Introduction to DNA Sequencing
Technologies
Advanced Genetic Epidemiology and
Statistical Molecular Genetics Workshop
October 22, 2010
Gregory A. Buck, Ph.D.
Director, Center for the Study of Biological Complexity
Professor, Microbiology and Immunology
Virginia Commonwealth University
Holy Grail: the Human Genome
Complexity (number of bases per haploid
genome) of the human genome:
-
3x109 base pairs (nucleotides)
Human Genome
How much does it cost to sequence?
- First genome: $3-5 billion
- James Watson: ~$300,000
- Today: $5,000 - $100,000
- Goal: $1000 (soon < $100?)
Human Genome
How much time to sequence?
- First genome sequenced (2004):
. Estimated - 15 years (1990’s)
. Actual - 13 years (capillary sequencing)
- James Watson (2008): ~ 2 months
. So-called ‘next generation’ sequencing
- Now: two weeks?
- Goal: tricorder (Star Trek)
X Prize:
$10 million award is set for faster DNA maps
(2006)
By Nicholas Wade
Published: THURSDAY, OCTOBER 5, 2006
A $10 million prize for cheap and rapid sequencing of the human
genome was announced by the X Prize Foundation of Santa Monica,
California.
The terms of the prize require competitors to sequence 100 human
genomes of their choice within 10 days, and within six months, those
of a further 100 people chosen by the foundation.
http://www.iht.com/articles/2006/10/05/news/genome.php
NHGRI Grants Support for 'Revolutionary'
Sequencing for $1,000 Genome
August 5, 2008
By a GenomeWeb staff reporter
Under one program, NHGRI may grant as much as $5 million in fiscal 2009 to between two and
seven awardees. Applicants for these funds may seek up to $1.5 million per year for a period of up
to five years.
A parallel grant program would give up to $2 million over three years to between two and seven
grantees, for direct costs of up to $200,000 per year.
A Small Business Innovation Research Grant from NHGRI will grant between four and six small
businesses up to a total of $3.6 million in fiscal 2009 to propose novel technologies to bring down
the cost of sequencing. Phase I of this program will give up to $250,000 of total costs per year for
up to two years, and Phase II applicants may seek up to $1.5 million total costs per year for up to
three years.
A parallel Small Business Technology Transfer program will spend up to $2 million in fiscal 2009
to support between two and five awards to small businesses investigating the development of new
sequencing methods. This program will award up to $250,000 total costs per year for up to two
years for Phase I programs, and it will support up to $1.5 million in total costs per year for up to
three years for Phase II programs.
Sequencing Technologies
1977: Fred Sanger (Cambridge, England) and Walter Gilbert
(Harvard University)
– Chemical sequencing (Gilbert)
– Dideoxy Nucleotide Triphosphate chain termination sequencing
(Sanger)
– Both used for 8-10 years (different strengths/drawbacks)
Chain termination sequencing proves most versatile, robust
– Applicable to automation
– First automated sequencers commercially available ~1985
H
H
5'
O
CH 2
BASE
H
H
H
H
H
BASE
H
BASE
H
BASE
H
H
H
H
BASE
CH 2
O
H
H
BASE
H
H
H
H
BASE
CH 2
O
H
O
O P O
O
H
H
H
O
O P O
O
O
CH 2
H
CH 2
O
O P O
O
H
H
O
O P O
O
O
CH 2
3'
H
H
O
H
H
H
H
O
O P O
O
H
H
O
O P O
O
O
CH 2
H
CH 2
O
H
H
Sanger or di-deoxymethod of sequencing
H
BASE
3'
H
O
O P O
H
O
H
O
O P O
O
O
CH 2
H
O
H
BASE
BASE
H
H
OH
H
H
CH 2
O P O
O
H
O
H
H
H
BASE
P
P
O
CH 2
H
O
CH 2
O
O P O
O
H
H
H
OH
H
H
BASE
BASE
H
H
O
H
P
H
H
H
H
CH 2
O
O
O P O
H
O
H
P
P
P
O
CH 2
H
O
BASE
H
H
H
H
H
H
BASE
H
H
O
H
CH 2
O
O P O
O
5'
Sequencing Technologies
Commercially available (1985):
- Dideoxy- (Sanger, enzymatic, termination method)
-
Applied Biosystems, Inc., uses fluorescent primers
Requires four primers (four dyes) per sequence read
Requires four reactions (one for each primer)
Works, but expensive, laborious
- DuPont: Genesis 1000 DNA Sequencer
-
Fluorescent chain termination sequencing
One primer, four terminators (one for each base, A, G, C, T)
One reaction per sequence read
Very efficient
Sells IP to ABI……….
Fluorescent chain termination
sequencing
See video
High Throughput Genome
Sequencing: The main player...
The PE/ABI 3700 Prism:
- automated, easy to use
- capillaries (not slab gel)
- 10 runs per day
- 96 sequences per run
- ~1000 sequences/day
- >300,000 sequences/ year
- >150 million bases/ year
- $300,000 per machine
First truly automated high
throughput sequencing
Sequenced the first human
genome…..
