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Next Generation Diagnostics: Potential Clinical Applications of Illumina’sTechnology

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Rob Kelley, MBA

Rob Kelley, MBA
Global Sales Manager
Translational Genomics
September 27, 2011

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    Next Generation Diagnostics: Potential Clinical Applications of Illumina’sTechnology Next Generation Diagnostics: Potential Clinical Applications of Illumina’sTechnology Presentation Transcript

    • Next Generation Diagnostics: Potential Clinical Applications of Illumina’s Technology Rob Kelley, MBA Global Sales Manager Translational Genomics September 27, 2011© 2010 Illumina, Inc. All rights reserved.Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iSelect, CSPro, GenomeStudio, Genetic Energy, and HiSeqare registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
    • Provider of Genomic Analysis Tools That Advance the Understanding of Genetics and Health From Genome-Wide Discovery to Targeted Validation and Screening Sequencing Arrays qPCR HiSeq HiScanSQ 1000 Unique Powerful, combination Flexible, of Scalable sequencing and arrays2
    • Markets Served Molecular Dx ~$3B Applied Markets ~$1B Life Sciences ~$2.8B Consumer3
    • Dx Strategy Vision To be the leader in translational diagnostics Venue Services Platforms Applications Objectives Accelerate Revenue Build Installed Base Create a novel dx Strategies Sequencing Services Partnering Internal Development Oncology Discovery Oncology High Discovery Platforms Return Sequencing Services Low Low Risk High4
    • Platforms BeadXpress FDA cleared instrument & Factor II/V assay Q2 10 FDA approved GPR beads Q2 10 CE Mark Q4 11 iScan Cytogenetics Post-Natal Q4 11 Cytogenetics Cancer TBD Cytogenetics PGS TBD Cytogenetics Pre-Natal TBD MiSeq Pre-IDE submission to FDA Q2 11 Carrier screen 2012 Somatic Mutation Panel 20125
    • Molecular Cytogenetics6
    • Cytogenetic Technologies and Resolution HiScanSQ Infinium HD Beadchips Genome-wide analysis Targeted/focused analysis7
    • ISCA (International Standards for Cytogenetic Arrays) Consensus Statement: AJHG, May 20108
    • ACMG (American College of Medical Genetics) revised guidelines: Arrays recommended first line9
    • Large & Growing Opportunity in Array-Based Cytogenetics $400 Molecular Cytogenetics Revenue $350 Forecasts by Market Segment (US) 100 $300 Percent of Revenues (%) 90 Microarray-Based 80 FISH $250 70 Revenue (Millions) $200 60 z 50 $150 40 $100 30 20 $50 10 0 $- 2007 2008 2009 2010 2011 2012 2013 2014 2015 2009 2010 2011 2012 Array-based cytology market growing A technology substitution is underway double digits (CAGR = 16–20%) ILLUMINA IS POSITIONED FOR GROWTH10 10
    • Detection of a Wide Range of AberrationsFor Research Use Only 11
    • HumanCytoSNP-12 BeadChip: Optimized for Efficient Cytogenetic Analysis ► Streamlined, most informative set of targeted and whole- genome SNP and non-polymorphic markers ► Uniform picket fence of entire genome (including ~92% of RefSeq genes) – 300,000 markers, mostly SNPs – 6.2kb median marker spacing yields ~30kb resolution ► Higher density in cyto high-value regions (~250 for ~40% of genome) – All pericentromeres and subtelomeres – Sex chromosomes – Common regions of interest (e.g., associated with known syndromes) – Regions contain ~9000 genes ► Higher density in ~400 “disease genes”For Research Use Only 12
    • BeadArrayTM Technology Specific Probe (50 base nt) Bead Identifier (30base nt) Oligo Mfg 2 μm Silica ~18-fold Redundancy Decoding = 100% QC Population in wells13
    • The Infinium HD Assay ► Unconstrained Marker Design – Freedom to select the best, most informative SNPs then fill-in with intensity-only probes ► Well-proven – High reproducibility (> 99.9%) – High call rates (> 99%) ► Streamlined, automatable ► PCR-free protocol ► No need to run a reference sample ► High locus selectivity and allele specificity – Two-step enzymatic discrimination14
