next generation diagnostics: potential clinical applications of illumina’stechnology

© 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.

Rob Kelley, MBAGlobal Sales Manager

Translational GenomicsSeptember 27, 2011

Next Generation Diagnostics: Potential Clinical Applications

of Illumina’s Technology

2

HiScanSQUnique

combination of

sequencing and arrays

Provider of Genomic Analysis ToolsThat Advance the Understanding of Genetics and Health

From Genome-Wide Discovery to Targeted Validation and Screening

Sequencing Arrays qPCR

HiSeq 1000

Powerful, Flexible, Scalable

3

Markets Served

Life Sciences~$2.8B

Consumer

Molecular Dx~$3B

Applied Markets

~$1B

Presenter

Presentation Notes

I use the term Markets As Illumina now Serves multiple - interconnected markets Each large and growing at a healthy rate Central to these and our first home = life science research market Established our strongest brand presence and a network of exceptionally strong customer relationships This market represents a value of approximately $2.8Bn today Even with the up and downs seen in 2009 = strong growth potential Just as committed to building successful businesses in the high value MolDx and consumer markets And as you will see later in my presentation Made great progress in setting Illumina as the premier supplier of genetic analysis tools in several applied markets With many new applied opportunities lying before us.

4

Dx Strategy

Risk HighLow

Oncology Discovery

Platforms

Objectives

Strategies

Venue

Low

High

Return

Partnering Internal Development

Build Installed Base

Oncology Discovery

Create a novel dx

Sequencing Services

Sequencing Services

Accelerate Revenue

Vision To be the leader in translational diagnostics

Platforms ApplicationsServices

5

Platforms

BeadXpressFDA cleared instrument & Factor II/V assay Q2 10FDA approved GPR beads Q2 10CE Mark Q4 11

iScanCytogenetics Post-Natal Q4 11 Cytogenetics Cancer TBDCytogenetics PGS TBDCytogenetics Pre-Natal TBD

MiSeqPre-IDE submission to FDA Q2 11Carrier screen 2012Somatic Mutation Panel 2012

6

Molecular Cytogenetics

7

Cytogenetic Technologies and Resolution

Genome-wide analysis

Targeted/focused analysis

HiScanSQ

Infinium HD Beadchips

Presenter

Presentation Notes

As Ness pointed out, there are a lot of cyto techniques currently used in the marketplace and options for customers to choose from. Deciding which product to use can be a dilemna for them. Ultimately, cytogeneticists will embrace technologies with greater diagnostic yield (or ability to detect causal aberrations). Resolution is one characteristic that can impact this by finding smaller abberrations. This slide does a good job of displaying the resolution of various methods.

8

ISCA (International Standards for Cytogenetic Arrays)Consensus Statement: AJHG, May 2010

9

ACMG (American College of Medical Genetics) revised guidelines: Arrays recommended first line

10

Large & Growing Opportunity in Array-Based Cytogenetics

10

A technology substitution is underway

ILLUMINA IS POSITIONED FOR GROWTH

$-

$50

$100

$150

$200

$250

$300

$350

$400

2009 2010 2011 2012

Rev

enue

(Mill

ions

)

Array-based cytology market growing double digits (CAGR = 16–20%)

Molecular Cytogenetics RevenueForecasts by Market Segment (US)

90

100

80

70

60

50

40

30

20

10

02007 2008 2009 2010 2011 2012 2013 2014 2015

Per

cent

of R

even

ues

(%)

z

Microarray-BasedFISH

11

Detection of a Wide Range of Aberrations

For Research Use Only

Presenter

Presentation Notes

The two leading methods of high-resolution copy number detection are array CGH (arrays of BAC, cDNA or oligonucleotide probes) and SNP genotyping arrays. In addition to providing a measurement of copy number, genotyping arrays can detect copy neutral LOH. Copy-neutral LOH is when you have events like gene conversion, in which genetic material is inherited from one parent (and not both). So, in a specific region of the genome, you will have what looks like LOH, but there are still two copies of DNA there. CN-LOH is known to cause Prader-Willi and many other syndromes. SNPs also provide genotypes, higher signal to noise ratios for detecting aberrations than intensity only and there are SNPs all over the genome, so we can target most relevant cyto regions.