Output from Fluorescent Chain Termination Sequencing
Fluorescent chain termination sequencing:
dominates market until ~ 2005:
Next Generation (NextGen) Sequencing
First out of the blocks:
Roche 454 FLX Genome Sequencer
Genome Sequencer FLX System
Customer Training Technical Overview
400 million bases/ day (5th floor, Sanger Hall)
(equal to 2 years output from cap sequencer!!)
www.roche-applied-science.com
Based on Pyrosequencing…..
Roche 454 Flx Technologies
- Based on Pyrosequencing –
- Pyrosequencing video
- Roche 454 FLX workflow video
Pyrosequencing – 454/Roche
http://454.com/products-solutions/how-it-works/sequencing-chemistry.asp
Roche 454 FLX Output:
- Based on Pyrosequencing –
- Currently:
-
~400 base maximum read length
~1 X 106 reads
~ 400 X 106 bases per run
1 run ~ 8 hours (1 day)
- [compare to 200 X 106 / year for capillary sequencing]
- Good for de novo sequencing, assembly
- Cost: $10,000 per run
Solexa/Illumina
Current Market Leader: Illumina Genome Analyzer
Reversible Terminator Chemistry
Solexa/Illumina
• All 4 labeled nucleotides in 1 reaction
O
O
cleavage
fluor
site
HN
O
N
HN
5’
DNA
O
N
O
O
O
PPP
3’
block
Incorporation
Detection
Deblock; fluor removal
3’
OH free 3’ end
Next cycle
X
Solexa/Illumina
3’ 5’
Cycle 1:
Add sequencing reagents
First base incorporated
Remove unincorporated bases
A
T
G
C
C
G
T
Cycle 2-n: Add sequencing reagents and repeat
T
A
C
A
Detect signal
C
G
A
T
T
A
G
A
•
C
T
C
C
G
A
G
C
T
C
G
A
T
5’
•
•
•
All four labelled nucleotides in
one reaction
High accuracy
Base-by-base sequencing
No problems with homopolymer
repeats
Base Calling
Solexa/Illumina
T G C TAC GAT …
1
2
3
4
5
6
7
8
TTTTTTTGT…
The identity of each base of a cluster is read off
from sequential images
(sequencing genomes with the Illumina video)
9
Illumina/Solexa Technologies
- Based on Sequencing by Synthesis (bridge PCR) –
- Currently:
- ~100 base maximum read length
- ~ 500 X 106 reads/run
- ~ 50 X 109 bases per run (100 X 109 in paired end reads)
- 1 run ~ 10 days
- Good for re-sequencing, CHiP Seq, RNA seq
- Cost: $10 – 20,000 per run
New Illumina HiSeq2000: 200 X 109 bases/run
- ~ 10 X 1012 bases/year
- 100,000 fold increase over fluor. chain termination seq
- >3,000 human genomes!
Applied Biosystems: Solid 4/HQ
Sequencing by Ligation…
See video…..
http://marketing.appliedbiosystems.com/images/Product_Microsites/Solid_Knowledge_MS/video/SOLiD_video_final.wmv
SOLiD Sequencing Technology
Currently:
-
50 base reads (75?)
Up to 400 Giga bases (billion bases) per run
>20 X 1012 bases per year (~2X Illumina)
Reduced costs (<50%/base cost)
Best for applications where short reads are sufficient:
CHiP seq, RNA Seq…. (not de novo sequencing)
Single Molecule Technologies
Holy Grail:
- No bias (due to replication, amplification)
- Should work with limiting amounts of template
- Long reads: for de novo sequencing
Contenders:
- true Single-Molecule Sequencing (tSMS) – Helicos
- SMRT (Single Molecule, Real Time Sequencing) –
Pacific BioSciences
see videos
Single Molecule Technologies
Advantages:
- No amplification, cloning biases
- Use small quantities of substrate (DNA)
- Fast (rate of replication)
Challenges
- Signal to noise ratios
- Sensitivity
- Error rates
To date: still largely experimental:
- Short reads (Helioscope)
- Low output; e.g., < 100,000 reads/run (Pac Bio)
Other Technologies
Looming:
- Ion Torrent: based on release of H+ ions
- requires emulsion PCR
- Inherent biases
- Current read length < 100 bp; high error rate
- Oxford Nanopore Technologies: passage of bases through
a nanopore in a lipid bilayer
- No data available
- Others coming
http://www.iontorrent.com/technology/
http://www.iontorrent.com/technology/
Nanopore
http://www.nanoporetech.com/sections/first/14
Wish list:
- Longer reads
- Today: 25 - 800 bases
- Looming: 1 – 20 Kbases?
- Ideal: entire chromosome [metagenomics]
- Low amounts DNA required
- No amplification bias
- No replication bias
- Can sequence hard to get DNA
- High accuracy and fidelity
- Rapid (currently over a week per run)
- Lower cost ($100/human genome?)
Thank you!