© 2010 Illumina, Inc. All rights reserved.
Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, Gold...
2
HiScanSQ
Unique
combination
of
sequencing
and arrays
Provider of Genomic Analysis Tools
That Advance the Understanding o...
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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
De...
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Platforms
BeadXpress
FDA cleared instrument & Factor II/V assay Q2 10
FDA approved GPR beads Q2 10
CE Mark Q4 11
iScan
C...
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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
10
A technology substitution is underway
ILLUMINA IS POSITIONED...
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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 ...
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BeadArrayTM Technology
Oligo Mfg 2 μm Silica
~18-fold Redundancy Decoding = 100% QC
Bead Identifier
(30base nt)
Specifi...
14
The Infinium HD Assay
► Unconstrained Marker Design
– Freedom to select the best, most
informative SNPs then fill-in wi...
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Copy-Neutral Cytogenetic Aberrations
Forms of copy-neutral cytogenetic aberrations
► Uniparental disomy
– Case in which...
16
SNPs Provide More Information to Detect Copy Number
► Also can detect:
– Amplification
– Unbalanced aberration
– Aneupl...
17
B allele frequency data
(AAB genotype)
A shift in the LogR value is
detectible, but the integration
of B allele data im...
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 s...
21
Found Regions Displayed in KaryoStudioDx
User‐defined 
Known Regions track
Found RegionsFound RegionsFound Regions 
Sam...
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Diagnostic PDF Report Generated From Aberrations
Report Includes:
• Sample and product information
• Aberration display...
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HumanCytoSNP‐12
Optimized for
cytogenetics
Plans for FDA Submission: iScan Platform
Package includes Q4 ’09 Illumina su...
24
Molecular Diagnostics
25
Load
MiSeq –
Finally a sequencer designed with Dx customers in mind
Go
Workflow
Fully integrated system
On-board cluste...
26
MiSeq – Prep, Run, Analyze
Sample to Data in as Little as Eight Hours*
08:00:00
MiSeq is the Only Personal Sequencing S...
27
TruSeq Exome Enrichment
Targets = 100,000s
TruSeq Custom Enrichment
Targets = 1000s
TruSeq Custom Amplicon
Targets = 10...
28
► Rapid & Economical
– Up to 384 amplicons per sample, 96 samples per
plate (36,864 reactions)
– Plate based processing...
29
Assay
Biochemistry
TruSeq Custom Amplicon Assay Time
96 samples & 384 targets: from DNA to called variants in ~2 days
8...
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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 c...
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MiSeq - Comparison to CE Sequencing
Example: TruSeq Custom Amplicon with 96 Samples x 384 TargetsTSCA
Assay
biochemistr...
32
MiSeq Applications
A huge variety of applications can be done on MiSeq*
Application Read Length Kit Config. Sample Prep...
33
MiSeq
Characterization of Genetic Variations in Tumor Tissues
► Deep sequencing of cancer samples to
detect somatic mut...
34
MiSeq FFPE Amplicon Data
KRAS Exon 2 – 76 Base Amplicon
1.1% variant in
normal adjacent
tissue > Assay
LOD of 0.5%
Rect...
© 2010 Illumina, Inc. All rights reserved.
Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, Gold...
36
37
38
39
40
41
42
43
How to Generate Library Plates
44
45
46
47
48
49
© 2010 Illumina, Inc. All rights reserved.
Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, Gold...
51
How to Start a MiSeq Run
The MiSeq Control Software (MCS) is a streamlined push-button user
interface designed to quick...
52
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 &
...
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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 perfo...
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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 status
of run
Output to current
BaseSpace
account
Name of run
Realtime run
metric...
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MiSeq Reporter
► MiSeq Reporter (MSR) is the onboard bioinformatics engine that
automatically processes MiSeq primary a...
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MiSeq Reporter
© 2010 Illumina, Inc. All rights reserved.
Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, Gold...
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Start Up Page
Currently run and progress
List of past runs
Run completion status
Area for Illumina messages
and 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/outScop...
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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
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Clinical
Whole
Genome
Sequencing
Illumina
CLIA Lab
2009
>30 Whole
genomes
83
• A patient treated with Erlotinib but lung
metastases are unresponsive
• Whole genome and transcriptome
sequencing of ...
84
Acute Promyelocytic Leukemia (APL)
Most APL patients have a characteristic translocation between
chromosomes 15 and 17 ...
