VeraCode TechnologyCytokine Sandwich Assay Overview PE PE PE Cytokine Antigens Biotinylated...

© 2009 Illumina, Inc.Illumina, Making Sense Out of Life, Sentrix, GoldenGate, DASL, Oligator, Infinium, GenomeStudio, BeadArray, Array of Arrays, BeadXpress, VeraCode, IntelliHyb, iSelect, CSPro, and Solexa are registered trademarks or trademarks of Illumina Inc.

VeraCode TechnologyFrom Research to DiagnosticsJune 22, 2009

Most Complete Genomics Assay Portfolio

Solexa Sequencing

PLATFORM

APPLICATIONValidation,

Screening &Standard Tests

Whole-Genome Targeted AnalysisSequence-Based Discovery

BeadArray BeadArray VeraCode

MARKER DENSITY

Infinium iSelectGenotyping Panels

6K-60K

GoldenGateGenotyping Panels

96-1536

Resequencing

HumanHap650Y

Human610-Quad

Human1M-Duo

Exon510-Duo

HumanCNV370-Duo

Human 6/Ref-8 Gex BovineSNP50

SNP & CNV Discovery

De novo Sequencing

CanineSNP20

African AmericanAdmixture GG

MHC GG

CNV-12

MethylationCancer Panel I

Linkage-12

DASL Expression Panels

384-1536

Infinium AssayDirect Hyb

Infinium Assay GoldenGate & DASL Assay

VeraCodeGoldenGate

96- & 384-plex

Universal Capture

Carboxyl - Protein

GoldenGate& DASL Assay

Sequencing bySynthesis

Custom Standard

PRODUCT

TECHNOLOGY

240K to 1M6K to 60K (iSelect)

96 to 1536 (GoldenGate)1 to 384-plex1base to >15GB Custom

BeadXpressiScaniScanGenome Analyzer

Tag Expression

Transcript Profiling

ChIP Sequencing

Mouse 6/Ref-8 Gex

Rat Ref-12 Gex

DASL Expression

GoldenGate Methylation Panels

384-1536Micro RNAMeDIP Sequencing

BiSulfite CpG

Methylation

2008 Release Genome-wideMethylation27

Illumina’s Solution for Diverse Applications

Cylindrical glass microbeads

240 μm length x 28 μm diameter

VeraCode® Technology

VeraCode Technology

CODE IMAGE

1 0 1 1 0 1 0 1 0 0 0 0 1 0 1

BINARY CODE DECIMAL CODE= 41133

CODE GENERATED BY CREATING EMBEDDED HOLOGRAPHIC DIFFRACTIVE ELEMENTS

High Density Codes Easily Imprinted (24 bit)

Virtually Unlimited Unique Bead Codes Available –626 unique codes used in launched products

Glass Surface of Beads Ideal for Bioassays –numerous surface chemistries available

Specific panels created easily with addition or removal of specific bead types

CONVENTIONALCCD CAMERA

“READING”BEAM

Easily adaptable to changing content requirements

+Beads Pooled in

96-well Plates

(48 to 384-plex/well)

Individual Tubes of VeraCode Beads

VeraCode Beads Pre-plated into standard

product

Customized panels created by pooling

different tubes of beads

BeadXpress™ Reader

INSTRUMENT SPECIFICATIONS

Regulatory Design ControlREGULATORY

Optional 1 or 2 color detectionDETECTION

120 samples/hr at 10-plex (two-color detection)80 samples/hr at 96-plex (two-color detection)THROUGHPUT

1 to 384MULTIPLEXING PER SINGLE WELL

Standard 96-well plate & Stripwell PlatesSAMPLE DELIVERY FORMAT

ASSAY BEAD KIT

SAMPLE PLATE

ASSAYCODE

LIBRARY

DATA & LOG FILES

VeraCode Technology

EIGHT WELLS ARE SAMPLED SIMULTANEOUSLY AND MICROBEADS ARE TRANSFERRED INTO A MULTI-SECTORED READER CELL.

