© 2013 Illumina, Inc. All rights reserved.

Illumina, IlluminaDx, BaseSpace, BeadArray, BeadXpress, cBot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium,

iSelect, MiSeq, Nextera, NuPCR, SeqMonitor, Solexa, TruSeq, TruSight, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks

of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

Applying MiSeq to

Pathogen Tracing

Susan Knowles

Sr. Manager, Market Development

Illumina, Inc.

March 2014

2

Introduction to Illumina

Next Generation Sequencing for Food Pathogens

Supporting the FDA Genome Trakr Network

Public Health 2.0

Goals and Objectives

© 2013 Illumina, Inc. All rights reserved.

Illumina, IlluminaDx, BaseSpace, BeadArray, BeadXpress, cBot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium,

iSelect, MiSeq, Nextera, NuPCR, SeqMonitor, Solexa, TruSeq, TruSight, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks

of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

Introduction to

Illumina

4

Introduction to Illumina

Founded in 1998

Initial Public Offering on July 27, 2000

Headquarters in San Diego, CA

3,000 employees worldwide, San Diego HQ

>$1.4B annual sales 2013

90% of the worlds DNA sequencing

Instruments generate opproximately 1PB/week of sequence data

IP portfolio of 135+ issued patents and 168 pending applications.

5

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)

Illumina

Cambridge

Illumina

Singapore

Illumina Hayward

(Hayward, CA)

Illumina Global

Headquarters

(San Diego, CA)

Australia

South Africa

Greece Turkey

Russia

Middle East

Israel

6

0

100

200

300

400

500

600

$-

$500

$1,000

$1,500

$2,000

$2,500

$3,000

Dec-07 Dec-08 May-09 Sep-09 Dec-09 Jan-10 Jan-10 Jul-11 Oct-14 Jan-14 Mar-14

OU

TP

UT P

ER

DA

Y (

GIG

AB

AS

ES)

CO

ST P

ER

GIG

AB

AS

E

HiSeq 2000 HiSeq 2500 HiSeq 2500 1Tb HiSeq 2000 v3 GAII GAIIx GAIIx 2x100 GAIIx 50Gb HiSeq X Ten GAI GAIIx 95Gb

Innovation: Making Sequencing Faster & Cheaper

7

The New Illumina Portfolio Sequencing Power for Every Scale

MiSeq

Focused Power

NextSeq 500

Flexible Power Regulated Power

MiSeqDx

Production Power

HiSeq 2500

Population Power

HiSeq X Ten

Speed and

simplicity for

whole-genome,

exome, and

transcriptome

sequencing.

Speed and

simplicity for

targeted and

small-genome

sequencing.

The world’s first

CE-IVD and FDA

cleared NGS

platform.

Power and

efficiency for large-

scale genomics.

$1,000 human genome

and extreme throughput

for population-scale

sequencing.

8

Throughput to Match Microbiology Applications

Shotgun metagenomics

Microbial diversity

Gene content and discovery

rRNA Metagenomics

Relative abundance of microbial

diversity

– 16S for bacteria and archaea

– 18S for eukaryotes

Microbial genomics

Detection

Identification

Antibiotic sensitivity testing

Molecular epidemiology

High

Throughput

Low

Throughput

9

Meet Miseq

On-board clustering

Fast SBS

On-board analysis

Integrating three concepts

10

MiSeq- A Closer Look

2 ft

11

Load

MiSeq Simple workflow

Go

VERY SIMPLE USER INTERACTION

Preloaded single use reagent cartridge

contains cluster generation, SBS & PE reagents

RFID based reagents & flowcell tracking

Auto flow cell positioning

Walkaway automation

12

MiSeq Instrument Options for output and read length

FLEXIBLE DATA OUTPUT

Multiple flow cell options

from 1M reads to 25M

from 300MB to 15GB

READ LENGTH

1x50

2x150

2x250

2x300

2x75

0

5

10

15

20

25

30

35

40

45

50

Nano Micro v2 v3

1 4

15

25

1 4

15

25 Read 2

Read 1

READ COUNT

13

Illumina’s cloud computing environment.

Most MSR workflows available on

BaseSpace

Free data storage

Data sharing with collaborators

Streamlined on-board analysis workflows

No intervention from sample loading to

report

All workflows generate FASTQ files that

can be analyzed by most 3rd party apps

Simplify Analysis MiSeq Reporter and BaseSpace

TruSeq

Amplicon

De Novo

Assembly Enrichment Generate

FASTQ LibraryQC Metagenomics PCR

Amplicon

Resequencing Small

RNA

MiSeq Reporter

(MSR)

14

Greater than 85% of desktop data generated on MiSeq

Analysis of data submissions to the NCBI Sequence Read Archive (SRA); *As of January 02, 2014

Most Widely Used Desktop NGS System

15

Adopted by Worldwide Public Health Agencies NGS Networks

FOOD-BORNE

PATHOGEN OUTBREAKS

PUBLIC HEALTH

GENOMIC

EPIDEMIOLOGY

GENOMIC

EPIDEMIOLOGY

16

NGS for Food Pathogens

PROOF OF PRINCIPLE STUDIES

PILOT PROGRAM

IMPACT

17

Food Safety Testing Leveraging NGS data to revolutionize pathogen analysis

Outbreak Detection

– Cluster determination

– Is the strain known,

related to a known

strain or novel?