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Nextera DNA Sample Prep
Sequencing’s fastest and easiest sample prep
► 90 min sample prep
► No Covaris required
► High ...
86
Overall positioning of Targeted Resequencing on MiSeq
PCR amplicons through TruSeq Custom Enrichment
Application
Target...
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TSCA
TSCE
CE
Nextera
Samples
96
1000s
50
Target Sequence (kb)
20 70010 Mb’s2001 100
Positioning of Targeted Resequencin...
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Nextera and MiSeq
Sequencing’s fastest time to answer for rapid variant analysis
08:00:00
15:00:00
*Based on 1 x 36 bp ...
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Ovarian Cancer
Deadly
Incurable
Difficult Dx
Patients present with a suspicious/palpable mass
Less than 40% are cured
2...
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TruSeq Targeted Resequencing
A broad suite of tools for discovery or validation experiments
Option
Amount of
sequence
B...
91
Meet MiSeq
52.3 cm
12 cm
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Oncology Biomarker Discovery
Targeted Seq
300
FFPE Tumors
Frozen
25
Tumor/Normals
WGS, Gex, Methyl
25
Tumor/Normals
10 ...
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Options for Targeted Resequencing with Illumina
From specific, customized regions of interest to the complete coding re...
94
Simplest Sequencing Workflow
Integrated workflow from sample to analyzed data
TruSeq Chemistry
Clustering & Sequencing
...
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MiSeq Workflow Guide
Pre-defined data analysis workflows
Targeted
Resequencing
TruSeq Custom
Amplicon
Nextera PCR
Ampli...
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MiSeq Applications
A huge variety of applications can be done on MiSeq*
Application Read Length Kit Config. Sample Prep...
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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 Diagn...
99
Comprehensive VeraCode product portfolio
Research
Use Only
VeraCode ADME
Core Panel
VeraCode Universal
Capture & Carbox...
100
Fixed Content: VeraCode ADME Core Panel
► VeraCode ADME include184 polymorphisms (34 genes)
– >95% Pharma ADME Core Li...
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Sequencing Services
Illumina Clinical Services Lab
Somatic Mutation Panel
Somatic Mutation Panel launch Q4 11
Content ...
102
Individual Genome Sequencing: Workflow
o Initial discussion & genetic counseling
o Informed Consent and Service Agreem...
103
Innovative Genotyping Approach
Make Target
Add Oligos
Extend/Ligate
PCR
Make ssDNA
Hyb VBS
[A/B]
b
B
A
Cy
Cy
Cy
c
b
b
104
► Summary Report
► Consensus sequence with quality scores of calls
► SNPs report, with dbSNP designation or novel
► Al...
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Illumina Sequencing Workflow
Fragment DNA
Repair ends
Add A overhang
Ligate adapters
Purify
Library Preparation1
Clust...
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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
T
C
A
T
C
A
C
C
T
A
G
C
G
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A...
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Simplest Sequencing Workflow
Parallel sample
processing
Automated cluster
generation
Automated
sequencing
SIMPLIFIED
S...
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MiSeq FFPE Cancer Sequencing
Summary
• MiSeq and SBS Chemistry is capable of generating high depth of
coverage suffici...
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Competitive Environment
TruSeq Custom 
Amplicon
Fluidigm Access 
Array
HaloPlex PCR PCR/Homebrew
Number of Amplicons 4...