VeraCode Technology

GROOVE PLATECROSS-SECTION

BEADS FALLINTO GROOVE

CAPILLARY FORCE ATTRACTS BEADS INTO GROOVES

BEADS ALIGN TIGHTLY FOR OPTIMAL SCANNING EFFICIENCY

120140

180200

0 20 40 60 80 100

406080

100120140160180200220240

0 20 40 60 80 100

Code DetectionBEAD

“READING”BEAM

CCD LINE ARRAY

VeraCode Technology

FOLLOWING READOUT, THE BEADS ARE FLUSHED FROM THE READER CELL USING A SECOND ACTUATION PRESSURE CYCLE.

Data Analysis Streamlined with BeadStudio

VeraCode Custom Products

Easily adaptable to changing content requirements

+Beads Pooled in

96-well Plates

Individual Tubes of VeraCode Beads

VeraCode Beads Pre-plated into standard

product

Customized panels created by pooling

different tubes of beads

Comprehensive VeraCode product portfolio

VeraCode Carboxyl Bead Sets

VeraCode Universal Capture Bead Sets

Custom GoldenGate Genotyping, 48 to 384-plex

DASL Custom Gene Expression, 96 & 384-plex

Custom GoldenGate Methylation, 96 & 384-plex

Available Products

CARBOXYL BEAD SETS (PROTEIN BASED ASSAYS)

Unique Bead Code Types in

Assay DesignGuidelines

CUSTOMER DEVELOPED ASSAY

GUIDELINESPRODUCT

Customer controls level of multiplexing and number of samples

Activate Surface Covalently BindEDC/Sulfo-NHSAntibodiesAntigensPeptides

Streptavidin

NUCLEIC ACID ATTACHMENT: ONE-STEP PROCESS

EDC/Sulfo-NHS plus NH2+Oligo NucleicAcids

PROTEIN ATTACHMENT: TWO-STEP PROCESS

Carboxyl Beads: Chemistry of Attachment

Cytokine Sandwich Assay Overview

CytokineAntigens

BiotinylatedSecondary Antibody

StreptavidinPhycoerythrin

PEPEPE

Antibodies immobilizedvia COOH surface and EDClinker chemistry

VeraCode Bead

Available Products

UNIVERSAL CAPTURE BEAD SETS

Unique Bead Code Types in

Assay DesignGuidelines

CUSTOMER DEVELOPED ASSAYS

GUIDELINESPRODUCT

Customer controls level of multiplexing and number of samplesVeraCode Primer

Design Software

Allele-Specific Primer Extension (ASPE)

Biotin-NTP’sIncorporated

Wild-Type

Variant

Primer Not ExtendedPrimer Extended

MismatchMatch

The VeraCode Assay Designer Solution

Access user-specific accounts

Input SNP targets

Choose workflow option

Customize design parameters

Select among possible assay designs

Download detailed ordering information

Assay Design Results

Example output of an assay design run

Summary Results

Primer Designs

Customer Feedback: Software Required for Multiplex Assays

VeraCode Luminex

Assay Designer Primer 3Primer Design

Multiplex Compatibility Testing mfold

Tag-ITUniversal Oligo Tagging

Order File Generation

SAP/EXO

Prepare Microtitre

PlateMultiplex

ASPEMultiplex

Multiplex ASPE Genotyping Protocol

Add ASPESample Hybridize Detection

User definedKitting Options

Total Hands On Time ~ 1.5 hr/plateTotal Time (Sample to Answer) ~ 6.5 hours

Bead Kitting Workflow – VeraCode vs. Luminex

Centrifuge

Sonicate

Vortex

Transfer to plate

Centrifuge; remove supernatant

Transfer to kit, add buffer & shake

Aspirate excess buffer, seal plate

Light Sensitive

Wash: add buffer; vortex; repeat

Low Plex ASPE Genotyping Results

0 10000 20000 30000 40000

Intensity (A)

0 2000 4000 6000 8000 1000012000 1400016000 180002000022000 2400026000

Intensity (A)

16000In

0 10000 20000 30000

Intensity (A)

