illumina gaiix for high throughput sequencing
TRANSCRIPT
Illumina Genome Analyzer IIx for
high throughput sequencing
Cosentino Cristian, PhD
Genomics and Bioinformatics unit
Filarete Foundation – Milan (IT)
15.4.2010
Summary
Towards NGS sequencing
Data management
1
3
Target enrichment4
NGS with Illumina GAIIx2
Genome Analyzer IIx
Summary
Towards NGS sequencing
Data management
1
3
Target enrichment4
NGS with Illumina GAIIx2
Approaching to NGS
Discovery of DNA structure(Cold Spring Harb. Symp. Quant. Biol. 1953;18:123-31)
1953
Sanger sequencing method by F. Sanger(PNAS ,1977, 74: 560-564)
1977
PCR by K. Mullis(Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73)
1983
Development of pyrosequencing(Anal. Biochem., 1993, 208: 171-175; Science ,1998, 281: 363-365)
1993
19
80
19
90
20
00
20
10
Single molecule emulsion PCR 1998
Human Genome Project(Nature , 2001, 409: 860–92; Science, 2001, 291: 1304–1351)
Founded 454 Life Science 2000
454 GS20 sequencer(First NGS sequencer)
2005
Founded Solexa 1998
Solexa Genome Analyzer(First short-read NGS sequencer)
2006
GS FLX sequencer(NGS with 400-500 bp read lenght)
2008
Hi-Seq2000(200Gbp per Flow Cell)
2010
Illumina acquires Solexa(Illumina enters the NGS business)
2006
ABI SOLiD(Short-read sequencer based upon ligation)
2007
Roche acquires 454 Life Sciences(Roche enters the NGS business)
2007
NGS Human Genome sequencing(First Human Genome sequencing based upon NGS technology)
2008
1
Next-generation sequencing platforms
Isolation and
purification of target
DNA
Sample preparation
Cluster generation
on solid-phaseEmulsion PCR
Sequencing by synthesis
with reversible
terminators
Pyrosequencing Sequencing by ligation
Sequencing by synthesis
with reversible
termintors
Am
pli
fic.
Ch
emis
try
Data analysis
Roche 454Illumina GAII ABi SOLiD Helicos HeliScope
1
Single moleculeClonally amplified DNAs
Summary
Towards NGS sequencing
Data management
1
3
Target enrichment4
NGS with Illumina GAIIx2
Cluster station
Genome Analyzer IIx Paired-end module Linux server
Bioanalyzer 2100
GAIIx instrumentsIllumina GAII
Flow Cell
2
ApplicationsIllumina GAII 2
de novo sequencing (whole-genome)
re-sequencing (whole-genome or targeted)
RNA-seq
smallRNA-seq
CHiP-seq
Single-read
Paired-end
Multiplexing
Sequencing by synthesis with reversible terminatorIllumina GAII 2
Parameter Performance
Amplification Bridge-PCR on solid-phase
Chemistry SBS with reversible terminators
Cost 2 $/Mbp
Advantages Disadvantages
•Most widely used platform (> 90 science/nature publication)•Sample preparation automatable•SBS, real-time analysis and base calling are performed simultaneously to the run•Automated cluster generation procedure
•Low multiplexing capability•Substitution errors
Coverage estimation 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
0.1
1
10
100
1000
10000
0.001 0.01 0.1 1 10
Co
vera
ge/l
ane
Input DNA (Gbp)
35 cycles
50 cycles
75 cycles
100 cycles
35x2 cycles
50x2 cycles
75x2 cycles
100x2 cycles
GAIIx sequencing workflowIllumina GAII 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Workbench
Cluster Station
Genome Analyzer
Linux Server
Library preparationSample
preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
gDNA Fragmented
DNA
Adaptor-
ligated DNAGel purification
2
Library validation
gDNA
2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
smallRNA
Total RNA
Bioanalyzer 2100
GAIIx sequencing workflowIllumina GAII 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Workbench
Cluster Station
Genome Analyzer
Linux Server
Flow cell
1 2 3 4 5 6 7 8
Lanes
120
tiles/lane
2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Cluster generation 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Amplification
Linearization, blocking and
primer hybridization
SBS sequencing
Sample preparation
Clusteramplification
Bridge amplification
Hybridize adapter-ligated forward
fragment and extend
Extension is completed
Denature dsDNA and wash original forward template;
