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

2

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

ASSAY

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

3

Illumina’s Solution for Diverse Applications

Cylindrical glass microbeads

240 μm length x 28 μm diameter

VeraCode® Technology

4

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

BEAD

“READING”BEAM

5

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

6

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

7

VeraCode Technology

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

8

VeraCode Technology

GROOVE PLATECROSS-SECTION

BEADS FALLINTO GROOVE

PLATE

CAPILLARY FORCE ATTRACTS BEADS INTO GROOVES

BEADS ALIGN TIGHTLY FOR OPTIMAL SCANNING EFFICIENCY

9

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Code DetectionBEAD

“READING”BEAM

CCD LINE ARRAY

10

VeraCode Technology

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

11

Data Analysis Streamlined with BeadStudio

12

VeraCode Custom Products

13

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

14

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

15

Available Products

CARBOXYL BEAD SETS (PROTEIN BASED ASSAYS)

Unique Bead Code Types in

Tubes

Assay DesignGuidelines

(RUO)

CUSTOMER DEVELOPED ASSAY

+

GUIDELINESPRODUCT

Customer controls level of multiplexing and number of samples

16

COOH-

COOH-

COOH-

COOH-

COOH-

COOH-

COOH-

COOH-

COOH-

COOH-

COOH-

COOH-

COOH-

COOH-

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

17

Cytokine Sandwich Assay Overview

PEPE

PE

CytokineAntigens

BiotinylatedSecondary Antibody

PE

StreptavidinPhycoerythrin

PEPEPE

Antibodies immobilizedvia COOH surface and EDClinker chemistry

VeraCode Bead

18

Available Products

UNIVERSAL CAPTURE BEAD SETS

Unique Bead Code Types in

Tubes

Assay DesignGuidelines

(RUO)

CUSTOMER DEVELOPED ASSAYS

+

GUIDELINESPRODUCT

Customer controls level of multiplexing and number of samplesVeraCode Primer

Design Software

19

Allele-Specific Primer Extension (ASPE)

Biotin-NTP’sIncorporated

Wild-Type

C B

B

Variant

AGG

C

AGG

C

Primer Not ExtendedPrimer Extended

MismatchMatch

A

20

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

21

Assay Design Results

Example output of an assay design run

Summary Results

Primer Designs

22

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

23

SAP/EXO

Prepare Microtitre

PlateMultiplex

ASPEMultiplex

PCR

Multiplex ASPE Genotyping Protocol

Add ASPESample Hybridize Detection

Scan

User definedKitting Options

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

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

24

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

25

Low Plex ASPE Genotyping Results

0 10000 20000 30000 40000

Intensity (A)

FII

0

10000

20000

Inte

nsi

ty (

B)

0 2000 4000 6000 8000 1000012000 1400016000 180002000022000 2400026000

Intensity (A)

FV

-2000

0

2000

4000

6000

8000

10000

12000

14000

16000In

ten

sity

(B

)

0 10000 20000 30000

Intensity (A)

MTHFR667

0

10000

Inte

nsi

ty (

B)

0 2000 4000 6000 8000 100001200014000160001800020000220002400026000

Intensity (A)

MTHFR1298

-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

Inte

nsi

ty (

B)

≥ 99.99% reproducibility

100% concordant genotypes

26

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

27

Available Products

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

Assay specific oligos

+Reagents

GoldenGate Genotyping User Manual

(RUO)

