© 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.
Next-Generation
Sequencing:
custom solutions
Julien Abriol & Olivier Lucas, Ph.D.
Territory Account Manager
Inside Sales Consultant
.
2
Founded in 1998
Headquarters in San Diego, CA
More than 850,000 sq. ft.
Facilities in 7 countries
Over 2,300 employees
>1200 R&D staff
>400 support personnel
IP portfolio of >235 patents
Added to the NASDAQ-100 listing in 2008
Forbes “Fastest Growing Technology Company” 2007 & 2009
Illumina at a Glance
3 3
Global Organization Expanded Manufacturing, R&D, Sales, Service & Support
Commercial
Mfg/R&D
Partners
Illumina KK (Tokyo) Jinan, China
Chengdu, China
Korea
India
Malaysia
Vietnam
Shanghai
New Zealand
Thailand
Taiwan
Illumina BV (The Netherlands)
Illumina China (Beijing)
Over 2300 Employees
>1200 R&D staff
>400 Support Personnel
Illumina Cambridge
Illumina Singapore
Illumina Hayward (Hayward, CA)
Illumina Global Headquarters (San Diego, CA)
Australia
South Africa
Greece Turkey
Russia
Middle East
Israel
Illumina Brazil
4
Serving Many Customers
Jay Flatley
President and CEO
Human Health
Genetics
Infectious Disease
Research Reproductive Health
Forensics
Consumer
Cancer
BioPharm
Agriculture
Human Health
Genetics
Infectious Disease
Research Reproductive Health
Forensics
Consumer
Cancer
BioPharm
Agriculture
5
Our Vision Innovating for the Future of Genetic Analysis
To be the leading provider of integrated solutions that
advance the understanding of genetics and health
To Targeted Validation and Beyond!
From Genome Wide Discovery!
6
Illumina Portfolio Overview – Innovation is in our DNA
From Genome-Wide Discovery to Targeted Validation and Screening
HiScanSQ
Unique combination of sequencing and
arrays
HiSeq 2500
Redefining the trajectory of sequencing
Genome Analyzer IIx
Most widely adopted NGS
platform
iScan
Speed, quality and versatility
for arrays
BeadXpress
Accuracy, versatility and flexibility for
molecular testing
Eco
Gold-standard qPCR made accessible
Sequencing Arrays qPCR
HiSeq 1500
Powerful, Flexible, Scalable
Miseq
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Illumina Genomics Power and flexibility
ChIP-Seq
Gene Expression / Regulation
– GEX
– Methylation
– ChIP-Seq
– Small RNA
DNA Analysis
– Whole-genome sequencing
– Metagenomics
– Targeted resequencing
– resequencing
– CNV
– de novo sequencing
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M a r i n e B i o t e c h n o l o g i e s
Industry
New enzymes
Natural products
Agricultural
Microalgae
Natural products
Acquaculture
Genetic selection
Feed traceability
Fisheries
Biodiversity
Traceability
(Barcoding/metagenomics)
Medical
Genetic engineering
Pharmacogenomics
Metagenomics Genetic analysis
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Woods Hole Marine Biological Lab now fully transitionned – 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)
Marine Genomics 4 Users (mg4u.eu): « for a targeted transfer of knowledge to Industry”
– Genomics in marine monitoring: New opportunities for assessing marine health status (2013)
Bourlat et al. Marine Pollution Bulletin
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.
Next Generation
Sequencing
Instruments
.
.
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Data Equivalence!.
60,000
HiSeq 2500
MiSeq MiSeq75
eq 25
1
ABI3730
12
Of MiSeq v2, Microbes, and Man
Organism Genome size n depth
Staphylococcus aureus (MRSA) 2.8 Mb 27 50x
Mycobacterium tuberculosis (TB) 4.4 Mb 18 50x
Escherichia coli 4.6 Mb 18 50x
Plasmodium falciparum 22.9 Mb 6 30x
Human Target size n depth
20 exons 3 kb 675 2000x
Targeted region 0.5 Mb 162 50x
All coding exons 25 Mb 3 40x
RNA, miRNA, ChIP-Seq, etc 6M tags 3 n.a.