12

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”


Presenter

Presentation Notes

This is our new cytogenetics BeadChip. 12-samples per BeadChip ~300k total markers (mostly SNPs) Infinium HD assay (with no changes) New reagent kit options for low-volume customers Full robot and LIMS support Priced as low as $125 per sample Content highly optimized for efficient identification of common cytogenetic abnormalities Pericentromeres, subtelomeres, sex chromosomes Content also highly-optimized for WGAS tagSNPs for CEU and Asian populations Fully supported analysis within KaryoStudio or GenomeStudio

13

BeadArrayTM Technology

Oligo Mfg 2 μm Silica

~18-fold Redundancy Decoding = 100% QC

Bead Identifier(30base nt)

Specific Probe(50 base nt)

Population in wells

Presenter

Presentation Notes

At ILMN in San Diego is a room solely for the purpose of creating Oligos. There are dozens of systems making our oligos 24hr/day 6 days a week. In fact, ILMN is the worlds largest producer of oligos and actually has the capacity to supply the entire worlds oligo needs 2x over. These Oligos are attached to 2micron beads. In addition to the oligo is a beadtype identifier. These beads are mixed into a large bead pool and these beads pools are stable for 2 years. What does this mean for you? Consistency. It is one Lot of beads and oligos so you limit potential variatiion in results. Also of note, when these beads are added to each chip, they are done so with 15-20x redundancy. That is, each bead with 100’s of thousands of oligos is represented approx 18x on the chip. So in reality, your 300k feature chip actually has 300k x 18 or >5M beads on it. That is the beauty of ILMN’s technology. To confirm the location of the beads on each chip, the identifier is used and a map file is made for each chip representing the location of each bead. The good news here is in doing so, you basically just QC’d every well on that chip to make sure the bead is there an functioning properly.

14

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 discrimination

Presenter

Presentation Notes

To summarize a cyto-array workflow: - We start with 200ng DNA. Afternoon of the first day: Genomic DNA amplified 1000-1500X fold - Fragmented ~300-600bp 2nd Day: Single Base Extension utilizes a single probe sequence ~50bp long designed to hybridize immediately adjacent to the SNP query site 3rd Day: Following targeted hybridization to the bead array, the arrayed SNP locus-specific primers (attached to beads) are extended with a single hapten-labeled dideoxynucleotide in the SBE reaction. The haptens are subsequently detected by a multi-layer immunohistochemical sandwich assay.

15

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 individualvulnerable to phenotypes that may be associated to the effected gene(s)


16

SNPs Provide More Information to Detect Copy Number

► Also can detect:– Amplification– Unbalanced aberration– Aneuploidy– Mosaicism

Normal (diploid)

Deletion (loss of one copy)

Duplication (gain of one copy)

Genotypes

Copy-Neutral LOH (UPD)

Log R Ratio B Allele Frequency

IntensityFor Research Use Only

Presenter

Presentation Notes

This slide will help visualize how SNP’s help provide this additional info. If you look at the display to the left… But what happens if there is a gene conversion event such as in Prader-Willi syndrome and the subject receives both copies of an allele from the same parent.?

17

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

Detected duplication

A profile of chromosome 3 of a cell line derivedfrom a breast tumor.

SNP-based Detection Provides More Information and Enables Better Characterization of Chromosomal Aberrations


Presenter

Presentation Notes

Duplications are much clearer and better-defined with both B-allele frequency and logR value to interrogate them. “Digital” nature of B allele frequency results in higher signal to noise ratio than intensity information would provide alone

18

Copy-Neutral Features Are Missed by Array CGH

IlluminaAgilent Agilent Illumina

Chromosome Zoom to position

19

KaryoStudioDx: Illumina’s custom‐designed Cytogenetics Software

20

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 met

21

Found Regions Displayed in KaryoStudioDx

User‐defined Known Regions track

Found RegionsFound RegionsFound Regions

Samples table with QC score

Found regions table

Link out to databases:UCSC, DGV, ENSEMBL, DECIPHER, etc.

