julien abriol & olivier lucas, ph.d. territory account ... · illumina portfolio overview –...

© 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

7

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

8

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

9

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

.

.

11

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

!"#$%"&'()"*+,-),.)"

/,.0,-+"

1$-$"

,23(04'-"

5$67$-4$"

!""""""""""""""""""""""""""

#"""""""""

8,30((,.9"5$67$-40-1" #&*"5$67$-40-1"

© 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

G

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

16

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

:"

;"

<::=" <::>" <:;:" <:;;" <:;<" <:;?"

@#!5$6" 204.',..,95"

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