MTHFR667

0 2000 4000 6000 8000 100001200014000160001800020000220002400026000

Intensity (A)

MTHFR1298

≥ 99.99% reproducibility

100% concordant genotypes

Customer Success

Sciona – Health and Wellness company offering genetic testing and personalized exercise, diet and lifestyle recommendations

– Validated assays presented in October 28, 2008 Webinar1

– GSTM1: Deletion of GSTM1 associated with compromised detoxification– GSTT1: Deletion of GSTT1 associated with compromised detoxification– ALDH2: 487 G->A associated with alcohol intolerance– CYP1A1: 462 A->G associated with increased metabolism of carcinogens

Children's Hospital of Eastern Ontario – Newborn screening laboratory testing 135,000 children per year

– Assay development presented at 2008 Illumina Seminar Series2

– HbC, HbE, HbSickle, HbO and HbF: Hemaglobinopathy panel – SMA – Spinal Muscular Atrophy assay looks at gene copy number variation

associated with severity of muscular disease

1: http://www.illumina.com/webinars/archives/archivesWebinars.ilmn?entry=50&w=20

2: http://www.illumina.com/seminars/archive/Crocker_0808_NewbornScreening.pdf

Available Products

GOLDENGATE GENOTYPING ASSAY (48, 144, 192 & 384-PLEX)

Assay specific oligos

+Reagents

GoldenGate Genotyping User Manual

GOLDENGATEGENOTYPING

+Beads Pooled in

96-well Plates

USER MANUALCustom GoldenGate

GoldenGate Assay Used for Majority of the International HapMap Project

10.0%3, 8p, 21The China HapMap ConsortiumChina

8q, 9, 18q, 22, X

4q, 7q, 18p, Y

Illumina

Baylor

UCSF & Washington University

United States

10.0%2, 4pThomas Hudson, McGill University

Canada

24.0%1, 6, 10, 13, 20David Bentley, Wellcome Trust Sanger Institute

United Kingdom

25.1%5, 11, 14, 15, 16, 17, 19Yusuke Nakamura, RIKEN/University of Tokyo

% of GenomeChromosome AssignmentResearch Group and LeadersCountry

>1,000,000 SNP assays developed285,000,000 genotype calls made~70% done with Illumina GoldenGate assay using the Sentrix BeadArray Reader

AddressAllele Specific Extension &Ligation

Universal PCR Sequence 1

Universal PCR Sequence 2

Universal PCR Sequence 3’