Presenter

Presentation Notes

Ability to filter CNV polymorphisms Cross-matching capabilities to the most popular cytogenetic databases

22

Diagnostic PDF Report Generated From Aberrations

Report Includes:• Sample and product information

• Aberration display

• Information on found region

• Cross‐matches to user‐defined Known Regions list

• Custom info fields

• ISCN nomenclature

Presenter

Presentation Notes

Sample and product information Aberration display Information on found region Cross-matches to user-defined Known Regions list

23

HumanCytoSNP‐12

Optimized forcytogenetics

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 usePost‐natalDD/ID/MCABroad, genome‐wide coverage

Simultaneously pursuing CE‐IVD marking (ISO 13485) – 2011/2012

23

iScan/KaryoStudioDx

Presenter

Presentation Notes

For that reason Illumina put a lot of energy and money for engaging a dialog with the FDA few years ago and we have submitted a complete cytogenetics package including, one specific SNPArray, a scanner and a dedicated software. Also in our effort to step in the Diagnostic Space, we have submitted our iScan platform, both arrays and KaryoStudio Dx to the FDA end of 2009. We aim to get the CE-IVD marking by end of this year and probably early in the last Quarter. KaryoStudio Dx will be a very different software than it is right now with a new major features like algorithm for mosaic detection, integration QC controls etc….

24

Molecular Diagnostics

25

Load

MiSeq –Finally a sequencer designed with Dx customers in mind

Go

Workflow Fully integrated systemOn-board cluster generation and data visualizationPreloaded reagent cartridge1 flow cell lane per run

PerformanceUp to 5M clusters2 x 150 bp in under 28 hoursRFID reagent & flow cell trackingAuto flow cell positioningWalk-away automation

Presenter

Presentation Notes

The product of you library prep – a single sample or multiplexed samples, is loaded into the preconfigured reagent cartridge. The consumables required for a run consist of a single channel flowcell, preconfigured reagent cartridge and buffer bottle. Run setup is fast and efficient: Flowcell loading is quick and easy, the flowcell is auto positioned in the holding area, and the system will provide feedback that the operation has been performed correctly The reagent cartridge is guided into a reagent chiller area while the buffer and waste bottles are placed into a separate area. Shut the chiller door and the main door and consumables loading is complete. Each consumable also is tagged with an RFID (Radio frequency ID chip) which allows for seemless tracking and traceability

26

MiSeq – Prep, Run, AnalyzeSample to Data in as Little as Eight Hours*

08:00:00

MiSeq is the Only Personal Sequencing System Capable of an 8 Hour Sample to Data Workflow

Amplicons

Clones

gDNA

*1x36bp run – 3 hr sequencing

Presenter

Presentation Notes

MiSeq is the Only Personal Sequencer capable of a workflow as fast as 8 hours sample to data. The workflow can accommodate multiple input types with a few examples shown here: amplicons, clones or gDNA. The input material is processed using a simple, rapid library prep that yields fragments with specific adapters attached. This product, which can also be multiplexed samples is loaded into the MiSeq reagent cartridge. A menu driven user interface prompts the user to load the necessary consumables on the instrument. Cluster generation, sequencing and paired end chemistry can progress in a fully automated fashion. Once the run is complete, the on-board computing hardware will perform all image processing yielding output of base calls and quality scores. The on-board computing hardware also performs variant detection and reporting. MiSeq is the only system that can perform standard image processing AND variant calling and reporting on a single instrument.

27

TruSeq Exome EnrichmentTargets = 100,000s

TruSeq Custom EnrichmentTargets = 1000s

TruSeq Custom AmpliconTargets = 100s

TruSeq Targeted Resequencing The simplest and most scalable targeted resequencing solutions

Nextera PCR AmpliconsTargets = 10s

28

► 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

►

Coming soon! TruSeq Custom Amplicon SequencingUnprecedented amplicon and sample multiplexing

29

Assay Biochemistry

TruSeq Custom Amplicon Assay Time96 samples & 384 targets: from DNA to called variants in ~2 days

8am – Day 1

HybridizationSetup

Oligos, universal reagents

Extension &Ligation, PCR

with index

Library Normalization

Create pooled library,

normalize

Cluster Gen &Sequencing

Pre-kittedsequencing

reagents

Real-timeAnalysis

Alignments, variant calling

2pm – Day 1 5pm – Day 2

<8 hr assay with <3 hr hands-on timeNo fragmentation requiredNo gel purification stepsNo additional hardware

30

► 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 coverage

TSCA throughput and coverage on MiSeqHow many samples can be run together?

