© 2011 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, HiScan, TruSeq, Eco, MiSeq and Nextera are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
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Julien Abriol & Olivier Lucas, Ph.D. Territory Account Manager Inside Sales Consultant
Next-Generation Sequencing: custom solutions
2
Facilities in 7 countries
Over 2,300 employees
More than 850,000 sq. ft.
Forbes “Fastest Growing Technology Company” 2007 & 2009
Added to the NASDAQ-100 listing in 2008
IP portfolio of >235 patents
>1200 R&D staff >400 support personnel
Headquarters in San Diego, CA
Founded in 1998
Illumina at a Glance
3
Commercial Mfg/R&D Partners
Illumina Global Headquarters (San Diego, CA)
Illumina Hayward (Hayward, CA)
Illumina Singapore
Australia
Malaysia
Taiwan Vietnam
New Zealand
Korea Illumina KK (Tokyo)
Illumina China (Beijing)
Shanghai Thailand
Over 2300 Employees >1200 R&D staff >400 Support Personnel
India
Turkey Jinan, China Israel Chengdu, China
Middle East
Greece
Russia
South Africa
Illumina Brazil
Illumina Cambridge
Illumina BV (The Netherlands)
Expanded Manufacturing, R&D, Sales, Service & Support
Global Organization
4
Research
Forensics
BioPharm
Agriculture
Consumer
Human Health
Jay Flatley President and CEO
Serving Many Customers
Infectious Disease
Genetics
Reproductive Health
Cancer
5
To Targeted Validation and Beyond!
To be the leading provider of integrated solutions that advance the understanding of genetics and health
From Genome Wide Discovery!
Innovating for the Future of Genetic Analysis
Our Vision
6
Powerful, Flexible, Scalable
Redefining the trajectory of sequencing
Miseq
HiSeq 1500
HiSeq 2500 Most widely adopted NGS platform
Genome Analyzer IIx
Sequencing
Unique combination of sequencing and arrays
HiScanSQ
BeadXpress Accuracy, versatility and flexibility for molecular testing
iScan Speed, quality and versatility for arrays
Arrays
From Genome-Wide Discovery to Targeted Validation and Screening
Gold-standard qPCR made accessible
Eco
qPCR
Illumina Portfolio Overview – Innovation is in our DNA
7
GEX Methylation ChIP-Seq Small RNA
– – – – – –
ChIP-Seq
Whole-genome sequencing Metagenomics Targeted resequencing resequencing CNV de novo sequencing
DNA Analysis
– – – –
Gene Expression / Regulation
Power and flexibility
Illumina Genomics
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Genetic analysis
Microalgae Natural products
Agricultural
(Barcoding/metagenomics)
Biodiversity Traceability
Fisheries
Genetic selection Feed traceability
Acquaculture
Marine Biotechnologies
Pharmacogenomics Metagenomics
Genetic engineering
Medical
Natural products
New enzymes
Industry
9
– Genomics in marine monitoring: New opportunities for assessing marine health status (2013) Bourlat et al. Marine Pollution Bulletin
Marine Genomics 4 Users (mg4u.eu): « for a targeted transfer of knowledge to Industry”
– Transcriptomics of microinvertebrates (Welch lab) – Marine biodiversity/Encyclopedia of Life (Zettler lab) – Functional genomics of microbial genomes (Serres lab) – Toxicology (Hamilton lab) – Metagenomes (Sogin, Huse, Morrisson, Eren labs)
Woods Hole Marine Biological Lab now fully transitionned
Illumina in marine biology and marine biotechnology
© 2011 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, HiScan, TruSeq, Eco, MiSeq and Nextera are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
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Next Generation Sequencing Instruments
11
ABI3730
60,000
Data Equivalence!.
MiSeq
75
1
HiSeq eq 25 2500
12
22.9 Mb
Plasmodium falciparum
0.5 Mb 25 Mb 6M tags
All coding exons
RNA, miRNA, ChIP-Seq, etc
3 kb
Targeted region
20 exons
4.6 Mb
Escherichia coli
Target size
4.4 Mb
Mycobacterium tuberculosis (TB)
Human
2.8 Mb
Genome size
Staphylococcus aureus (MRSA)
Organism
Of MiSeq v2, Microbes, and Man
3
3
162
675
n
6
18
18
27
n
n.a.
40x
50x
2000x
depth
30x
50x
50x
50x
depth
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© 2011 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, HiScan, TruSeq, Eco, MiSeq and Nextera are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
. .
Workflow
15
4
5
Image Acquisition
7
8
6
3
1
2
Cluster Growth
Single molecule array
Library Preparation
DNA (0.05-1.0 µg)
Illumina Sequencing Workflow
9
T
G
A
T
G
A
C
5’
Sequencing
G
T G T A C G A T C A C C C G A T C G A A
Base Calling
TG TACGAT!