reverse template stays covalently attached to the array
2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Bridge amplification of the reverse fragment
Double-strand bridge is formed
Double strand bridge is denatured and reverse as wel as
forward fragments are covalently attached to the array
Bridge amplification 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Bridge amplification is repeated to enlarge the cluster
Double-strand bridges are denatured
Reverse strands fragments are cleaved and washed away
Bridge amplification 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Cluster with forward strands only,
covalently attached to the array
Sequencing primers start the
SBS process
Bridge amplification 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Sequencing workflowIllumina GAII 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Workbench
Cluster Station
Genome Analyzer
Linux Server
SBS technology 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Two colour excitation
Four colour emission detection
GAIIx optical path 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Install prism
Install flow-cell
Apply oil
First-base incorporation
Adjust focus
Check quality metrics
36-100 cycles sequencing run
for Read 1
Cluster amplified FlowCell
Read 1 analysis pipeline
Single-read sequencing workflow 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Paired-end sequencing workflow 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Install prism
Install flow-cell
Apply oil
First-base incorporation
Adjust focus
Check quality metrics
36-100 cycles sequencing run
for Read 1
Cluster amplified FlowCell
Read 1 and 2 analysis pipeline
Prepare Read 2
36-100 cycles sequencing run
for Read 2
Paired-end strategy
Paired-end sequencing works into GA and uses chemicals from PE
module to perform cluster amplification of the reverse strand
2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Sin
gle
-rea
d
(CS
)
Pai
red
-end
(PE
M)
Gene 1 Gene 2Reference
sequence
Sequence
reads
Mapped
read
Unmapped
read
Gene 1 Gene 2Reference
sequence
Sequence
reads
Mapped
read
Mapped
read
Paired-end (read 1 & read 2)
Single-read (read 1)
Paired-end strategy 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Sequencing workflowIllumina GAII 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Workbench
Cluster Station
Genome Analyzer
Linux Server
Firecrest and CASAVA
Firecrest
From image
to intensity
Bustard
From intensity
to reads
CASAVA
Consensus assembly
Gerald/ELAND
Alignment to genome
Base calls filesIntensity filesImage files
AssemblyAlignment files
2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
GenomeStudio
Data visualization
Sequence
ANALYSIS
GenomeStudio viewer 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
Small RNA sequencing
GenomeStudio viewer 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
RNA sequencing
GenomeStudio viewer 2
Sample preparation
Clusters amplification
Sequencing by synthesis
Analysis pipeline
Illumina GAII
DNA sequencing
Summary
Towards NGS sequencing
Data management
1
3
Target enrichment4
NGS with Illumina GAIIx2
Genotyping units
Sequencing unit
0.5 – 14 GB/Chip
1 – 6 Tb/FlowCell
High throughput data storage 3
1 Gbit LAN dedicated
Tape recording unit
for offline backup
Data storage
management server250 Tb storage
capacity
Sequencing pipeline
Genotyping applications
Database server
External services
Component Availability
CPU 40 core equivalent
RAM 1 Tb
Storage on-line (HD) 250 Tb
Storage off-line (Tape) 80 Tb normal160 Tb compressed
High throughput data analysis 3
Summary
Towards NGS sequencing
Data management
1
3
Target enrichment4
NGS with Illumina GAIIx2
High throughput sample preparation
Nature Methods, 2010, 7: 111-118
4
Locus lenght or #loci
SureSelect target enrichment
Agilent SureSelect
Solution-phase capture with
streptavidin-coated magnetic beads
Reported 60-80% of capture efficiency
4