    • Copy-Neutral Cytogenetic Aberrations Forms of copy-neutral cytogenetic aberrations ► Uniparental disomy – Case in which individual receives two copies of a chromosomal region from one parent, none from the other ► Copy-neutral loss of heterozygosity (or “acquired uniparental disomy”) – Case in which one allele of a gene in a heterozygote is already inactivated and the second, “good” allele is lost without a net change in copy number. This can occur through a gene conversion event in which the chromosome region containing the inactivated allele is used as a template to repair a gap occurring in the corresponding region of the other chromosome In either case, the absence a functional allele leaves the individual vulnerable to phenotypes that may be associated to the effected gene(s)For Research Use Only 15
    • SNPs Provide More Information to Detect Copy Number Normal (diploid) Deletion (loss of one copy) Duplication (gain of one copy) Copy-Neutral LOH (UPD) ► Also can detect: – Amplification – Unbalanced aberration Log R Ratio B Allele Frequency – Aneuploidy – Mosaicism Intensity GenotypesFor Research Use Only 16
    • SNP-based Detection Provides More Information and Enables Better Characterization of Chromosomal Aberrations Detected duplication B allele frequency data (AAB genotype) A shift in the LogR value is detectible, but the integration of B allele data improves the signal to noise ratio A profile of chromosome 3 of a cell line derived from a breast tumor.For Research Use Only 17
    • Copy-Neutral Features Are Missed by Array CGH Chromosome Zoom to position Agilent Illumina Agilent Illumina18
    • KaryoStudioDx:  Illumina’s custom‐designed Cytogenetics Software19
    • KaryoStudioDx Features Highlights ► Accepts data from any Infinium HD array* ► Automated data importation ► Automated scanning for aberrations ► User permissions and authentication – Input validation ► Mosaic detection ► Inheritance confirmation (non‐paternity) ► UPD parent of origin / inheritance calculation ► % consanguinity ► Quality metric: Pass or Fail ► Integrated Chromosome Browser ► Ability to filter CNV polymorphisms ► Cross‐matching capabilities to the most popular cytogenetic databases * Certain computer requirements must be met20
    • Found Regions Displayed in KaryoStudioDxFound regions table User‐defined  Known Regions trackLink out to databases: Found Regions Found Regions  UCSC, DGV, ENSEMBL, DECIPHER, etc.Samples table with QC score21
    • Diagnostic PDF Report Generated From AberrationsReport Includes:• Sample and product information• Aberration display• Information on found region• Cross‐matches to user‐defined Known  Regions list• Custom info fields• ISCN nomenclature22
    • Plans for FDA Submission: iScan Platform Package includes Q4 ’09 Illumina submitted Pre‐IDE for Cytogenetics, followed by  additional discussions with FDA  iScan platform, arrays, reagents and software Intended use Post‐natal DD/ID/MCA Broad, genome‐wide coverage Simultaneously pursuing CE‐IVD marking (ISO 13485) – 2011/2012  iScan/KaryoStudioDx HumanCytoSNP‐12 Optimized for cytogenetics 2323
    • Molecular Diagnostics24
    • MiSeq – Finally a sequencer designed with Dx customers in mind Workflow Fully integrated system On-board cluster generation and data visualization Preloaded reagent cartridge 1 flow cell lane per run Performance Up to 5M clusters 2 x 150 bp in under 28 hours RFID reagent & flow cell tracking Auto flow cell positioning Walk-away automation Go Load25
    • MiSeq – Prep, Run, Analyze Sample to Data in as Little as Eight Hours* Amplicons Clones 08:00:00 gDNA MiSeq is the Only Personal Sequencing System Capable of an 8 Hour Sample to Data Workflow26 *1x36bp run – 3 hr sequencing
