© 2016 Illumina, Inc. All rights reserved.
Illumina Next-Generation Sequencing in MicrobiologyPernille Albertus
Senior Marketing Technical Specialist - Illumina
3
Illumina | Our mission
To improve human health byunlocking the power of the genome
For Research Use Only. Not for use in diagnostic procedures.
4
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
5
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
6
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.
7
Illumina | Who we serve
Research
Forensics Agriculture
Complex Disease
Reproductive Health Oncology
Consumer
Genetic Health BioPharm
Infectious Disease
Population Sequencing
10
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
12
1 ng input gDNA
designed for small genomes
fast workflow
low investment in ancillary
equipment
Prepare library Sequence Analyze
Nextera XT library preparation kit
13
LoadGo
preloaded reagent cartridge
auto flow cell positioning
walkaway automation
MiSeq Sequencer
Prepare library Sequence Analyze
14
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
15
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
16
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.
19
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
20
newly
synthesized
strandoriginal
template
● The double-stranded molecule is
denatured
● The original template is washed
away
Denature double-stranded DNA
discard
21
● The newly synthesized strand is
covalently attached to the flow cell
surface
Anchor the template to the surface
22
● 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
24
Denature the double-stranded bridge
● The double-stranded bridge is
denatured
● Result: Two copies of covalently
bound single-stranded templates
25
● Single-stranded molecules flip over to hybridize to adjacent primers
● Hybridized primer is extended by polymerase
Bridge amplification
28
● .. 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
30
● A sequencing primer
is introduced to the
flow cell and
hybridized to the
adapter sequence
annealing site
Read 1 sequencing primer hybridization
Sequencing
primer
31
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
32
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
33
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
35
Original
forward
strand
● Bridges are linearized and the
original forward template is
cleaved off
Paired-end sequencing
36
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
40
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
41
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