13
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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
Library Preparation
DNA
(0.05-1.0 µg)
1 2 3 7 8 9 4 5 6
T G T A C G A T !
Illumina Sequencing Workflow
C
C
C
C C C C C C C C C C C C C C
A
A
A
T
T
G
G
G
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Sequencing
Single molecule array
Cluster Growth
Image Acquisition Base Calling
5’
5’ 3’
T G T A C G A T C A C C C G A T C G A A A A A A G G C C
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MiSeq System
17
MiSeq – Single Instrument Workflow The World’s Most Widely Adopted Sequencing Technology Just Got Personal
Included On-Instrument:
Cluster Generation
Paired-End Fluidics
Computing for Primary and Secondary Analysis
2007: GA launch, the year of the 1st Gb 2010: HiSeq launch, 3 genomes/run 2011: MiSeq launch, NGS in routine
18
Illumina Sequencing Workflow
Fragment DNA Repair ends Add A overhang
Ligate adapters Purify
Fragment DNA Repair ends Add A overhang
Ligate adapters Purify
Library Preparation 1
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
19
MiSeq: for all you seek Integrated. Optimized. Simplified.
Amplicon Sequencing
Custom Amplicon
De novo sequencing
Resequencing
16S Metagenomics
RNA-Seq ChIP-Seq
Targeted Resequencing
Custom Enrichment
Library QC
Small genome
Plasmid
Clone checking
Regulation
Small RNA sequencing
RNA sequencing
20
2Q11 3Q11 4Q11 1Q12 2Q12 3Q12 4Q12 1Q13 2Q13 3Q13 4Q13
MiSeq – Continuous Performance Improvements Path towards 15Gb per run; enabling broader range of applications
1
10
20
Ou
tpu
t -
Gb
Output >1.5 Gb
Clusters ~7M
Read length 2 x 150 bp
Output >8 Gb
Clusters >15M
Read length 2 x 250 bp
Output 15 Gb
Clusters 25M
Read length 2 x 300 bp
Faster chemistry Dual surface imaging
August 2013
Output >1.5 Gb
Clusters ~7M
Read length 2 x 150 bp
Read length 2 x 250 bp
Faster chemistry Dual surface imaging D
“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
this functionality. The development, release, and timing of any features or functionality described for our products remains at our sole discretion.”
21
MiSeq – Continuous Performance Improvements Path towards 15Gb per run; enabling broader range of applications
22
HiSeq 2500 Sequencing System
23
HiSeq 2500 System Combining innovation
• Clustering on-board
• Fast Chemistry
• Longer Reads
• Data rate
• TDI scanning
• Larger flow cell
• Rapid turnaround
• Clustering on-board
• Complete walk-away workflow
• Longer 2x150 reads
MiSeq HiSeq 2500 HiSeq 2000
24
HiSeq 2500 Sequencing System Fast turnaround and highest output in a single instrument
1 human genome
in a day
High Output Mode
600 Gb in ~10.5 days
3 billion clusters
cBot required
Rapid Run Mode
120Gb in ~1 day
600 million clusters
No cBot required
1 Instrument – 2 Run Modes
User configurable
5 human genomes
in 10.5 days
Highest output Fastest turnaround
25
Acquisition of Moleculo Enabling synthetic read lengths up to 10Kb from Illumina short reads
Proprietary technology for phased synthetic long reads (>10Kb) from
short reads
– Novel library prep & analysis
algorithm
Complete solution including
sample prep, sequencing w/
Illumina systems, cloud-based informatics
Obviates need for long read
systems
– High accuracy from Illumina SBS
Anticipated availability
– Services already available!
– Commercial kits Q1 2014
26
Applications
27
Industry
New enzymes
Natural products
Agricultural
Microalgae
Natural products
Acquaculture
Genetic selection
Feed traceability
Fisheries
Biodiversity
Traceability
(Barcoding/metagenomics)
Medical
Genetic engineering
Pharmacogenomics
Metagenomics MiSeq/
HiSeq
M a r i n e B i o t e c h n o l o g i e s
(Ba
ng
cs
G
P
ics)
© 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.