    • TruSeq Targeted Resequencing The simplest and most scalable targeted resequencing solutions TruSeq Exome Enrichment Targets = 100,000s TruSeq Custom Enrichment Targets = 1000s TruSeq Custom Amplicon Targets = 100s Nextera PCR Amplicons Targets = 10s27
    • Coming soon! TruSeq Custom Amplicon Sequencing Unprecedented amplicon and sample multiplexing ► Rapid & Economical – Up to 384 amplicons per sample, 96 samples per plate (36,864 reactions) – Plate based processing – <8 hrs from DNA to sequencing‐ready library – No gels, no fragmentation – uses standard lab equipment ► Fully customized target probes and capture – Extension and ligation based assay ► Interactive probe design and ordering – Personalized and easy to use design tool  – Rapid design turnaround – as little as 10 days from  design to assay shipment ►28
    • TruSeq Custom Amplicon Assay Time 96 samples & 384 targets: from DNA to called variants in ~2 days Hybridization Assay Library Cluster Gen & Real-time Setup Biochemistry Normalization Sequencing Analysis Oligos, Extension & Create pooled Pre-kitted Alignments, universal Ligation, PCR library, sequencing variant calling reagents with index normalize reagents 8am – Day 1 2pm – Day 1 5pm – Day 2 <8 hr assay with <3 hr hands-on time No fragmentation required No gel purification steps No additional hardware29
    • TSCA throughput and coverage on MiSeq How many samples can be run together? ► Coverage Uniformity spec: >80% bases covered at 0.2x mean coverage – e.g. if mean coverage is 100x, then >80% bases covered at 20x ► Users need to carefully plan how many samples are sequenced together based on number of amplicons, to achieve desired coverage30
    • MiSeq - Comparison to CE SequencingExample: TruSeq Custom Amplicon with 96 Samples x 384 Targets Dispense, PCR, Cleanup Quant Dye-terminator sequencing, cleanup 3730xl CE 96 x 384 well 96 x 384 well 5-6 weeks plates plates ~$3/amplicon One plate of gDNA+oligos Assay Biochemistry Pool Libraries and for all 384 amplicons Sequence TSCA TruSeq Custom 96-well plate AssayAmplicon MiSeq biochemistry •Highly multiplexed 4 days •Integrated sample indexing •Standard lab equipment <$0.75/amplicon31
    • MiSeq Applications A huge variety of applications can be done on MiSeq* Application Read Length Kit Config. Sample Prep Kit TruSeq Custom Amplicon 2 x 150 300PE TSCA Nextera Amplicon 1 x 36 50PE NXT Standard Amplicon 2 x 150 300PE TS DNA or HB Small Genome - De Novo 2 x 150 300PE TS DNA Small Genome - Reseq., plasmids 1 x 36, 2 x 150 50PE / 300PE TS DNA or NXT 16S Metagenomics (amplicon) 2 x 150 300PE TS DNA, NXT, HB Library QC 2 x 25 50PE Open Small RNA 1 x 36 50PE TS smRNA RNA-Seq (human, mammalian) 2 x 50 50PE TS RNA RNA-Seq (bacterial, viral) 2 x 150 300PE RZ + TS RNA TruSeq Custom Enrichment 2 x 50 300PE TS DNA ChIP-Seq 1 x 50 50PE TS DNA TSCA = TruSeq Custom Amplicon TS DNA = TruSeq DNA Sample Prep NXT = Nextera TS RNA = TruSeq RNA Sample Prep TS smRNA = TruSeq Small RNA Sample Prep HB = Homebrew RZ + TS RNA = RiboZero + TruSeq RNA Sample Prep *Applications can be performed, but MiSeq platform may not be the most optimal solution for a particular application. There will be situations where HiSeq, GA or HiScan SQ are better suited to a particular application.32
    • MiSeq Characterization of Genetic Variations in Tumor Tissues ► Deep sequencing of cancer samples to detect somatic mutations, gene amplifications and germline variants that influence patient treatment decisions ► Genes under consideration include AKT1, ALK, BRCA1, BRCA2, BRAF, COMT, CYP17A1, CYP2A6, CYP2C8, CYP2D6, CYP3A4, DPYD, EGFR, ERBB2, FGFR2, GNAQ, KIT, KRAS, MET, MTHFR, NRAS, PDGFRA, PDGFRB, PIK3CA, PTEN, TP53, TPMT, TYMS, UGT1A1, VEGFA, VEGFR33
    • MiSeq FFPE Amplicon Data KRAS Exon 2 – 76 Base Amplicon Ovarian Rectal Gastric No-FFPE Gastric Rectal Sample Tumor Normal Tumor Control Normal Tumor Coverage 151695 X 178667 X 176530 X 179630 X 161866 X 178900 X 1.1% variant in 27.5% G C normal adjacent variant at Chr12: tissue > Assay 25398284 in rectal LOD of 0.5% tumour34