GOLDENGATEGENOTYPING

+Beads Pooled in

96-well Plates


USER MANUALCustom GoldenGate

28

GoldenGate Assay Used for Majority of the International HapMap Project

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

15.5%

9.1%

4.4%

1.9%

8q, 9, 18q, 22, X

4q, 7q, 18p, Y

12

7p

Illumina

Broad

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

Japan

% 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

29

AG

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

30

A Address #561Amplification Template

PCR with Common Primers

GoldenGate Genotyping AssayAmplification

Cy3 Universal Primer 1

Cy5 Universal Primer 2

Universal Primer P3

31

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

32

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

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

48 134 104

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs3136329

0

0.20

0.40

0.60

0.80

1

No

rm R

111 131 420 0.20 0.40 0.60 0.80 1

Norm Theta

rs6748810

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

1.80N

orm

R

97 145 420 0.20 0.40 0.60 0.80 1

Norm Theta

rs1800937

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

1.80

No

rm R

2 42 2410 0.20 0.40 0.60 0.80 1

Norm Theta

rs1005056

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

No

rm R

252 33 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs2059520

0

0.20

0.40

0.60

0.80

1

No

rm R

97 137 450 0.20 0.40 0.60 0.80 1

Norm Theta

rs4148217

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

10 96 1770 0.20 0.40 0.60 0.80 1

Norm Theta

rs13320360

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

No

rm R

268 13 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs1498311

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

28 106 1520 0.20 0.40 0.60 0.80 1

Norm Theta

rs13282019

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

No

rm R

0 44 2390 0.20 0.40 0.60 0.80 1

Norm Theta

rs673548

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

12 77 1960 0.20 0.40 0.60 0.80 1

Norm Theta

rs4784744

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

44 152 900 0.20 0.40 0.60 0.80 1

Norm Theta

rs3825585

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

No

rm R

0 38 246

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs4289236

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

19 82 1840 0.20 0.40 0.60 0.80 1

Norm Theta

rs10514805

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

No

rm R

0 33 2510 0.20 0.40 0.60 0.80 1

Norm Theta

rs4953020

0

0.20

0.40

0.60

0.80N

orm

R

27 105 1530 0.20 0.40 0.60 0.80 1

Norm Theta

rs6720173

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

8 84 1940 0.20 0.40 0.60 0.80 1

Norm Theta

rs3136245

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

29 75 1820 0.20 0.40 0.60 0.80 1

Norm Theta

rs1799898

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

13 61 2120 0.20 0.40 0.60 0.80 1

Norm Theta

rs820299

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

98 147 41

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs3258

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

135 101 500 0.20 0.40 0.60 0.80 1

Norm Theta

rs5927

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

1.80

2.00

No

rm R

16 95 1730 0.20 0.40 0.60 0.80 1

Norm Theta

rs8110695

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

11 70 2030 0.20 0.40 0.60 0.80 1

Norm Theta

rs9326954

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

94 140 520 0.20 0.40 0.60 0.80 1

Norm Theta

rs3791981

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

No

rm R

224 59 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs4952887

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

11 44 2290 0.20 0.40 0.60 0.80 1

Norm Theta

rs1042031

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

1.80

2.00

No

rm R

8 103 1750 0.20 0.40 0.60 0.80 1

Norm Theta

rs2072447

0

0.20

0.40

0.60

0.80

1

No

rm R

173 89 24

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs2569538

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

No

rm R

1 31 2540 0.20 0.40 0.60 0.80 1

Norm Theta

rs4647247

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

0 13 2700 0.20 0.40 0.60 0.80 1

Norm Theta

rs10515198

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

0 40 2400 0.20 0.40 0.60 0.80 1

Norm Theta

rs1801702

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

No

rm R

3 11 2710 0.20 0.40 0.60 0.80 1

Norm Theta

rs4647229

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

276 6 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs1800947

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

23848 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs265431

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

225 59 1

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs2645424

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

No

rm R

51 136 940 0.20 0.40 0.60 0.80 1

Norm Theta

rs9353019

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

1.80

2.00

No

rm R

1 21 2640 0.20 0.40 0.60 0.80 1

Norm Theta

rs2303152

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

0 33 2500 0.20 0.40 0.60 0.80 1

Norm Theta

rs5882

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

117 119 500 0.20 0.40 0.60 0.80 1

Norm Theta

rs1616534

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

83 153 470 0.20 0.40 0.60 0.80 1

Norm Theta

rs1736058

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

1.80

No

rm R

4 96 1800 0.20 0.40 0.60 0.80 1

Norm Theta

rs1800932

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

196 81 90 0.20 0.40 0.60 0.80 1

Norm Theta

rs3805586

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

30 139 1170 0.20 0.40 0.60 0.80 1

Norm Theta

rs13425206

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

0 19 2650 0.20 0.40 0.60 0.80 1

Norm Theta

rs4987188

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

4 12 2700 0.20 0.40 0.60 0.80 1

Norm Theta

rs289741

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

128 112 45

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs9326953

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

168 111 70 0.20 0.40 0.60 0.80 1

Norm Theta

rs748766

0

0.20

0.40

0.60

0.80

1

No

rm R

91 114 800 0.20 0.40 0.60 0.80 1

Norm Theta

rs12056593

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

No

rm R

280 6 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs1146634

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

78 157 510 0.20 0.40 0.60 0.80 1

Norm Theta

rs5930

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

1.80

2.00

No

rm R

63 127 930 0.20 0.40 0.60 0.80 1

Norm Theta

rs4647221

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

253 31 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs4236927

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

263 23 00 0.20 0.40 0.60 0.80 1

Norm Theta

rs7607312

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

70 159 560 0.20 0.40 0.60 0.80 1

Norm Theta

rs267307

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

No

rm R

21963 20 0.20 0.40 0.60 0.80 1

Norm Theta

rs916042

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

1.80

No

rm R

19 35 2300 0.20 0.40 0.60 0.80 1

Norm Theta

rs6741393

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

1.80

No

rm R

0 23 263

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs6718187

0

1

2

No

rm R

64 138 84

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs5880

0

0.20

0.40

0.60

0.80

1

No

rm R

3 24 259

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs533617

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

1.60

1.80

No

rm R

261 24 0

0 0.20 0.40 0.60 0.80 1

Norm Theta

rs17320429

-0.20

0

0.20

0.40

0.60

0.80

1

1.20

1.40

No

rm R

257 19 10

33

Inter-User Variability

9696OPA multiplex

100%

100%99.97%

100%100%99.96%

144

95

User A

100%

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

Setup

TargetSpecification

34

Available Products

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


+Reagents


(RUO)

DASLGENE EXPRESSION

+Beads Pooled in

96-well Plates

(96 & 384-plex/well)

USER MANUALCustom DASL

35

The DASL Assay

/WV/WV

Product capture by hybridization to array

PCR with common primers

Address’P1

P1

P3’

P3

Address

b

b

Total RNA

cDNA synthesis

Query oligo annealing, extension, and ligation

P2

P2

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.