GoldenGate Genotyping AssayAllele Specific Extension and Ligation

Genomic DNA [T/C] Ligase [T/A]Polymerase

A Address #561Amplification Template

PCR with Common Primers

GoldenGate Genotyping AssayAmplification

Cy3 Universal Primer 1

Cy5 Universal Primer 2

Universal Primer P3

Hybridization to VeraCode Beads – one SNP per bead type

GoldenGate Assay – two color detection

/\/\/\

illumiCode #561

illumiCode #217

illumiCode #1024

/\/\/\

A/A G/G C/T

SNP #561 SNP #217 SNP #1024

Bead Code 6457

Bead Code 6002

Bead Code 5433

GoldenGate Assay Results

41,184

Total # of Data Points

100%100%99.9%28696

HeritabilityReproducibilityCall RateSamples Tested

Reaction Multiplex

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs2020911

48 134 104

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs3136329

111 131 420 0.20 0.40 0.60 0.80 1

Norm Theta

rs6748810

97 145 420 0.20 0.40 0.60 0.80 1

Norm Theta

rs1800937

2 42 2410 0.20 0.40 0.60 0.80 1

Norm Theta

rs1005056

252 33 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs2059520

97 137 450 0.20 0.40 0.60 0.80 1

Norm Theta

rs4148217

10 96 1770 0.20 0.40 0.60 0.80 1

Norm Theta

rs13320360

268 13 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs1498311

28 106 1520 0.20 0.40 0.60 0.80 1

Norm Theta

rs13282019

0 44 2390 0.20 0.40 0.60 0.80 1

Norm Theta

rs673548

12 77 1960 0.20 0.40 0.60 0.80 1

Norm Theta

rs4784744

44 152 900 0.20 0.40 0.60 0.80 1

Norm Theta

rs3825585

0 38 246

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs4289236

19 82 1840 0.20 0.40 0.60 0.80 1

Norm Theta

rs10514805

0 33 2510 0.20 0.40 0.60 0.80 1

Norm Theta

rs4953020

27 105 1530 0.20 0.40 0.60 0.80 1

Norm Theta

rs6720173

8 84 1940 0.20 0.40 0.60 0.80 1

Norm Theta

rs3136245

29 75 1820 0.20 0.40 0.60 0.80 1

Norm Theta

rs1799898

13 61 2120 0.20 0.40 0.60 0.80 1

Norm Theta

rs820299

98 147 41

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs3258

135 101 500 0.20 0.40 0.60 0.80 1

Norm Theta

rs5927

16 95 1730 0.20 0.40 0.60 0.80 1

Norm Theta

rs8110695

11 70 2030 0.20 0.40 0.60 0.80 1

Norm Theta

rs9326954

94 140 520 0.20 0.40 0.60 0.80 1

Norm Theta

rs3791981

224 59 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs4952887

11 44 2290 0.20 0.40 0.60 0.80 1

Norm Theta

rs1042031

8 103 1750 0.20 0.40 0.60 0.80 1

Norm Theta

rs2072447

173 89 24

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs2569538

1 31 2540 0.20 0.40 0.60 0.80 1

Norm Theta

rs4647247

0 13 2700 0.20 0.40 0.60 0.80 1

Norm Theta

rs10515198

0 40 2400 0.20 0.40 0.60 0.80 1

Norm Theta

rs1801702

3 11 2710 0.20 0.40 0.60 0.80 1

Norm Theta

rs4647229

276 6 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs1800947

23848 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs265431

225 59 1

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs2645424

51 136 940 0.20 0.40 0.60 0.80 1

Norm Theta

rs9353019

1 21 2640 0.20 0.40 0.60 0.80 1

Norm Theta

rs2303152

0 33 2500 0.20 0.40 0.60 0.80 1

Norm Theta

rs5882

117 119 500 0.20 0.40 0.60 0.80 1

Norm Theta

rs1616534

83 153 470 0.20 0.40 0.60 0.80 1

Norm Theta

rs1736058

4 96 1800 0.20 0.40 0.60 0.80 1

Norm Theta

rs1800932

196 81 90 0.20 0.40 0.60 0.80 1

Norm Theta

rs3805586

30 139 1170 0.20 0.40 0.60 0.80 1

Norm Theta

rs13425206

0 19 2650 0.20 0.40 0.60 0.80 1

Norm Theta

rs4987188

4 12 2700 0.20 0.40 0.60 0.80 1

Norm Theta

rs289741

128 112 45

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs9326953

168 111 70 0.20 0.40 0.60 0.80 1

Norm Theta

rs748766

91 114 800 0.20 0.40 0.60 0.80 1

Norm Theta

rs12056593

280 6 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs1146634

78 157 510 0.20 0.40 0.60 0.80 1

Norm Theta

rs5930

63 127 930 0.20 0.40 0.60 0.80 1

Norm Theta

rs4647221

253 31 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs4236927

263 23 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs7607312

70 159 560 0.20 0.40 0.60 0.80 1

Norm Theta

rs267307

21963 20 0.20 0.40 0.60 0.80 1

Norm Theta

rs916042

19 35 2300 0.20 0.40 0.60 0.80 1

Norm Theta

rs6741393

0 23 263

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs6718187

64 138 84

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs5880

3 24 259

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs533617

261 24 0

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs17320429

257 19 10

Inter-User Variability

9696OPA multiplex

100%99.97%

100%100%99.96%

User A

98.94%99.74%

100%100%99.92%

User B

99.97%> 99.0%Call Rate100%> 95%Sample Success Rate

100%> 99.5%Heritability (Parent/Parent/Child)

