next generation diagnostics: potential clinical applications of illumina’stechnology
DESCRIPTION
Rob Kelley, MBAGlobal Sales ManagerTranslational GenomicsSeptember 27, 2011TRANSCRIPT
© 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
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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
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Markets Served
Life Sciences~$2.8B
Consumer
Molecular Dx~$3B
Applied Markets
~$1B
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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
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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
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Molecular Cytogenetics
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Cytogenetic Technologies and Resolution
Genome-wide analysis
Targeted/focused analysis
HiScanSQ
Infinium HD Beadchips
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ISCA (International Standards for Cytogenetic Arrays)Consensus Statement: AJHG, May 2010
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ACMG (American College of Medical Genetics) revised guidelines: Arrays recommended first line
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Large & Growing Opportunity in Array-Based Cytogenetics
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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
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Detection of a Wide Range of Aberrations
For Research Use Only
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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
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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
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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
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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)
For Research Use Only
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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
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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
For Research Use Only
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Copy-Neutral Features Are Missed by Array CGH
IlluminaAgilent Agilent Illumina
Chromosome Zoom to position
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KaryoStudioDx: Illumina’s custom‐designed Cytogenetics Software
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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
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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.
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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
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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
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iScan/KaryoStudioDx
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Molecular Diagnostics
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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
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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
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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
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► 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
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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
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► 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?
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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
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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.
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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
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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
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How to Generate Library Plates
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© 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.
Run
Starting and Monitoring a MiSeq Run
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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
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MiSeq Instrument User Interface
Begin sequencing by selecting “SEQUENCE” button
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[OPTIONAL] Log on to BaseSpace
BaseSpace is a cloud option to store, analyse, and share your MiSeq data.
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[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.
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Animation Instructions to Load Flow Cell
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Instructions to Load Reagent Cartridge and Waste Bottles
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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
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[OPTIONAL] User-specified Sample Sheet
User may select sample sheet from alternate folder or attached USB key
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Review of MiSeq Run
User has opportunity to review parameters of run before submitting
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MiSeq Performs Pre-Run Check
MCS checks for dependencies to ensure run success
Click here to start run
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MiSeq Performs Sequencing Run
Current statusof run
Output to currentBaseSpaceaccount
Name of run
Realtime runmetrics
Connectivity statusWith BaseSpace
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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.
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MiSeq Reporter
© 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.
MiSeq Reporter Screenshots
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Start Up Page
Currently run and progress
List of past runs
Run completion status
Area for Illumina messagesand updates
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General Summary Report
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Resequncing Details Report
Coverage and error
Q score
SNP/indel + annotation
Table of samples/variants
Zoom in/outScope of view
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Amplicon Detail Report
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De Novo Details Report
Syntenic dot plot
De novo metrics
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Small RNA Summary Page
Trimmed read lengths
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Small RNA Details Report
Distribution of RNA species
Top 10 most abundant species
Tabular information
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Metagenomics Summary Page
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Metagenomics Details Report
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Run Status
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Run Sample Sheet
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Run Log
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Run Errors
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Change Repository
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Requeue Run
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Questions
81
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Clinical Whole Genome Sequencing
IlluminaCLIA Lab2009
>30 Whole genomes
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• 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
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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
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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
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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
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TSCA
TSCE
CENextera
Samples
96
1000s
50
Target Sequence (kb)20 70010 Mb’s2001 100
Positioning of Targeted Resequencing products
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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
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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
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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!
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Meet MiSeq
52.3 cm
12 cm
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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
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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.
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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
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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
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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.
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ILMN vs. Moore’s Law
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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
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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)
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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
For Research Use Only
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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
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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
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Innovative Genotyping Approach
Make Target
Add Oligos
Extend/Ligate
PCR
Make ssDNA
Hyb VBS
[A/B]b
BA
Cy
Cy
Cy
c
b
b
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► 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
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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
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Sample Prep Workflow
DNA RNA
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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
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Simplest Sequencing Workflow
Parallel sample processing
Automated clustergeneration
Automated sequencing
SIMPLIFIED SAMPLE PREP
cBot CLUSTER GENERATION
SEQUENCING DATA PROCESSING & ANALYSIS
Simple, efficient data analysis
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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
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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