© 2016 Illumina, Inc. All rights reserved.

Illumina Next-Generation Sequencing in MicrobiologyPernille Albertus

Senior Marketing Technical Specialist - Illumina

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Introduction to the company

Illumina

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Illumina | Our mission

To improve human health byunlocking the power of the genome

For Research Use Only. Not for use in diagnostic procedures.

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Illumina | Our background

FINANCIALS

$2.40B 8%2016 Revenue

REVENUE

GROWTH YOY

AWARDS

TOP 3 SMARTEST COMPANIES2016 MIT TECHNOLOGY REVIEW

10 BREAKTHROUGH TECHNOLOGIES

2016 MIT TECHNOLOGY REVIEW

12 MOST DISRUPTIVE NAMES IN BUSINESS

2013 FORBES

TOP 10 INNOVATIONS

2015 THE SCIENTIST MAGAZINE

TOP 10 INNOVATIONS

2012 THE SCIENTIST MAGAZINE

FASTEST GROWING TECH COMPANIES

2010 FORBES

COMPANY

Fall, 1998FOUNDED

July 27, 2000IPO

~5,500EMPLOYEES

San Diego, CAHEADQUARTERS 20

Francis deSouzaPresident and CEO

OFFICES

GLOBALLY

TOP 10 MOST INNOVATIVE COMPANIES

IN BIOTECH

2017 FAST COMPANY

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Illumina | Platforms

SEQUENCING SYSTEMS | Sequencing-by-Synthesis (SBS)

FLEXIBLE POWER

NextSeq® 500/550

POPULATION POWER PRODUCTION POWER

HiSeq® 2500/3000/4000/X

CUTTING-EDGE ARRAY SCANNER

iScan®

ARRAY SCANNER | Infinium

* For In Vitro Diagnostic Use

FOCUSED POWER

MiSeq®, MiSeqDx®*, MiSeqFGx™ MiniSeq™

For Research Use Only. Not for use in diagnostic procedures.

NovaSeq™ 5000/6000

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2014$1,000 1st $1,000 genome

2010$10,000 1st sub-10K genome

2008$200,000 1st 30x genome

2007$2,000,000 1st NGS Genome

2006$20,000,000 1st individual genome

2003$3,000,000,000 Human Genome Project

Illumina | Driving down the cost of sequencing

For Research Use Only. Not for use in diagnostic procedures.

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Illumina | Who we serve

Research

Forensics Agriculture

Complex Disease

Reproductive Health Oncology

Consumer

Genetic Health BioPharm

Infectious Disease

Population Sequencing

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Illumina | Groundbreaking technology

Expert Rev. Mol. Diagn. 13(3), 00–00 (2013)

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Libraries – Sequencing – Analysis

The NGS workflow in microbiology

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Four core methods in microbiology

Whole Genome

Sequencing

Generate accurate

reference genomes,

for microbial

identification

Shotgun

Metagenomics

Detect very low

abundance members

of the microbial

community

16S rRNA

Sequencing

Identify and compare

bacteria present

within a given sample

Virology

Determine the

sources of infection,

route of transmission,

and molecular

pathways

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Prepare library Sequence Analyze

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1 ng input gDNA

designed for small genomes

fast workflow

low investment in ancillary

equipment

Prepare library Sequence Analyze

Nextera XT library preparation kit

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LoadGo

preloaded reagent cartridge

auto flow cell positioning

walkaway automation

MiSeq Sequencer

Prepare library Sequence Analyze

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Prepare library Sequence Analyze

BaseSpace is a hosted platform for storage,

analysis, and sharing of NGS data

Tight instrument

integration

Public and private

analysis tools

Easy sharing and

collaboration

- setup and monitor runs

from a web portal

- automatically stream data

from the instrument to

BaseSpace

- eliminate need for server

hardware and IT support

with BaseSpace Cloud

- over 60 published Apps

- deploy your own private

Apps on BaseSpace

Cloud

- share data with anyone in

the world

- no need to set up FTP

site or mail HDs

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Prepare library Sequence Analyze

BaseSpace Apps for bacterial NGS data

Velvet de novo Assemblyoptimized for bacterial Mate Pair Nextera libraries

SPAdes Genome Assemblerstandard isolates and single-cell MDA bacterial assenblies

DNASTAR de novo assembly of bacterial genomes

Rescafprovides an improved set of scaffold sequences using bacterial assemblies

SRST2reports the presence of STs (sequence types) from a MLST database, and more

PEDANT Sequence Analyzerbacterial genome gene prediction, protein annotation, and functional classification of genes

LoFreq Rare Variant Callercall rare variants in viral and bacterial sequencing datasets

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BaseSpace | Tiers

BASIC PROFESSIONAL* ENTERPRISE

FREE $4,995/yearStarts at

$29,995/year*

Storage 1T limit expandable expandable

Analysis** 250-iCredit limit expandable expandable

Run Setup ✔ ✔ ✔

Unlimited Monitoring ✔ ✔ ✔

Unlimited Sharing ✔ ✔ ✔

Multi-user access ✔ ✔

Bioinformatics support 8 hours 24 hours

Multiple organizations ✔

Private domain & single sign-on ✔

PHI Security/Privacy compliance ✔

Audit Trail ✔

Additional storage $360/TB $360/TB

Additional compute $1 per iCredit $1 per iCredit

* Academic version available at $ 995/year excl. 8 hours of bioinformatics support

