Post on 11-Oct-2020
© 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