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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
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
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
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Large & Growing Opportunity in Array-Based Cytogenetics
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
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.
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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)
► 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
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
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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
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.
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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
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.
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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
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.?
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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
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
Simultaneously pursuing CE‐IVD marking (ISO 13485) – 2011/2012
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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….
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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
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
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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
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.
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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
*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
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.
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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
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.
• 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.
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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
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
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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
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
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.
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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
*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
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
The pharmaADME working group is a consotium of pharmaceutical companies that got together to determine what genes they would ideally want to investigate.
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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
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
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)
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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
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
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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
Presenter
Presentation Notes
Steps of Illumina Sequencing Workflow
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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.
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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
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.
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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