31

MiSeq - Comparison to CE SequencingExample: TruSeq Custom Amplicon with 96 Samples x 384 Targets

TSC

A

Assay biochemistry

CE

One plate of gDNA+oligosfor all 384 amplicons

Quant

96 x 384 wellplates

3730xl5-6 weeks

~$3/amplicon

96-w

ell p

late TruSeq Custom

Amplicon•Highly multiplexed•Integrated sample indexing•Standard lab equipment

Dispense, PCR, Cleanup

Assay Biochemistry Pool Libraries and Sequence

Dye-terminator sequencing, cleanup

96 x 384 wellplates

MiSeq4 days

<$0.75/amplicon

32

MiSeq ApplicationsA 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 = NexteraTS RNA = TruSeq RNA Sample Prep TS smRNA = TruSeq Small RNA Sample Prep HB = HomebrewRZ + 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.

33

MiSeqCharacterization 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, VEGFR

Presenter

Presentation Notes

MiSeq™, a low-cost personal sequencing system that leverages the company’s proven TruSeq Sequencing chemistry to offer individual researchers a platform with rapid turnaround time, unmatched accuracy, and the easiest to use workflow.

34

MiSeq FFPE Amplicon DataKRAS Exon 2 – 76 Base Amplicon

1.1% variant in normal adjacent tissue > Assay LOD of 0.5%

Rectal Normal

OvarianTumor

GastricTumor

No-FFPEControl

GastricNormal

RectalTumorSample

Coverage 178667 X151695 X 176530 X 179630 X 161866 X 178900 X

27.5% G C variant at Chr12:

25398284 in rectal tumour

© 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 and HiScan are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

Illumina Experiment Manager

September 2011

43

How to Generate Library Plates


Run

Starting and Monitoring a MiSeq Run

51

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) account

52

MiSeq Instrument User Interface

Begin sequencing by selecting “SEQUENCE” button

53

[OPTIONAL] Log on to BaseSpace

BaseSpace is a cloud option to store, analyse, and share your MiSeq data.

54

[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.

55

Animation Instructions to Load Flow Cell

56

Instructions to Load Reagent Cartridge and Waste Bottles

57

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 Sheet

58

[OPTIONAL] User-specified Sample Sheet

User may select sample sheet from alternate folder or attached USB key

59

Review of MiSeq Run

User has opportunity to review parameters of run before submitting

60

MiSeq Performs Pre-Run Check

MCS checks for dependencies to ensure run success

Click here to start run

61

MiSeq Performs Sequencing Run

Current statusof run

Output to currentBaseSpaceaccount

Name of run

Realtime runmetrics

Connectivity statusWith BaseSpace

62

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.

63

MiSeq Reporter


MiSeq Reporter Screenshots

65

Start Up Page

Currently run and progress

List of past runs

Run completion status

Area for Illumina messagesand updates

66

General Summary Report

67

Resequncing Details Report

Coverage and error

Q score

SNP/indel + annotation

Table of samples/variants

Zoom in/outScope of view

68

Amplicon Detail Report

69

De Novo Details Report

Syntenic dot plot

De novo metrics

70

Small RNA Summary Page

Trimmed read lengths

71

Small RNA Details Report

Distribution of RNA species

Top 10 most abundant species

Tabular information

72

Metagenomics Summary Page

73

Metagenomics Details Report

74

Run Status

75

Run Sample Sheet

76

Run Log

77

Run Errors

78

Change Repository

79

Requeue Run

80

Questions

82

Clinical Whole Genome Sequencing

IlluminaCLIA Lab2009

>30 Whole genomes

83

• A patient treated with Erlotinib but lung metastases are unresponsive

• Whole genome and transcriptomesequencing 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/R82

Sequencing Informs Therapy

Presenter

Presentation Notes

Shifting paradigms now to cancer diagnostics and prognostics.