Results

100%> 90%Locus Success Rate

100%100%

User C

> 99.0%Reproducibility> 99.5%Concordance

144Bead Multiplex

95Number of DNA Samples

TargetSpecification

Available Products

VERACODE DASL CUSTOM GENE EXPRESSION (32 to 384-transcripts)

+Reagents

DASLGENE EXPRESSION

+Beads Pooled in

96-well Plates

(96 & 384-plex/well)

USER MANUALCustom DASL

The DASL Assay

/WV/WV

Product capture by hybridization to array

PCR with common primers

Address’P1

Address

Total RNA

cDNA synthesis

Query oligo annealing, extension, and ligation

cDNA synthesis includes random priming for fragmented RNAs.

Sequences of ~ 50 nt are targeted for analysis by addressed query oligos.

PCR primers are shared among all targets, and amplicons are a uniform size.

Reproducible across a wide range of RNA degradation.

VeraCode DASL Assay Workflow

2-day assay from sample to answer

100 ng for intact RNA; 200 ng for FFPE RNA

Pre-kitted VeraCode bead plates in 96 or 384-plex (32 to 384 transcripts)

Same controls as found in DASL on BeadArray

Each bead plate contains 30-fold redundancy for each unique VeraCode bead code

Data Analysis performed on BeadStudio GX module

Fresh Frozen and FFPE RNA Reproducibility on VeraCode

Fresh Frozen Prostate Normal

0 10000 20000 30000 40000 50000 60000

Intensity Replicate 1In

FFPE Prostate Normal

0 10000 20000 30000 40000 50000 60000

Intensity Replicate 1

Fresh Frozen Prostate Tumor

0 10000 20000 30000 40000 50000 60000

FFPE Prostate Tumor

0 10000 20000 30000 40000 50000 60000

Fresh Frozen Tumor vs Normal

0 10000 20000 30000 40000 50000 60000

Intensity Normal

FFPE Tumor vs Normal

0 10000 20000 30000 40000 50000 60000

Intensity Normal

RNAs derived from fresh frozen or FFPE human tissues were analyzed.

FFPE RNAsdemonstrated nearly equivalent reproducibility compared to intact RNAs (r2 > 0.98).

Performance Summary

r2 > 0.98Overall reproducibility of technical replicates (FFPE RNA)

ValueMetric

< 1%False positive rate

> 92%Concordance with BeadArray for Number of Probes Detected

r2 > 0.99Reproducibility between VeraCode bead lots

r2 > 0.98Reproducibility between operators

r2 > 0.99Overall reproducibility of technical replicates (intact RNA)

Available Products

VERACODE GOLDENGATE METHYLATION (48 to 384-promoter regions)

+Reagents

DASLGENE EXPRESSION

+Beads Pooled in

96-well Plates

(96 & 384-plex/well)

USER MANUALCustom DASL

GoldenGate Methylation Assay

1. Bisulfite treatment of DNA● Convert unmethylated cytosine to uracil

2. GoldenGate Assay• DNA Activation

• Allele-Specific Extension (ASE) & Ligation

• PCR Amplification

• Hybridize to Bead Substrate

• Scan

GoldenGate Methylation AssayHybridization & Detection

IllumiCode

CpG locus 1 CpG locus 2

CpG locus 3

Unmethylated

Methylated

Semi-methylated

VeraCode Methylation Data Analysis

Data analysis performed on BeadStudio Methylation module

Internal replication experiments show an r2

correlation of 0.997

Experiments comparing methylation results for the same loci on BeadArray and VeraCode show high concordance (r2 = 0.97)

Ability to Analyze Multiple Applications

100000

10.1 1 10 100 1000 10000

Conc (pM)

AnalyteFit3-sigma LOD

GENE REGULATION PROTEIN EXPRESSION

GENOTYPING

0 1 2 3 4 5

Log10(Conc pg/ml)

IL-6 (6152)