NexteraXT DNA Sample
Preparation Kit Sequencing’s fastest and
easiest sample prep
From DNA to sequencing-ready library in as little as 90min
29
Step 1: Tagmentation of template DNA
30
Step 2: PCR to add adapters and indices
31
Step 3: Cleanup and Sequence
32
Sample Normalization is included No library quantification or qPCR is required – go straight to MiSeq!
Completed libraries, range of yields
Index CV for
20-sample pools
Quant in triplicate with
qPCR
Calculate dilutions
Manually dilute and pool
Pool A Pool B
15.8% 18.2%
Nextera XT sample pooling is as simple as pipetting 5 µl!
Bead-based Normalization:
Bind, Wash,
Elute
13.5% 15.5%
5 µl of each desired library
33
Streamlined Library Preparation
~4-6 hours 100ng-5 µg
1 or 50 ng <2 hours
Nextera
34
Microbial profiling with 16S
35
16S ribosomal DNA for microbial profiling
V1 V2 V3 V4 V5 V6 V7 V8 V9
C1 C2 C3 C4 C5 C6 C7 C8 C9
• 16S rRNA forms part of bacterial ribosomes. • Contains regions of highly conserved and highly variable sequence. • Large public 16S databases available for comparison • Conserved regions can be targeted to amplify broad range of bacteria from environmental samples. • Not quantitative due to copy number variation
36
16S rRNA Metagenomics
37
Small genome (re)sequencing
38
MiSeq Applications Small Genome Resequencing
Resequencing of 5.2Mb B. cereus in a single workday
5.4 million reads yielded 175Mb of data
which aligned to ATCC10987 with
mismatch rate of 0.06%
>98% of genome with average coverage
of 30x
Clustering + Sequencing
4.5 hours
(20 mins hands on)
gDNA Prep ! Library
1.5 hours
(15-30 min hands on)
Align to reference/Call
SNPs
2 hours
On Board Instrument
*1x36bp
39
MiSeq Applications Small Genome Resequencing
Resequencing of 3 algae strains
Genome size: 50Mb
Desired coverage 30X
4.5Gb of sequencing (2x150bp)
Indicative costs:
– 4*40=160€
– 780€
– 313€/genome
Clustering + Sequencing
24 hours
(20 mins hands on)
gDNA Prep ! Library
1.5 hours
(15-30 min hands on)
Align to reference/Call
SNPs
>2 hours
On Board Instrument
40
RNA seq
For Research Use Only
41
RNAseq adoption
Mortazavi et al., (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods
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Publications in Pubmed relative to RNAseq or microarrays (September 30th, 2013; normalized to max)
Data pulled from NIH database, represents grants in 1st year of support
42
RNA Seq Description
Quantitate levels of RNA expression
Discover and profile mRNA in any eukaryotic species
Obtain full sequence from any poly-A tailed RNA to analyze novel transcripts,
novel isoforms, alternative splice sites, rare transcripts, and cSNPs in one
experiment.
Profile small RNA in any organism without any prior assumptions
Find novel microRNAs, characterize mutations, and analyze the differential
expression of all small RNAs
Generate reads that maintain information about the strandedness of the
transcript for transcriptome annotation or bacterial transcriptome profiling
Discover and profile non-coding RNA (ncRNA) in the transcriptome
43
Experimental Design Required Read depth
Required Reads Per Sample for RNA-Seq (Human)
44
TruSeq RNA Workflow Overview
mRNA kit Total RNA kit
45
Qualitative and quantitative RNA analysis
Any species - even when reference
genome not available
No prior knowledge required
– Alternative transcripts
– Gene fusions
– cSNPs, allele-specific expression
RNA-Seq Benefits A Sequencing-based Technology to Profile the Transcriptome
46
Illumina’s Suite of Sample Prep Solutions
DNA Sample Prep
Nextera
Nextera XT
TruSeq DNA LT & HT
TruSeq DNA PCR-free
Nextera Mate Pair
Targeted
Resequencing
TruSeq Custom Amplicon
TruSeq Exome & Custom
Enrichment
Nextera Exome & Custom
Enrichment
RNA & Regulation
Sample Prep
TruSeq Small RNA
TruSeq RNA
TruSeq Stranded RNA
(FFPE)
TruSeq ChIP
47
Thank You!
48