    • Illumina Experiment Manager September 2011© 2010 Illumina, Inc. All rights reserved.Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iSelect, CSPro, GenomeStudio, Genetic Energy, HiSeq andHiScan are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
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    • How to Generate Library Plates43
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    • Run Starting and Monitoring a MiSeq Run© 2010 Illumina, Inc. All rights reserved.Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iSelect, CSPro, GenomeStudio, Genetic Energy, HiSeq andHiScan are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
    • How to Start a MiSeq Run The MiSeq Control Software (MCS) is a streamlined push-button user interface designed to quickly start a sequencing & analysis run ► Minimal user input required to start a run ► A sample sheet is required to initiate a run ► User has the option to save data locally or to a BaseSpace (cloud) account51
    • MiSeq Instrument User Interface Begin sequencing by selecting “SEQUENCE” button52
    • [OPTIONAL] Log on to BaseSpace BaseSpace is a cloud option to store, analyse, and share your MiSeq data.53
    • [OPTIONAL] First Time BaseSpace User If this is the first time user is using BaseSpace, they must agree the to terms & conditions of BaseSpace use.54
    • Animation Instructions to Load Flow Cell55
    • Instructions to Load Reagent Cartridge and Waste Bottles56
    • Sample Sheet Required for MiSeq Run Sample Sheet is required for each MiSeq run ► Contains instructions on how to perform sequencing chemistry ► Also contains instructions on how to perform bioinformatics secondary analysis: – Resequencing – amplicon resequencing – de novo – small RNA – Metagenomics – library QC ► By default, MiSeq will use the Sample Sheet (in Sample Sheet repository) that matches the reagent cartridge RFID ► User may override MCS to select a user-specified Sample Sheet57
    • [OPTIONAL] User-specified Sample Sheet User may select sample sheet from alternate folder or attached USB key58
    • Review of MiSeq Run User has opportunity to review parameters of run before submitting59
    • MiSeq Performs Pre-Run Check MCS checks for dependencies to ensure run success Click here to start run60
    • MiSeq Performs Sequencing Run Current status of run Output to current BaseSpace account Name of run Realtime run metrics Connectivity status With BaseSpace61
    • MiSeq Reporter ► MiSeq Reporter (MSR) is the onboard bioinformatics engine that automatically processes MiSeq primary analysis (image and basecall) data. ► At launch, MSR supports the following reports: – Resequencing – Amplicon resequencing – de novo assembly (using Velvet) – Small RNA – 16S metagenomics – Library QC ► MSR contains a webserver so users may point browser to MiSeq instrument to view reports. ► MSR outputs may be stored on the instrument or on network folders.62
    • MiSeq Reporter63
    • MiSeq Reporter Screenshots© 2010 Illumina, Inc. All rights reserved.Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iSelect, CSPro, GenomeStudio, Genetic Energy, HiSeq andHiScan are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
    • Start Up Page Area for Illumina messages Currently run and progress and updates List of past runs Run completion status65
    • General Summary Report66
    • Resequncing Details Report Coverage and error Scope of view Zoom in/out Q score SNP/indel + annotation Table of samples/ variants67
    • Amplicon Detail Report68
    • De Novo Details Report Syntenic dot plot De novo metrics69
    • Small RNA Summary Page Trimmed read lengths70
    • Small RNA Details Report Distribution of RNA species Top 10 most abundant species Tabular information71
    • Metagenomics Summary Page72
    • Metagenomics Details Report73