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Next Generation Diagnostics: Potential Clinical Applications of Illumina’sTechnology

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Rob Kelley, MBA
Global Sales Manager
Translational Genomics
September 27, 2011

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Next Generation Diagnostics: Potential Clinical Applications of Illumina’sTechnology

  1. 1. © 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 HiSeq are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Rob Kelley, MBA Global Sales Manager Translational Genomics September 27, 2011 Next Generation Diagnostics: Potential Clinical Applications of Illumina’s Technology
  2. 2. 2 HiScanSQ Unique combination of sequencing and arrays Provider of Genomic Analysis Tools That 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. 3. 3 Markets Served Life Sciences ~$2.8B Consumer Molecular Dx ~$3B Applied Markets ~$1B
  4. 4. 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. 5. 5 Platforms BeadXpress FDA cleared instrument & Factor II/V assay Q2 10 FDA approved GPR beads Q2 10 CE Mark Q4 11 iScan Cytogenetics Post-Natal Q4 11 Cytogenetics Cancer TBD Cytogenetics PGS TBD Cytogenetics Pre-Natal TBD MiSeq Pre-IDE submission to FDA Q2 11 Carrier screen 2012 Somatic Mutation Panel 2012
  6. 6. 6 Molecular Cytogenetics
  7. 7. 7 Cytogenetic Technologies and Resolution Genome-wide analysis Targeted/focused analysis HiScanSQ Infinium HD Beadchips
  8. 8. 8 ISCA (International Standards for Cytogenetic Arrays) Consensus Statement: AJHG, May 2010
  9. 9. 9 ACMG (American College of Medical Genetics) revised guidelines: Arrays recommended first line
  10. 10. 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 Revenue(Millions) Array-based cytology market growing double digits (CAGR = 16–20%) Molecular Cytogenetics Revenue Forecasts by Market Segment (US) 90 100 80 70 60 50 40 30 20 10 0 2007 2008 2009 2010 2011 2012 2013 2014 2015 PercentofRevenues(%) z Microarray-Based FISH
  11. 11. 11 Detection of a Wide Range of Aberrations For Research Use Only
  12. 12. 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” For Research Use Only
  13. 13. 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
  14. 14. 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
  15. 15. 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 individual vulnerable to phenotypes that may be associated to the effected gene(s) For Research Use Only
  16. 16. 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 Intensity For Research Use Only
  17. 17. 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 derived from a breast tumor. SNP-based Detection Provides More Information and Enables Better Characterization of Chromosomal Aberrations For Research Use Only
  18. 18. 18 Copy-Neutral Features Are Missed by Array CGH IlluminaAgilent Agilent Illumina Chromosome Zoom to position
  19. 19. 19 KaryoStudioDx:  Illumina’s custom‐designed Cytogenetics Software
  20. 20. 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. 21. 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.
  22. 22. 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
  23. 23. 23 HumanCytoSNP‐12 Optimized for cytogenetics 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 use Post‐natal DD/ID/MCA Broad, genome‐wide coverage Simultaneously pursuing CE‐IVD marking (ISO 13485) – 2011/2012  23 iScan/KaryoStudioDx
  24. 24. 24 Molecular Diagnostics
  25. 25. 25 Load MiSeq – Finally a sequencer designed with Dx customers in mind Go Workflow Fully integrated system On-board cluster generation and data visualization Preloaded reagent cartridge 1 flow cell lane per run Performance Up to 5M clusters 2 x 150 bp in under 28 hours RFID reagent & flow cell tracking Auto flow cell positioning Walk-away automation
  26. 26. 26 MiSeq – Prep, Run, Analyze Sample 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
  27. 27. 27 TruSeq Exome Enrichment Targets = 100,000s TruSeq Custom Enrichment Targets = 1000s TruSeq Custom Amplicon Targets = 100s TruSeq Targeted Resequencing The simplest and most scalable targeted resequencing solutions Nextera PCR Amplicons Targets = 10s
  28. 28. 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 Sequencing Unprecedented amplicon and sample multiplexing
  29. 29. 29 Assay Biochemistry TruSeq Custom Amplicon Assay Time 96 samples & 384 targets: from DNA to called variants in ~2 days 8am – Day 1 Hybridization Setup Oligos, universal reagents Extension & Ligation, PCR with index Library Normalization Create pooled library, normalize Cluster Gen & Sequencing Pre-kitted sequencing reagents Real-time Analysis Alignments, variant calling 2pm – Day 1 5pm – Day 2 <8 hr assay with <3 hr hands-on time No fragmentation required No gel purification steps No additional hardware
  30. 30. 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 MiSeq How many samples can be run together?
  31. 31. 31 MiSeq - Comparison to CE Sequencing Example: TruSeq Custom Amplicon with 96 Samples x 384 TargetsTSCA Assay biochemistry CE One plate of gDNA+oligos for all 384 amplicons Quant 96 x 384 well plates 3730xl 5-6 weeks ~$3/amplicon 96-wellplate 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 well plates MiSeq 4 days <$0.75/amplicon
  32. 32. 32 MiSeq Applications A 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 = Nextera TS RNA = TruSeq RNA Sample Prep TS smRNA = TruSeq Small RNA Sample Prep HB = Homebrew RZ + 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. 33. 33 MiSeq Characterization 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
  34. 34. 34 MiSeq FFPE Amplicon Data KRAS Exon 2 – 76 Base Amplicon 1.1% variant in normal adjacent tissue > Assay LOD of 0.5% Rectal Normal Ovarian Tumor Gastric Tumor No-FFPE Control Gastric Normal Rectal Tumor Sample Coverage 178667 X151695 X 176530 X 179630 X 161866 X 178900 X 27.5% G C variant at Chr12: 25398284 in rectal tumour
  35. 35. © 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
  36. 36. 36
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  38. 38. 38
  39. 39. 39
  40. 40. 40
  41. 41. 41
  42. 42. 42
  43. 43. 43 How to Generate Library Plates
  44. 44. 44
  45. 45. 45
  46. 46. 46
  47. 47. 47
  48. 48. 48
  49. 49. 49
  50. 50. © 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
  51. 51. 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. 52. 52 MiSeq Instrument User Interface Begin sequencing by selecting “SEQUENCE” button
  53. 53. 53 [OPTIONAL] Log on to BaseSpace BaseSpace is a cloud option to store, analyse, and share your MiSeq data.