C
G
C C
A
3’ 5’
16
MiSeq System
17
Included On-Instrument: Cluster Generation Paired-End Fluidics Computing for Primary and Secondary Analysis
2007: 2010: 2011: GAMiSeq HiSeq launch, launch, the year 3NGS genomes/run ofin the routine 1st Gb
The World’s Most Widely Adopted Sequencing Technology Just Got Personal
MiSeq – Single Instrument Workflow
18
4 Data Analysis
Images Intensities Reads Alignments
Perform sequencing Generate base calls
Hybridize to flow cell Extend hybridized template Perform bridge amplification Prepare flow cell for sequencing
Fragment DNA Repair ends Add A overhang Ligate adapters Purify
3 Sequencing
2 Cluster Generation
1 Library Preparation
Illumina Sequencing Workflow
Plasmid
Library QC
RNA sequencing
ChIP-Seq
Small genome
19
Targeted Resequencing
Custom Amplicon
Amplicon Sequencing
Integrated. Optimized. Simplified.
MiSeq: for all you seek
Regulation
Custom Enrichment
De novo sequencing
RNA-Seq
Small RNA sequencing
16S Metagenomics
Resequencing
Clone checking
3Q11
4Q11
2 x 150 bp
Read length
2Q11
~7M
>1.5 Gb
Clusters
Output
1Q12
2Q12
3Q12
4Q12
Faster chemistry Dual surface imaging D
2 x 250 bp
>15M
Clusters Read length
>8 Gb
Output
1Q13
2Q13
3Q13
4Q13
2 x 300 bp
Read length
August 2013
25M
Clusters
“This information is intended to outline general product direction and it should not be relied on in making a purchasing decision. This material is for information purposes only and may not be incorporated into any contract. This information is not a commitment, promise, or legal obligation to deliver 20 this functionality. The development, release, and timing of any features or functionality described for our products remains at our sole discretion.”
1
10
20
15 Gb
Output
Path towards 15Gb per run; enabling broader range of applications
MiSeq – Continuous Performance Improvements
Output - Gb
21
Path towards 15Gb per run; enabling broader range of applications
MiSeq – Continuous Performance Improvements
22
HiSeq 2500 Sequencing System
23
• Longer Reads
• Clustering on-board • Fast Chemistry
MiSeq
Combining innovation
HiSeq 2500
• Longer 2x150 reads
• Rapid turnaround • Clustering on-board • Complete walk-away workflow
HiSeq 2500 System
• Data rate • TDI scanning • Larger flow cell
HiSeq 2000
24
120Gb in ~1 day 600 million clusters No cBot required
600 Gb in ~10.5 days 3 billion clusters cBot required
1 human genome in a day
Fastest turnaround
5 human genomes in 10.5 days
Highest output
User configurable
Rapid Run Mode
High Output Mode
1 Instrument – 2 Run Modes
Fast turnaround and highest output in a single instrument
HiSeq 2500 Sequencing System
25
– Services already available! – Commercial kits Q1 2014
Anticipated availability
– High accuracy from Illumina SBS
Obviates need for long read systems
Complete solution including sample prep, sequencing w/ Illumina systems, cloud-based informatics
– Novel library prep & analysis algorithm
Proprietary technology for phased synthetic long reads (>10Kb) from short reads
Enabling synthetic read lengths up to 10Kb from Illumina short reads
Acquisition of Moleculo
26
Applications
27
MiSeq/ HiSeq
Microalgae Natural products
Agricultural
(Barcoding/metagenomics) (Ba ics)
Biodiversity Traceability
Fisheries
Genetic selection Feed traceability
Acquaculture
Marine Biotechnologies
P cs Pharmacogenomics Metagenomics
Genetic engineering G ng
Medical
Natural products
New enzymes
Industry
© 2011 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, HiScan, TruSeq, Eco, MiSeq and Nextera are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
From DNA to sequencing-ready library in as little as 90min
Sequencing’s fastest and easiest sample prep
NexteraXT DNA Sample Preparation Kit
29
Step 1: Tagmentation of template DNA
30
Step 2: PCR to add adapters and indices
31
Step 3: Cleanup and Sequence
32
Quant in triplicate with qPCR Manually dilute and pool
5 µl of each desired library
Calculate dilutions
Nextera XT sample pooling is as simple as pipetting 5 µl!
Bead-based Normalization: Bind, Wash, Elute
Completed libraries, range of yields
13.5%
15.8%
Pool A
15.5%
18.2%
Pool B
Index CV for 20-sample pools
No library quantification or qPCR is required – go straight to MiSeq!
Sample Normalization is included
33
100ng-5 µg
Nexter a
1 or 50 ng
Streamlined Library Preparation
98% of genome with average coverage of 30x
5.4 million reads yielded 175Mb of data which aligned to ATCC10987 with mismatch rate of 0.06%
Resequencing of 5.2Mb B. cereus in a single workday
MiSeq Applications Small Genome Resequencing
*1x36bp
Align to reference/Call SNPs 2 hours On Board Instrument
Clustering + Sequencing 4.5 hours (20 mins hands on)
(15-30 min hands on)
gDNA Prep ! Library 1.5 hours
Desired coverage 30X
4.5Gb of sequencing (2x150bp)
Indicative costs:
39
Genome size: 50Mb
– 4*40=160€ – 780€ – 313€/genome
Resequencing of 3 algae strains
MiSeq Applications Small Genome Resequencing
Align to reference/Call SNPs >2 hours On Board Instrument
Clustering + Sequencing 24 hours (20 mins hands on)
(15-30 min hands on)
gDNA Prep ! Library 1.5 hours
For 40Research Use Only
RNA seq
41