84

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. unpublished

Presenter

Presentation Notes

The results demonstrate the feasibility of systematic, genome-wide characterization of rearrangements in complex human cancer genomes, raising the prospect of a new harvest of genes associated with cancer using this strategy

85

Nextera DNA Sample PrepSequencing’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

~1.5 hours

~12 hours

Enables effective use of single 36bp reads

86

Overall positioning of Targeted Resequencing on MiSeqPCR amplicons through TruSeq Custom Enrichment

Application Target Sequence

Number samples/ project

Price / sample (Prep and Seq) Key Benefit

CE + Amplicons < 10 kb <100 $50 Small sample/content

projects

Nextera + Amplicons < 20 kb 100s $80 Long contiguous

amplicons and speed

TSCA 10 – 96 kb 100s – 1000s $50 - 300 Multiplexing and speed

TSCE 700 kb – 2 Mb 100s – 1000s $260 - 800 More target content

87

TSCA

TSCE

CENextera

Samples

96

1000s

50

Target Sequence (kb)20 70010 Mb’s2001 100

Positioning of Targeted Resequencing products

88

Nextera and MiSeqSequencing’s fastest time to answer for rapid variant analysis

08:00:00

15:00:00

*Based on 1 x 36 bp reads

1 – 8 samples

1 – 96 samples

89

Ovarian Cancer

Deadly

Incurable

Difficult DxPatients present with a suspicious/palpable mass

Less than 40% are cured

204,449 New cases annually; 124,860 deaths

Early Dx is KeyFive year survival is good if diagnosed early, but most patients are diagnosed late stage

Illumina SolutionDevelop a diagnostic assay which will diagnose ovarian cancer at an early stage

19%22%

30%33%

37%40%

47%

54%

72%

100%

92%

85%82%

69%

56%51%

39%

26%

17%12%

-10%

10%

30%

50%

70%

90%

110%

Ia Ib Ic Iia Iib Iic IIIa IIIb IIIc IV

Cum % of Cases

5 Yr Survival

90

TruSeq Targeted ResequencingA broad suite of tools for discovery or validation experiments

Option Amount of sequence Best for Availability

TruSeq ExomeEnrichment ~62 Mb

Mendelian disease: case-control exome studies, rarer

variants, causal variantsexome-wide linkage analysis

Now!

TruSeq CustomEnrichment ~1 to ~10 Mb GWAS follow-up: validation of

variants, variant discovery, pathways Now!

TruSeq Custom Amplicon Sub-500 Kb

Amplicon sequencing: high-throughput CE experiments, ultra deep seq, variant disc, screening

2H2011

Nextera + PCR Amplicons

100’s of bptargets

Amplicon sequencing: ultra-deep sequencing, validation, screening, CE

replacementNow!

91

Meet MiSeq

52.3 cm

12 cm

Presenter

Presentation Notes

MiSeq™, a low-cost personal sequencing system that leverages the company’s proven TruSeq Sequencing chemistry to offer individual researchers a platform with rapid turnaround time, unmatched accuracy, and the easiest to use workflow.

92

Oncology Biomarker Discovery

Targeted Seq300

FFPE Tumors

Frozen25

Tumor/Normals

WGS, Gex, Methyl25

Tumor/Normals

10 Cases/20 ControlsBlood, Tumor, Pap,

Proximal Fluids

CLIA Lab Servicefor

Early Detection

IVDDiagnostics

25/25 40 Biomarkers20 Novel

10 @ 1,000X58 @ 300X

Ovarian

23/23 141 Biomarkers

Gastric

25/25 6/6 Sequenced30 by Q1

Colon

Samples Discovery Validation Translation Dx Service Dx Product

http://bit.ly/ncghc

93

Options for Targeted Resequencing with IlluminaFrom specific, customized regions of interest to the complete coding region

Option Amount of sequence Best for Availability

TruSeq ExomeEnrichment ~62 Mb

Mendelian disease: case-control studies, rarer variants,

causal variants, linkage analysisNow!