  54. 54. 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. 55. 55 Animation Instructions to Load Flow Cell
  56. 56. 56 Instructions to Load Reagent Cartridge and Waste Bottles
  57. 57. 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. 58. 58 [OPTIONAL] User-specified Sample Sheet User may select sample sheet from alternate folder or attached USB key
  59. 59. 59 Review of MiSeq Run User has opportunity to review parameters of run before submitting
  60. 60. 60 MiSeq Performs Pre-Run Check MCS checks for dependencies to ensure run success Click here to start run
  61. 61. 61 MiSeq Performs Sequencing Run Current status of run Output to current BaseSpace account Name of run Realtime run metrics Connectivity status With BaseSpace
  62. 62. 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. 63. 63 MiSeq Reporter
  64. 64. © 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
  65. 65. 65 Start Up Page Currently run and progress List of past runs Run completion status Area for Illumina messages and updates
  66. 66. 66 General Summary Report
  67. 67. 67 Resequncing Details Report Coverage and error Q score SNP/indel + annotation Table of samples/ variants Zoom in/outScope of view
  68. 68. 68 Amplicon Detail Report
  69. 69. 69 De Novo Details Report Syntenic dot plot De novo metrics
  70. 70. 70 Small RNA Summary Page Trimmed read lengths
  71. 71. 71 Small RNA Details Report Distribution of RNA species Top 10 most abundant species Tabular information
  72. 72. 72 Metagenomics Summary Page
  73. 73. 73 Metagenomics Details Report
  74. 74. 74 Run Status
  75. 75. 75 Run Sample Sheet
  76. 76. 76 Run Log
  77. 77. 77 Run Errors
  78. 78. 78 Change Repository
  79. 79. 79 Requeue Run
  80. 80. 80 Questions
  81. 81. 81
  82. 82. 82 Clinical Whole Genome Sequencing Illumina CLIA Lab 2009 >30 Whole genomes
  83. 83. 83 • A patient treated with Erlotinib but lung metastases are unresponsive • Whole genome and transcriptome sequencing 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
  84. 84. 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
  85. 85. 85 Nextera DNA Sample Prep Sequencing’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. 86. 86 Overall positioning of Targeted Resequencing on MiSeq PCR 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. 87. 87 TSCA TSCE CE Nextera Samples 96 1000s 50 Target Sequence (kb) 20 70010 Mb’s2001 100 Positioning of Targeted Resequencing products
  88. 88. 88 Nextera and MiSeq Sequencing’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. 89. 89 Ovarian Cancer Deadly Incurable Difficult Dx Patients present with a suspicious/palpable mass Less than 40% are cured 204,449 New cases annually; 124,860 deaths Early Dx is Key Five year survival is good if diagnosed early, but most patients are diagnosed late stage Illumina Solution Develop 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. 90. 90 TruSeq Targeted Resequencing A broad suite of tools for discovery or validation experiments Option Amount of sequence Best for Availability TruSeq Exome Enrichment ~62 Mb Mendelian disease: case-control exome studies, rarer variants, causal variants exome-wide linkage analysis Now! TruSeq Custom Enrichment ~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 bp targets Amplicon sequencing: ultra-deep sequencing, validation, screening, CE replacement Now!
