Recent Technological Advances in
Next-Generation Sequencing
Kirby Siemering PhD
Australian Genome Research Facility
RCPA
June, 2011
Whole Genome Sequencing (WGS)
Where have we been?
1953 – Structure of DNA solved
1977 – Sanger sequencing invented
– First genome sequenced – ФX174 (5 kb)
1986 – First automated sequencing machine
1990 – Human Genome Project started
1992 – First “sequencing factory” at TIGR
Whole Genome Sequencing (WGS)
Where have we been?
1995 – First bacterial genome – H. influenzae (1.8 Mb)
1998 – First animal genome – C. elegans (97 Mb)
2003 – Completion of Human Genome Project (3 Gb)
– 13 years
– $2.7 bn
2005 – First “next-generation” sequencing instrument
2008 – 2356 genome sequences in NCBI database
Whole Genome Sequencing (WGS)
Where have we been?
Where are we now?
“Next Generation” Sequencing
• Massively parallel DNA sequencing (not Sanger)
• Up to 600 Gb sequence per run (1-2 weeks)
= 6 human genomes at 30X coverage!!
• $10-30K per run reagent cost
• Limitations in read length and accuracy
• 3 systems dominate market
- Roche GS FLX (454)
- Illumina HiSeq 2000 (Solexa)
- AB SOLiD (Agencourt)
Roche GS FLX sequencing technology
gDNA
fragment or PCR
adapter ligation
emulsion PCR
sequence generation
Illumina sequencing technology
Robust Reversible Terminator Chemistry Foundation
3’ 5’
DNA
(0.1-1.0 ug)
A
G
C
T
G
C
T
A
C
G
A
T
A
C
C
C
G
A
T
C
G
A
T
A
T
C
G
A
T
G
C
T
Sample
preparation
Cluster growth
5’
Sequencing
1
2
3
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6
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T G C T A C G A T …
Image acquisition
Base calling
Rapid Innovation Driving Cost Down
Evolution of NGS system output
Throughput
(GB)
300
300GB
120
100
80
60
40
20
20GB
3GB
6GB
2007
2008
0
2009
2010
Cost per Human Genome
Where are we now?
Where are we going?
The end of the current revolution?
Where are we going?
Archon X PRIZE
Where are we going?
Archon X PRIZE
100 human genomes at 99.999% accuracy in less
than 10 days at a cost of <$10,000 per genome
$10 million
=> Ultimate holy grail of “$1,000 genome”
How to get there?
Non-optical detection – Ion Torrent
How to get there?
Single Molecule Sequencing – Pacific Biosciences
How to get there?
Non-optical SMS – Oxford Nanopore
So you can sequence human genomes
But what does it all mean?
NGS – feeding the discovery pipeline
Discovery
Validation
Application
MDx
Where might this technology take us?
Information convergance
Where might this technology take us?
GATTACA – food for thought…