrix-Roche CYP450 Capillary sequencing driven Realtime PCR very 1st FDA available kit still technical demanding not fully automated Custom Multiplex Flexibility Low-Cost solutions (manual) ABI-SNPlex Greiner BioOne CE-IVD chips Core facility oriented task Sequenom-iPlex-MassArray Eppendorf BioChip Beckman Coulter-SNPStream LGLife Sciences Whole Genome Sequencing- DrChip NextGeneration Sequencing Low to None Flexibility but very technologies high Multiplexing Low-Cost solutions (semi-automated) lot of interest iLLumina-Golden Gate ClonDiag-Genomica CE-IVD chips future of modern medicine, personalized iLLumina-Infinium Autogenomics IVD, RUO medicine Affymetrix-GeneChip Osmetech IVD Hybribio Still work to do on: BioCore cost/patient process automation Newest alternatives (fully automated) BioIT process Nanosphere CE-IVD GeneFluidics RUO 23 23
    • Increasing interest for automated micro-array processing 24 Osmetech Autogenomics Greiner BioOne ClonDiag Genomica Nanosphere 24
    • Roche GS-FLXti Roche GS-FLXti: 0.4 Gb/run Setup time: 3-4 d 1m reads @ 400b 0.4Gb/run Run time: 10 hrs images: 27 GB $7768/run 0.4Gb Primary Analysis: 15 GB $19.41/Mb PA CPU time: 220 hrs $648k/inst. Final file size: 4 GB AB Solid 3.0 AB Solid 3.0: 10-20 Gb/run Setup time: 3-5 d NextGen 100m reads @ 50b 5-12.5 Gb/run/slide Run time: 3.5-10 d Sequencing images: 2.5 TB $6873/run 5+5Gb Primary Analysis: 750 GB $0,69/Mb PA CPU time: in run time $599k/inst. Final file size: 140 GB Illumina GA2: Illumina GA2 5-10 Gb/run Setup time: 2-3 d the future 60m reads @ 50b 6-11 Gb/run Run time: 3-6 d of MDx? images: 900 GB $8250/run 5Gb Primary Analysis: 350 GB $0,33/Mb PA CPU time: 100 hrs $460k/inst. Final File Size: 75 GB 25 25
    • Roche GS-FLXti Roche GS-FLXti: 0.4 Gb/run Setup time: 3-4 d 1m reads @ 400b 0.4Gb/run Run time: 10 hrs images: 27 GB $7768/run 0.4Gb Primary Analysis: 15 GB $19.41/Mb PA CPU time: 220 hrs $648k/inst. Final file size: 4 GB AB Solid 3.0 AB Solid 3.0: 10-20 Gb/run Setup time: 3-5 d NextGen 100m reads @ 50b 5-12.5 Gb/run/slide Run time: 3.5-10 d Sequencing images: 2.5 TB $6873/run 5+5Gb Primary Analysis: 750 GB $0,69/Mb PA CPU time: in run time $599k/inst. Final file size: 140 GB Illumina GA2: Illumina GA2 5-10 Gb/run Setup time: 2-3 d the future 60m reads @ 50b 6-11 Gb/run Run time: 3-6 d of MDx? images: 900 GB $8250/run 5Gb Primary Analysis: 350 GB $0,33/Mb PA CPU time: 100 hrs $460k/inst. Final File Size: 75 GB 25 25
    • Numerous fields of applications 26
    • HIV Ultra Deep Sequencing for drug resistance monitoring 4000X coverage minority populations detection 27
    • NextGen Roche GS-FLX: Sequencers Workflow 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) 3. Data generation via sequencing-by-synthesis (9 hours) 4. Image and Base calling analysis (~8 hours) GS-FLX Software 5. Data analysis using different bioinformatics tools ▪GS Reference Mapper •Long Single Reads / Standard Shotgun (required input = 3–5μg,5μg recommended) ~1,000,000 single reads with an average read length of 400 bases ▪GS De Novo Assembler •Paired End Reads (required input = 5μg @25 ng/μl or above, in TE; >10kb) ▪GS Amplicon Variant ◦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 Analyzer 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. Third Party Software •Amplicon Sequencing (1-5ng or 10-50ng) ◦The DNA-sample preparation for Amplicon Sequencing with the GS FLX System consists of a simple PCR amplification reaction with special Fusion Primers. The Fusion Primer consists of a 20-25 bp target-specific sequence (3' end) and a 19 bp fixed sequence (Primer A or Primer B on the 5' end). 28
    • NextGen Roche GS-FLX: Sequencers Workflow 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) 3. Data generation via sequencing-by-synthesis (9 hours) 4. Image and Base calling analysis (~8 hours) GS-FLX Software 5. Data analysis using different bioinformatics tools ▪GS Reference Mapper •Long Single Reads / Standard Shotgun (required input = 3–5μg,5μg recommended) ~1,000,000 single reads with an average read length of 400 bases ▪GS De Novo Assembler •Paired End Reads (required input = 5μg @25 ng/μl or above, in TE; >10kb) ▪GS Amplicon Variant ◦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 Analyzer 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. Third Party Software •Amplicon Sequencing (1-5ng or 10-50ng) ◦The DNA-sample preparation for Amplicon Sequencing with the GS FLX System consists of a simple PCR amplification reaction with special Fusion Primers. The Fusion Primer consists of a 20-25 bp target-specific sequence (3' end) and a 19 bp fixed sequence (Primer A or Primer B on the 5' end). 28
    • Conclusion 29 Realtime PCR has been a revolution for MDx New procedures and lab organization A 2 step process: NA extraction and PCR/detection Bringing a new capacity for simple automation Huge development and options for NA extraction mag. beads procedures as a standard Integration of realtime PCR step through full automated instrument 29
    • Conclusion 30 Development of fully integrated instrument for MDx is bringing new concept and technologies Microfluidics, bioelectronics, nanotechnologies Telecommunication integration vs clinical chemistry settings integration The needed evolution of SNP procedures Finally low cost micro-array platforms with options for semi and full automation The next upcoming revolution in MDx: making use of NextGeneration instrumentation in routine clinical diagnostic settings 30