In-depth Analysis

– Pathogenicity –

identify genes

associated with toxicity

and virulence

– Taxonomy

Outbreak

Management

– Epidemiology

– Traceback and source

attribution

– Recalls

18

Listeria outbreak in cantaloupes

Proof of Principle Retrospective Study NGS Analysis of Listeria Outbreak Samples*

July 2011, Listeria-contaminated

cantaloupe outbreak spread to 28

states, infected 146 people, killing

30.

The outbreak was tracked by

PulseNet US, national molecular

subtyping surveillance system of

foodborne pathogens.

Pulse field gel electrophoresis

(PFGE) used to subtype Listeria

isolates from human cases and

cantaloupe samples and track the

outbreak.

Is NGS a more effective way to way

of performing bacterial typing?

*Collaboration with US CDC PulseNet

19

Ease of Use: Bacterial Genome Sequencing from Isolate Efficient workflow and quality data for resequencing

Nextera XT Library Prep

2.5 hours

Prepped Library thru Sequencing

27 hours

(20 minutes hands on)

Resequencing Alignment and Variant Calling

2 hours

(fully automated)

31:30 gDNA

20

Sample Homozygous

SNPs

A01 194

A02 131

A03 14,312

A04 55,857

A05 136

A06 57,526

A07 14,775

A08 193

MiSeq Reporter Resequencing Workflow SNPs accurately measure variation from the reference genome

MiSeq Reporter - on-board analysis

workflows

Resequencing – reconstruction of a

genome sequencing from reads

mapped to a previously sequenced

reference genome.

Outbreak samples yielded > 92%

alignment with the reference genome.

A04 and A06 - highly divergent

from the reference.

A03 and A07 – Divergent (to a

lesser extent) from the reference.

21

Assess Concordance with PFGE Data SNP-Based clustering illustrates genomic relatedness

Hierarchical clustering of samples

based on SNP calls.

Sample 1, 2,5 and 8 seem closely

related

Samples 4 and 6 and 3 and 7 seem

to form outlier groups, respectively.

The results matched what

PulseNet obtained viewing the

results generated by PFGE.

SNP-based analysis – differentiates

by as little as one SNP

22

Pilot Network: FDA CFSAN Selects MiSeq to Identify

Foodborne Pathogens

7 State health departments

and 10 FDA-ORA labs

Genome Trakr Network1

A pilot network and coordinated effort

across state and federal labs.

Sequencing pathogens collected from

foodborne outbreaks, contaminated

food products and environmental

sources.

Illumina’s role

– MiSeq instruments

– Library prep and sequencing

reagents

– Installation and training

– Service and support

Source:

http://www.fda.gov/Food/FoodScienceResearch/WholeGenome

SequencingProgramWGS/ucm363134.htm

23

FDA Protocol Application: Bacterial WGS from culture

Sample Prep Nextera

Library Prep

MiSeq &

Primary

Analysis

• Nextera XT

• ~12 samples

per run

• Sequencing kit:

500 cycle kit,

2 x 250 bp

paired-end

sequencing.

• 20x-30x

coverage

• MiSeq Reporter

workflow:

Generate FASTQ

• Lyse cells from

cultured

isolate

• Genomic DNA

extraction • Data sent to FDA

or BaseSpace for

storage and

sharing with FDA

and upload to NCBI

SRA database and

analysis.

• Grow

culture

24

Impact: NGS Used to Assess Food Pathogen Outbreak in

Food and Clinical Samples

Compared with pulsed-field gel

electrophoresis (PFGE), WGS provides

clearer distinction between cases and

foods that are likely part of a given

outbreak and those that are not.

Whole-genome sequences of the Listeria strains

isolated from Roos Foods cheese products were

available after the recall and were found to be

highly related to sequences of the Listeria strains

isolated from the patients.

25

Technical Applications

Scientists

Field Application Scientists

Field Service Engineers

Territory Account

Managers

Project Management

Supporting the GenomeTrakr Network

Illumina’s service and support infrastructure

“Customers don’t expect

you to be perfect. They

do expect you to fix

things when they go

wrong.” Donald Porter

26

Supporting the GenomeTrakr Network Tech Support Group (TS)

TAS – Technical Applications

Scientists

First line call

Email and phone support

All network accounts flagged

Technical Applications

Scientists

Field Application Scientists

Field Service Engineers

Territory Account

Managers

Project Management

27

FDA CFSAN Network Illumina Support Team Field Application Specialists (FAS)

Deliver on-site trainings and

trouble-shooting

Typically brought into the

picture by TS

Helps with chemistry and

software based troubleshooting

Escalate complex cases

Technical Applications

Scientists

Field Application Scientists

Field Service Engineers

Territory Account

Managers

Project Management

28

FDA CFSAN Network Illumina Support Team Project Management

Track outstanding support

issues via dashboard

Meet regularly with FDA

management

Organize new network lab

trainings

Advance warning of significant

changes to software and

hardware

Technical Applications

Scientists

Field Application Scientists

Field Service Engineers

Territory Account

Managers

Project Management

29

Public Health 2.0 Changing the paradigm

Detect Analyze/Report Share Update

Methods

Simplify

Analysis Connectivity

Faster diagnosis and response times – detection, identification and containment

Improved accuracy and methods – high resolution, high throughput, automated

Cloud-based data storage/exchange and sharing

Functional and geographic connectivity for analysis and communications

30

Thank You