    • Run Status74
    • Run Sample Sheet75
    • Run Log76
    • Run Errors77
    • Change Repository78
    • Requeue Run79
    • Questions80
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    • Clinical Whole Genome Sequencing Illumina CLIA Lab 2009 >30 Whole genomes82
    • Sequencing Informs Therapy • A patient treated with Erlotinib but lung metastases are unresponsive • Whole genome and transcriptome sequencing of the cancer on the GA reveals that • The drug target was mutated, hence the unresponsiveness • 17 genetic disruptions of a key cancer pathway • A target for the alternative FDA approved drug sunitinib was over- expressed • Sunitinib treatment was successful and the tumours regressed. Jones et al. Genome Biology 2010, 11:R82 http://genomebiology.com/2010/11/8/R8283
    • Acute Promyelocytic Leukemia (APL) Most APL patients have a characteristic translocation between chromosomes 15 and 17 that fuses the PML1 and RARA genes – These patients respond well to treatment with ATRA but treatment requires prior demonstration of the translocation or fusion gene ► A targeted PCR test of the patient’s DNA did not reveal the characteristic fusion gene ► Whole genome sequencing (in just 1 week) revealed a novel 77 kb insertion that recapitulates the translocation ► Rearrangement event confirmed by PCR ► ATRA treatment prescribed Mardis, E, Wilson R et al. unpublished84
    • Nextera DNA Sample Prep Sequencing’s fastest and easiest sample prep ► 90 min sample prep ► No Covaris required ► High throughput ► Super low 50ng input unlocks access to precious samples ► Enables a range of CE applications: amplicons, plasmids, small genomes Enables effective use of single 36bp reads ~12 hours ~1.5 hours85
    • Overall positioning of Targeted Resequencing on MiSeq PCR amplicons through TruSeq Custom Enrichment Target Number samples Price / sample Application Key Benefit Sequence / project (Prep and Seq) CE + Small sample/content < 10 kb <100 $50 Amplicons projects Nextera + Long contiguous < 20 kb 100s $80 Amplicons amplicons and speed Multiplexing and TSCA 10 – 96 kb 100s – 1000s $50 - 300 speed TSCE 700 kb – 2 Mb 100s – 1000s $260 - 800 More target content86
    • Positioning of Targeted Resequencing products TSCE TSCA 1000sSamples 96 50 Nextera CE 1 10 20 100 200 700 Mb’s Target Sequence (kb)87
    • Nextera and MiSeq Sequencing’s fastest time to answer for rapid variant analysis 1 – 8 samples 15:00:00 1 – 96 samples 08:00:00 *Based on 1 x 36 bp reads88
    • Ovarian Cancer Deadly 110% 204,449 New cases annually; 124,860 deaths Cum % of Cases 100% 5 Yr Survival 90% 92% Incurable 85% 82% Less than 40% are cured 70% 72% 69% Difficult Dx 56% 54% 50% 51% Patients present with a suspicious/palpable mass 47% 40% 39% 37% 33% 30% 30% Early Dx is Key 26% 22% 19% 17% Five year survival is good if diagnosed early, 10% 12% but most patients are diagnosed late stage Ia Ib Ic Iia Iib Iic IIIa IIIb IIIc IV -10% Illumina Solution Develop a diagnostic assay which will diagnose ovarian cancer at an early stage89
    • TruSeq Targeted Resequencing A broad suite of tools for discovery or validation experiments Amount of Option Best for Availability sequence Mendelian disease: TruSeq Exome case-control exome studies, rarer ~62 Mb Now! Enrichment variants, causal variants exome-wide linkage analysis TruSeq Custom GWAS follow-up: validation of ~1 to ~10 Mb Now! Enrichment variants, variant discovery, pathways Amplicon sequencing: high- TruSeq Custom Sub-500 Kb throughput CE experiments, ultra 2H2011 Amplicon deep seq, variant disc, screening Amplicon sequencing: ultra-deep Nextera + PCR 100’s of bp sequencing, validation, screening, CE Now! Amplicons targets replacement90
    • Meet MiSeq 52.3 cm12 cm 91