Bridging from the lab to the clinic

BeadXpress

• Collaborative approach to research

• Constantly improving throughput

• Excellent cost position

• Highly multiplexed

• Multiple markers on one system

Laboratory Translate Clinic

Genome Analyzer

Services

Oncology Discovery – Identify novel biomarkers

Sequencing Services – Utilize service lab for rapid translation

Platform Partnering – Build a large installed base

Illumina Diagnostics StrategyTo be the leader in cancer translational diagnostics

• Identify novel biomarkers for the early detection of ovarian cancer• Identify novel biomarkers to indicate response to platinum therapy in late stage ovarian cancer

• Establish a CLIA laboratory• Begin to offer services in the oncology area based on the ovarian biomarkers

• Partner with content providers• Enable broad adoption with removal of royalty burden• Internally develop products to create breadth of menu

To Be the Leader in Cancer Translational Diagnostics

Illumina Diagnostics Vision

Next Generation Sequencing

Whole Transcriptome Analysis

Methylation Profiling

Focus Core Technologies on Cancer

Initial Targets

Source: WHO

Stomach

Colon/ rectum

Breast

Percentage of All Cancers

0 6 8 10 12 140

KidneyCervix/ Uteri

Melanoma

Bladder

Testis

Thyroid

Prostate

Esophagus

MyelomaLeukemia

Pancreas

Nasopharynx

Ovarian Cancer Will be Our Entry Point

Deadly

Leading cause of death from gynecological cancerFifth leading cause of cancer mortality in womenMortality: 15,520 (US Only)

Laparotomy/Hysterectomy/Remove ovariesFollowed with six cycles of chemotherapyLess than 40% are cured

Incurable

Abdominal/pelvic exam, Ultrasound, CT, CA-125Patients present with a suspicious/palpable massBRCA 1/2 positive in a limited number of cases

Difficult Dx

Early Detection for Ovarian Cancer is Critical

Cumulative % of Cases

19% 22%

30% 33%37% 40%

Ia Ib Ic Iia Iib Iic IIa IIIb IIIc IV

Stage at Diagnosis

Late stage diagnosis …

5 Year Survival92%

85%82%

56%51%

17%12%

Ia Ib Ic Iia Iib Iic IIa IIIb IIIc IV

… results in poor prognosis

Gastric Cancer will be Our Second Target

Cardia 15% of Gastric CancersIncreasing due to obesity

Normal

Superficial Gastritis

Atrophic Gastritis

Small intestine Metaplasia

Colonic Metaplasia

Dysplasia

Carcinoma

High pH

Bacterial Growth

N=O Mutagens

NO3-Ascorbic Acid

NaClBeta Carotene

Gastric Carcinogenesis

Sarcoma

Bladder

Cervical

Stomach

Head/Neck

Myeloma

Ovarian

Kidney

Cancer-Related Conditions

Pancreatic

Other Cancers

Lymphoma

ColoRectal

Breast

Prostate

Leukemia

Unspecified Cancers

Solid Tumors

Transplantation

Growth Disorders

Eye Conditions

Skin Disorders

Genetic Disorders

Blood Disorders

Respiratory Disease

Digestive Disorders

Diabetes

Neurological Disease

AutoImmune

Infectious Disease

Cancer

More Therapies Means More ChoicesDrug Pipeline

Source: PhRMA: 2006 Medicines in Development Biotechnology

Discovery, Strategy & AdvantagesFocused Approach Addresses Unmet Medical Needs

Begin with 25 Tumor/Normal pairs

Comprehensive genetic analysis

Sequence to 30x coverage whole genome

Analyze full methylome and transcriptome

Validate findings on larger sample size

Implement on flexible platform

Blood TypingStrategy Illumina Advantage

Low cost internal sequencing

Significant throughput improvements

- Currently 1 genome/week internally

Unique bioinformatics capability - Multiple genome analysis

Capable of analyzing across methods

Ability to deploy content on arrays

BeadXpress optimal for multiplemethods

Illumina’s Diagnostic Division Branding

Discovery

Services

VeraCode TechnologyCytokine Sandwich Assay Overview PE PE PE Cytokine Antigens Biotinylated...

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