  91. 91. 91 Meet MiSeq 52.3 cm 12 cm
  92. 92. 92 Oncology Biomarker Discovery Targeted Seq 300 FFPE Tumors Frozen 25 Tumor/Normals WGS, Gex, Methyl 25 Tumor/Normals 10 Cases/20 Controls Blood, Tumor, Pap, Proximal Fluids CLIA Lab Service for Early Detection IVD Diagnostics 25/25 40 Biomarkers 20 Novel 10 @ 1,000X 58 @ 300X Ovarian 23/23 141 Biomarkers Gastric 25/25 6/6 Sequenced 30 by Q1 Colon Samples Discovery Validation Translation Dx Service Dx Product
  93. 93. 93 Options for Targeted Resequencing with Illumina From specific, customized regions of interest to the complete coding region Option Amount of sequence Best for Availability TruSeq Exome Enrichment ~62 Mb Mendelian disease: case-control studies, rarer variants, causal variants, linkage analysis Now! TruSeq Custom Enrichment ~700Kb to ~15 Mb GWAS follow-up: validation of variants, variant discovery, pathways Now! Nextera + PCR Amplicons 100’s of bp targets 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. 94. 94 Simplest Sequencing Workflow Integrated workflow from sample to analyzed data TruSeq Chemistry Clustering & Sequencing TruSeq DNA Simple, scalable and cost effective TruSeq RNA Optimized, gel-fee, low input TruSeq Small RNA Hi throughput miRNA discovery & profiling TruSeq Exome Lowest cost and most scalable exome sequencing
  95. 95. 95 MiSeq Workflow Guide Pre-defined data analysis workflows Targeted Resequencing TruSeq Custom Amplicon Nextera PCR Amplicon Metagenomics 16S 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. 96. 96 MiSeq Applications A 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 = Nextera TS RNA = TruSeq RNA Sample Prep TS smRNA = TruSeq Small RNA Sample Prep HB = Homebrew RZ + 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. 97. 97 ILMN vs. Moore’s Law
  98. 98. 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
  99. 99. 99 Comprehensive VeraCode product portfolio Research Use Only VeraCode ADME Core Panel VeraCode Universal Capture & Carboxyl Beads Custom GoldenGate Genotyping, 48, 96, 144, 192 & 384-plex DASL Custom Gene Expression, 32 to 384-plex Custom GoldenGate Methylation, 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. 100. 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 For Research Use Only
  101. 101. 101 Sequencing Services Illumina Clinical Services Lab Somatic Mutation Panel Somatic Mutation Panel launch Q4 11 Content will include genes with proven/anticipated clinical utility: KRAS EGFR BRAF TP53 VEGF-A ERBB2 ESR1 PGR TYMS UGT1A1 TPMT COMT CYP2D6 NRAS EML4/ALK WGS 30+ Whole genomes sequenced Q4 10 First clinical case reimbursed Q4 10 Cancer, genetic diseases, SCID cases Infrastucture CLIA certified for high complexity molecular diagnostics Q1 09 CAP Accredited Q2 09 CA State CLS training program Q1 10
  102. 102. 102 Individual Genome Sequencing: Workflow o Initial discussion & genetic counseling o Informed Consent and Service Agreement o 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 match o Whole genome sequencing of blood DNA o Analyze and QC sequence and called variants o Check sequence and genotype ID match o Archive full dataset Physician orders IGS for patient Genome sequencing and QC Clinical lab delivers data to physician
  103. 103. 103 Innovative Genotyping Approach Make Target Add Oligos Extend/Ligate PCR Make ssDNA Hyb VBS [A/B] b B A Cy Cy Cy c b b
  104. 104. 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
  105. 105. 105 Illumina Sequencing Workflow Fragment DNA Repair ends Add A overhang Ligate adapters Purify Library Preparation1 Cluster Generation Hybridize to flow cell Extend hybridized template Perform bridge amplification Prepare flow cell for sequencing 2 Sequencing Perform sequencing Generate base calls 3 Data Analysis Images Intensities Reads Alignments 4
  106. 106. 106 Sample Prep Workflow DNA RNA
  107. 107. 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 T C A T C A C C T A G C G T A G T Illumina Sequencing Technology Robust Reversible Terminator Chemistry Foundation Sequencing
  108. 108. 108 Simplest Sequencing Workflow Parallel sample processing Automated cluster generation Automated sequencing SIMPLIFIED SAMPLE PREP cBot CLUSTER GENERATION SEQUENCING DATA PROCESSING & ANALYSIS Simple, efficient data analysis
  109. 109. 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. 110. 110 Competitive Environment TruSeq Custom  Amplicon Fluidigm Access  Array HaloPlex PCR PCR/Homebrew Number of Amplicons 48 – 384 48 ‐ 480[1] < 2,000 1 ‐ 5 Target 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
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