    • Oncology Biomarker Discovery Samples Discovery Validation Translation Dx Service Dx Product Frozen WGS, Gex, Methyl Targeted Seq 10 Cases/20 Controls CLIA Lab Service IVD 25 25 300 Blood, Tumor, Pap, for Diagnostics Tumor/Normals Tumor/Normals FFPE Tumors Proximal Fluids Early Detection Ovarian 25/25 40 Biomarkers 10 @ 1,000X 20 Novel 58 @ 300X Gastric 23/23 141 Biomarkers Colon 25/25 6/6 Sequenced 30 by Q192
    • Options for Targeted Resequencing with Illumina From specific, customized regions of interest to the complete coding region Amount of Option Best for Availability sequence Mendelian disease: TruSeq Exome ~62 Mb case-control studies, rarer variants, Now! Enrichment causal variants, linkage analysis TruSeq Custom ~700Kb to ~15 GWAS follow-up: validation of variants, Now! Enrichment Mb variant discovery, pathways Nextera + PCR 100’s of bp Amplicon sequencing: ultra-deep seq, Now! Amplicons targets validation, screening, CE replacement TruSeq Custom Amplicon seq: high-throughput CE, ~100 Kb 2H2011 Amplicon ultra deep seq, variant disc, screening The only company with the complete end-to-end TRS workflow solution.93
    • Simplest Sequencing Workflow Integrated workflow from sample to analyzed data TruSeq Exome Lowest cost and most scalable exome sequencing TruSeq DNA Simple, scalable and cost effective TruSeq RNA Optimized, gel-fee, low input TruSeq Small RNA Hi throughput miRNA discovery & profiling TruSeq Chemistry Clustering & Sequencing94
    • MiSeq Workflow Guide Pre-defined data analysis workflows Primary Targeted Small Genome RNA Library QC workflows Resequencing Sequencing Sequencing TruSeq Custom Amplicon De novo Nextera PCR Small RNA Amplicon Secondary Metagenomics 16S rRNA workflows Resequencing Library QC Clone Checking TruSeq Custom Enrichment RNA-Seq Automated Plasmids Semi-Automated ChIP-Seq95
    • MiSeq Applications A huge variety of applications can be done on MiSeq* Application Read Length Kit Config. Sample Prep Kit TruSeq Custom Amplicon 2 x 150 300PE TSCA Nextera Amplicon 1 x 36 50PE NXT Standard Amplicon 2 x 150 300PE TS DNA or HB Small Genome - De Novo 2 x 150 300PE TS DNA Small Genome - Reseq., plasmids 1 x 36, 2 x 150 50PE / 300PE TS DNA or NXT 16S Metagenomics (amplicon) 2 x 150 300PE TS DNA, NXT, HB Library QC 2 x 25 50PE Open Small RNA 1 x 36 50PE TS smRNA RNA-Seq (human, mammalian) 2 x 50 50PE TS RNA RNA-Seq (bacterial, viral) 2 x 150 300PE RZ + TS RNA TruSeq Custom Enrichment 2 x 50 300PE TS DNA ChIP-Seq 1 x 50 50PE TS DNA TSCA = TruSeq Custom Amplicon TS DNA = TruSeq DNA Sample Prep NXT = Nextera TS RNA = TruSeq RNA Sample Prep TS smRNA = TruSeq Small RNA Sample Prep HB = Homebrew RZ + TS RNA = RiboZero + TruSeq RNA Sample Prep *Applications can be performed, but MiSeq platform may not be the most optimal solution for a particular application. There will be situations where HiSeq, GA or HiScan SQ are better suited to a particular application.96
    • ILMN vs. Moore’s Law97
    • Illumina’s First FDA Cleared In-Vitro Diagnostic Device ► The BeadXpress System is an FDA 510(k) cleared In-Vitro Diagnostic Device ► FDA Cleared BeadXpress System includes: – BeadXpress Reader – VeraScan Software ► The Intended Use Statement: – The BeadXpress® System is an In-Vitro Diagnostic Device intended for the simultaneous detection of multiple analytes in a DNA sample utilizing VeraCode holographic microbead technology. The BeadXpress System consists of the BeadXpress Reader and VeraScan software. – It is cleared for use only with FDA cleared VeraCode tests. BeadXpress Reader and VeraScan Software98
    • Comprehensive VeraCode product portfolio Research Regulated Use Only Products VeraCode GPR VeraCode ADME Universal Capture Core Panel Beads VeraCode Universal VeraCode GPR Capture & Carboxyl Carboxyl Beads Beads Custom GoldenGate Genotyping, VeraCode PGx Panel 48, 96, 144, 192 & (in development) 384-plex DASL Custom Gene VeraCode GI Panel Expression, (in development) 32 to 384-plex Custom GoldenGate Methylation, 48 to 384-plex99