36

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

37

Fresh Frozen and FFPE RNA Reproducibility on VeraCode

Fresh Frozen Prostate Normal

0

10000

20000

30000

40000

50000

60000

0 10000 20000 30000 40000 50000 60000

Intensity Replicate 1In

tens

ity R

eplic

ate

2

FFPE Prostate Normal

0

10000

20000

30000

40000

50000

60000

0 10000 20000 30000 40000 50000 60000

Intensity Replicate 1

Inte

nsity

Rep

licat

e 2

Fresh Frozen Prostate Tumor

0

10000

20000

30000

40000

50000

60000

0 10000 20000 30000 40000 50000 60000


Inte

nsity

Rep

licat

e 2

FFPE Prostate Tumor

0

10000

20000

30000

40000

50000

60000

0 10000 20000 30000 40000 50000 60000


Inte

nsity

Rep

licat

e 2

Fresh Frozen Tumor vs Normal

0

10000

20000

30000

40000

50000

60000

0 10000 20000 30000 40000 50000 60000

Intensity Normal

Inte

nsity

Tum

or

FFPE Tumor vs Normal

0

10000

20000

30000

40000

50000

60000

0 10000 20000 30000 40000 50000 60000

Intensity Normal

Inte

nsity

Tum

or

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

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

38

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)

39

Available Products

VERACODE GOLDENGATE METHYLATION (48 to 384-promoter regions)


+Reagents


(RUO)

DASLGENE EXPRESSION

+Beads Pooled in

96-well Plates

(96 & 384-plex/well)

USER MANUALCustom DASL

40

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

41

GoldenGate Methylation AssayHybridization & Detection

IllumiCode

IllumiCode

IllumiCode

CpG locus 1 CpG locus 2

CpG locus 3

Unmethylated

U/U

Methylated

C/C

Semi-methylated

C/U

42

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)

43

Ability to Analyze Multiple Applications

100000

10000

1000

100

10

10.1 1 10 100 1000 10000

Conc (pM)

Cou

nts

AnalyteFit3-sigma LOD

GENE REGULATION PROTEIN EXPRESSION

GENOTYPING

0 1 2 3 4 5

Log10(Conc pg/ml)

IL-6 (6152)

2

3

4

5

Lo

g1

0(N

et R

FU

)

44

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

iScan

Services

45

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

46

To Be the Leader in Cancer Translational Diagnostics

Illumina Diagnostics Vision

Next Generation Sequencing

Whole Transcriptome Analysis

Methylation Profiling

Focus Core Technologies on Cancer

47

Initial Targets

Source: WHO

Stomach

Colon/ rectum

Breast

Lung

Percentage of All Cancers

Prob

abili

ty o

f Cur

e (%

)100

0 6 8 10 12 140

10

20

30

40

50

60

70

80

90

2 4

KidneyCervix/ Uteri

Melanoma

Bladder

Oral

NHL

Testis

Thyroid

Prostate

Esophagus

MyelomaLeukemia

Brain

Pancreas

Ovary

CNS

Nasopharynx

Liver

48

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

49

Early Detection for Ovarian Cancer is Critical

Cumulative % of Cases

19% 22%

30% 33%37% 40%

47%

54%

72%

100%

Ia Ib Ic Iia Iib Iic IIa IIIb IIIc IV

Stage at Diagnosis

Late stage diagnosis …

5 Year Survival92%

85%82%

69%

56%51%

39%

26%

17%12%

Ia Ib Ic Iia Iib Iic IIa IIIb IIIc IV

Stage

… results in poor prognosis

50

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

51

10

11

16

17

23

25

31

36

37

37

39

42

46

51

54

66

79

79

89

96

97

145

Sarcoma

Bladder

Cervical

Stomach

Liver

Head/Neck

Myeloma

Ovarian

Brain

Kidney

Cancer-Related Conditions

Pancreatic

Skin

Other Cancers

Lymphoma

ColoRectal

Breast

Prostate

Leukemia

Lung

Unspecified Cancers

Solid Tumors

4

4

6

7

9

10

13

14

15

17

18

22

22

44

50

210

Transplantation

Growth Disorders

Eye Conditions

Skin Disorders

Genetic Disorders

Blood Disorders

Respiratory Disease

Digestive Disorders

Diabetes

Neurological Disease

Other

HIV

CVD

AutoImmune

Infectious Disease

Cancer

More Therapies Means More ChoicesDrug Pipeline

Source: PhRMA: 2006 Medicines in Development Biotechnology

52

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

53

Illumina’s Diagnostic Division Branding

Discovery

Services

veracode technologycytokine sandwich assay overview pe pe pe cytokine antigens biotinylated...

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