**Price depends on # of instruments connected.

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Deep dive of SBS chemistry

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Hybridize fragment and extend

adapter

sequence

3’

extension

surface of flow cell

coated with a lawn

of oligo pairs

● Single DNA libraries are hybridized

to the primer lawn

● Bound libraries are then extended

by polymerases

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newly

synthesized

strandoriginal

template

● The double-stranded molecule is

denatured

● The original template is washed

away

Denature double-stranded DNA

discard

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● The newly synthesized strand is

covalently attached to the flow cell

surface

Anchor the template to the surface

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● The single-stranded molecule flips over and forms a bridge by hybridizing to

an adjacent, complementary primer

● The hybridized primer is extended by polymerases

Bridge amplification

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● A double-stranded bridge is formed

Bridge amplification

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Denature the double-stranded bridge

● The double-stranded bridge is

denatured

● Result: Two copies of covalently

bound single-stranded templates

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● Single-stranded molecules flip over to hybridize to adjacent primers

● Hybridized primer is extended by polymerase

Bridge amplification

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● Bridge amplification cycle repeated until multiple bridges are formed

Bridge amplification

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● ds-DNA bridges

are denatured

● Reverse strands

are cleaved..

Linearization

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● .. and washed away,

leaving a cluster

with forward strands

only

Reverse strand cleavage

g c

a t

t a

t a

a t

c g

g c

t a

g c

a t

t a

t a

a t

c g

g c

t a

g c

a t

t a

t a

a t

c g

g c

t a

g c

a t

t a

t a

a t

c g

g c

t a

g

a

t

t

a

c

g

t

g

a

t

t

a

c

g

t

g

a

t

t

a

c

g

t

g

a

t

t

a

c

g

t

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● Free 3’ ends are

blocked to prevent

unwanted DNA

priming

Blocking

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● A sequencing primer

is introduced to the

flow cell and

hybridized to the

adapter sequence

annealing site

Read 1 sequencing primer hybridization

Sequencing

primer

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Sequencing by synthesis (SBS) close-up

C

G

A

T

fluorescently

labeled ddNTPs

are added together

with a polymerase

a base is

incorporated;

unincorporated

nucleotides are

washed away laser excites

fluorophore

camera captures

emission colour

T

fluorophore

is cleaved off

and terminator

is removed

A

the next

cycle begins...

T

T

12

3

4

5

6

TGCTACGAT

T T

G

CYCLE 1 CYCLE 2 CYCLE 3 CYCLE 4 CYCLE 5 CYCLE 6 CYCLE 7 CYCLE 8 CYCLE 9

BASE CALLING

C

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Sequenced

strandBlocked

3’-ends● After completion of

sequencing of the

forward strand, the

sequenced product is

stripped off

● 3’-ends of template

strands and lawn

primer are unblocked

Paired-end sequencing

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Bridge

formation

3’ extension

● Single-stranded template loops over to form a bridge by hybridizing with a

lawn primer

● 3’-ends of lawn primer is extended (double-stranded stretch allows the

polymerase to build the complementary strand)

Paired-end sequencing

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Double

stranded

DNA

Paired-end sequencing

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Original

forward

strand

● Bridges are linearized and the

original forward template is

cleaved off

Paired-end sequencing

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Reverse

strand

template

Blocked

3’-ends

Sequencing

primer

● Free 3’-ends of the reverse

template and lawn primers are

blocked to prevent unwanted DNA

priming

● The Read 2 sequencing primer is

introduced to the flow cell and

hybridized to the adapter

sequence annealing site

Paired-end sequencing

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Pooling samples in a single run

● Pooling samples depends on genome size, depth, and read length

organismgenome

sizedepth

read

length

no of samples

MiSeq NextSeq HiSeq*

Staphylococcus aureus

(MRSA)2.8 Mb 100x 2x125 19 267 167

Mycobacterium

tuberculosis (TB)4.4 Mb 100x 2x125 12 170 106

E. coli 4.6 Mb 100x 2x125 11 163 101

Plasmodium falciparum

(malaria)22.9 Mb 100x 2x125 2 43 27

*1 lane HO

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Pooling samples in a single run

● Pooling samples depends on genome size, depth, and read length

organismgenome

sizedepth

read

length

no of samples

MiSeq NextSeq HiSeq*

Staphylococcus aureus

(MRSA)2.8 Mb 100x 2x300 45

Mycobacterium

tuberculosis (TB)4.4 Mb 100x 2x300 29

E. coli 4.6 Mb 100x 2x300 27

Plasmodium falciparum

(malaria)22.9 Mb 100x 2x300 5

*1 lane HO

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Illumina | Driving the future of genomics

palbertus@illumina.com