TruSeq CustomEnrichment

~700Kb to ~15 Mb

GWAS follow-up: validation of variants, variant discovery, pathways Now!

Nextera + PCR Amplicons

100’s of bptargets

Amplicon sequencing: ultra-deep seq, validation, screening, CE replacement Now!

TruSeq Custom Amplicon ~100 Kb Amplicon seq: high-throughput CE,

ultra deep seq, variant disc, screening 2H2011

The only company with the complete end-to-end TRS workflow solution.

94

Simplest Sequencing WorkflowIntegrated workflow from sample to analyzed data

TruSeq ChemistryClustering & Sequencing

TruSeq DNASimple, scalable and cost

effective

TruSeq RNAOptimized, gel-fee, low input

TruSeq Small RNAHi throughput miRNA discovery & profiling

TruSeq ExomeLowest cost and most

scalable exome sequencing

95

MiSeq Workflow GuidePre-defined data analysis workflows

Targeted Resequencing

TruSeq Custom Amplicon

Nextera PCR Amplicon

Metagenomics16S rRNA

Clone Checking

TruSeq Custom Enrichment

ChIP-Seq

Small Genome Sequencing

De novo

Resequencing

Plasmids

RNA Sequencing

Small RNA

RNA-Seq

Library QC

Library QC

Primary workflows

Secondary workflows

Automated

Semi-Automated

96

MiSeq ApplicationsA 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 = NexteraTS RNA = TruSeq RNA Sample Prep TS smRNA = TruSeq Small RNA Sample Prep HB = HomebrewRZ + 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.

97

ILMN vs. Moore’s Law

98

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 Software

Presenter

Presentation Notes

The BeadXpress system is Illumina’s first FDA cleared In-Vitro Diagnostic Device. It was submitted to and cleared under the FDA’s Premarket Notification 510(k) - 21 CFR Part 807 Subpart E guideline. This is more commonly referred to as a 510(k) and requires that a device demonstrate substantial equivalence to a device that has already been cleared by the FDA. The FDA cleared BeadXpress System includes the BeadXpress Reader and the VeraScan Software. All medical devices are required to have a clearly defined intended use, which appears on product documentation. The intended use of the BeadXpress System is: 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 the VeraScan Software

99

Comprehensive VeraCode product portfolio

Research Use Only

VeraCode ADME Core Panel

VeraCode Universal Capture & Carboxyl

Beads

Custom GoldenGateGenotyping,

48, 96, 144, 192 & 384-plex

DASL Custom Gene Expression,

32 to 384-plex

Custom GoldenGateMethylation,

48 to 384-plex

Regulated Products

VeraCode GPR Universal Capture

Beads

VeraCode GPR Carboxyl Beads

VeraCode PGx Panel (in development)

VeraCode GI Panel(in development)

100

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 SLCO2B1


Presenter

Presentation Notes

The pharmaADME working group is a consotium of pharmaceutical companies that got together to determine what genes they would ideally want to investigate.

101

Sequencing ServicesIllumina Clinical Services Lab

Somatic Mutation PanelSomatic Mutation Panel launch Q4 11Content will include genes with proven/anticipated clinical utility:KRAS EGFR BRAF TP53 VEGF-AERBB2 ESR1 PGR TYMS UGT1A1TPMT COMT CYP2D6 NRAS EML4/ALK

WGS30+ Whole genomes sequenced Q4 10First clinical case reimbursed Q4 10Cancer, genetic diseases, SCID cases

InfrastuctureCLIA certified for high complexity molecular diagnostics Q1 09CAP Accredited Q2 09CA State CLS training program Q1 10

102

Individual Genome Sequencing: Workflow

o Initial discussion & genetic counseling o Informed Consent and Service Agreemento Saliva and blood sample taken (DNA possible)o Cooling-off period (7+ days); order confirmed

o Barcode samples for confidentiality o Saliva and blood genotype for ID matcho Whole genome sequencing of blood DNAo Analyze and QC sequence and called variantso Check sequence and genotype ID matcho Archive full dataset