    • Fixed Content: VeraCode ADME Core Panel ► VeraCode ADME include184 polymorphisms (34 genes) – >95% Pharma ADME Core List www.pharmaadme.org ADME Core (34 genes, 184 markers) ABCB1 CYP2C9 NAT1 SULT1A1 ABCC2 CYP2D6 NAT2 TPMT ABCG2 CYP2E1 SLC15A2 UGT1A1 CYP1A1 CYP3A4 SLC22A1 UGT2B15 CYP1A2 CYP3A5 SLC22A2 UGT2B17 CYP2A6 DPYD SLC22A6 UGT2B7 CYP2B6 GSTM1 SLCO1B1 VKORC1 CYP2C19 GSTP1 SLCO1B3 CYP2C8 GSTT1 SLCO2B1For Research Use Only 100
    • Sequencing Services Illumina Clinical Services Lab Infrastucture CLIA certified for high complexity molecular diagnostics Q1 09 CAP Accredited Q2 09 CA State CLS training program Q1 10 WGS 30+ Whole genomes sequenced Q4 10 First clinical case reimbursed Q4 10 Cancer, genetic diseases, SCID cases Somatic Mutation Panel Somatic Mutation Panel launch Q4 11 Content will include genes with proven/anticipated clinical utility: KRAS EGFR BRAF TP53 VEGF-A ERBB2 ESR1 PGR TYMS UGT1A1 TPMT COMT CYP2D6 NRAS EML4/ALK101
    • Individual Genome Sequencing: Workflow Physician orders IGS for patient o Initial discussion & genetic counseling o Informed Consent and Service Agreement o Saliva and blood sample taken (DNA possible) o Cooling-off period (7+ days); order confirmed Genome sequencing and QC o Barcode samples for confidentiality o Saliva and blood genotype for ID match o Whole genome sequencing of blood DNA o Analyze and QC sequence and called variants o Check sequence and genotype ID match o Archive full dataset Clinical lab delivers data to physician102
    • Innovative Genotyping Approach [A/B] Make Target b A B Add Oligos b Extend/Ligate b Cy PCR Cy Make ssDNA Cy c Hyb VBS103
    • Delivery of Individual Genome Sequence ► Summary Report ► Consensus sequence with quality scores of calls ► SNPs report, with dbSNP designation or novel ► All individual reads, aligned to the human genome reference sequence ► GenomeStudio genome browser installed on an encrypted hard drive with variants annotated104
    • Illumina Sequencing Workflow 1 Library Preparation Fragment DNA Repair ends Add A overhang Ligate adapters Purify 2 Cluster Generation Hybridize to flow cell Extend hybridized template Perform bridge amplification Prepare flow cell for sequencing 3 Sequencing Perform sequencing Generate base calls 4 Data Analysis Images Intensities Reads Alignments105
    • Sample Prep Workflow DNA RNA106
    • Illumina Sequencing Technology Robust Reversible Terminator Chemistry Foundation 3’ 5’ DNA (0.1-1.0 ug) A G T C A G C T T A C C G G A T A A C T C C C G G A T T C G A T 5’ Sample preparation Cluster growth Sequencing107
    • Simplest Sequencing Workflow SIMPLIFIED cBot CLUSTER SEQUENCING DATA PROCESSING SAMPLE PREP GENERATION & ANALYSIS Parallel sample Automated cluster Automated Simple, efficient data processing generation sequencing analysis108
    • MiSeq FFPE Cancer Sequencing Summary • MiSeq and SBS Chemistry is capable of generating high depth of coverage sufficient enough to detect rare variants even in highly degraded DNA • The limit of detection for these types of assays is approximately 0.5%, MiSeq was able to easily detect a variant close to the limit of detection • MiSeq has the bandwidth to cover 48 amplicons from 48 samples at an average coverage depth of over 2000x109
    • Competitive Environment TruSeq Custom  Fluidigm Access  HaloPlex PCR PCR/Homebrew Amplicon Array Number of Amplicons 48 – 384 48 ‐ 480[1] < 2,000 1 ‐ 5 Target Genomic  < 96 kb < 120 kb < 400 kb < 3 kb Sequence Panel Design DesignStudio FLDM service Design Wizard Primer3/Manual Total Assay Time  7 hr (2.5 hr) 5 hr (<1 hr) 24 hr (2.5 hr est) 2 hr (0.5 hr) (hands on) Manual Batch Size  192 48 48 < 384 (samples) Order to Ship Time 10 days 3 weeks < 3 weeks[2] < 5 days Special Hardware None $75K ??? ‐ Price per amplicon < $1.00 $0.50 ‐ 0.70 $0.50 – 1.10 $0.50 – 1.00 Input DNA 0.25 µg 0.05 µg 0.9 µg < 0.25 µg End to end solution Yes No No No llumina supported  Yes No No No assay [1] Promising 10‐plex PCR   [2]  Estimated110