Physician orders IGS for patient

Genome sequencing and QC

Clinical lab delivers data to physician

103

Innovative Genotyping Approach

Make Target

Add Oligos

Extend/Ligate

PCR

Make ssDNA

Hyb VBS

[A/B]b

BA

Cy

Cy

Cy

c

b

b

Presenter

Presentation Notes

The upfront targeting step with the VeraCode ADME Core assay chemistry provides two benefits to the panel. 1 – The specific region of the gene targeted for analysis is enriched with a single round of extension with a biotinylated primer. That product is extracted from the rest of the genome on a paramagnetic particle for subsequent analysis with allele specific primer extension and ligation. Because of the added round of specificity provided by this enrichment step, the assay is reduced to just one day. 2 – It is possible to isolate complex regions of the genome for subsequent analysis using targeting. We leverage this property here to analyze genes with high levels of homology. (next slide)

104

► 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 annotated

Delivery of Individual Genome Sequence

Presenter

Presentation Notes

Delivery includes: Overview report All reported calls, with their quality scores All reported SNPs with coordinates and dbSNP All raw reads, assembled in the genome viewer, Raw reads can be further analyzed in other software

105

Illumina Sequencing Workflow

Fragment DNARepair endsAdd A overhangLigate adaptersPurify

Library Preparation1

Cluster Generation Hybridize to flow cellExtend hybridized templatePerform bridge amplificationPrepare flow cell for sequencing

2

SequencingPerform sequencingGenerate base calls

3

Data AnalysisImagesIntensitiesReadsAlignments

4

Presenter

Presentation Notes

Steps of Illumina Sequencing Workflow

106

Sample Prep Workflow

DNA RNA

Presenter

Presentation Notes

- On fragmente l’ADN de préférence avec un automate comme le Covaris On répare les extrémités des fragments, phosporylation On procède à une étape de A-tailing On passe à la ligation des adapteurs: chaque adapteur est composé d’un primer d’amplification (utile à l’étape cluster suivante) appelé P5 et P7 et d’un primer pour amorcer le séquençage (Rd1 SP et Rd2 SP). Dans le cas du multiplexing: on a une séquence index qui fait 6 nucléotides en plus.

107

DNA(0.1-1.0 ug)

Sample preparation Cluster growth

5’

5’3’

G

T

C

A

G

T

C

A

G

T

C

A

C

A

G

TC

A

T

C

A

C

C

TAG

CG

TA

GT

Illumina Sequencing TechnologyRobust Reversible Terminator Chemistry Foundation

Sequencing

108

Simplest Sequencing Workflow

Parallel sample processing

Automated clustergeneration

Automated sequencing

SIMPLIFIED SAMPLE PREP

cBot CLUSTER GENERATION

SEQUENCING DATA PROCESSING & ANALYSIS

Simple, efficient data analysis

Presenter

Presentation Notes

With the advent of the HiSeq family, Illumina has even further simplified the entire sequencing workflow. We have a new simplified sample prep solution that will be available in Q2, cBot for cluster generation launched in Q4 2009, and we have also further simplified the data processing and analysis.

109

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 2000x

110

Competitive Environment

TruSeq Custom Amplicon

Fluidigm Access Array

HaloPlex PCR PCR/Homebrew

Number of Amplicons 48 – 384 48 ‐ 480[1] < 2,000 1 ‐ 5Target Genomic Sequence

< 96 kb < 120 kb < 400 kb < 3 kb

Panel Design DesignStudio FLDM service Design Wizard Primer3/Manual

Total Assay Time (hands on)

7 hr (2.5 hr) 5 hr (<1 hr) 24 hr (2.5 hr est) 2 hr (0.5 hr)

Manual Batch Size (samples)

192 48 48 < 384

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 assay

Yes No No No

[1] Promising 10‐plex PCR [2] Estimated

next generation diagnostics: potential clinical applications of illumina’stechnology

Business

copy number

copyneutral features

inactivated allele

research use

arraybased cytogenetics

allele specificity

functional allele

good allele