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.
Gregory A. Buck, Ph.D.
Director, Center for the Study of Biological Complexity
Professor, Microbiology and Immunology
Virginia Commonwealth University
Complexity (number of bases per haploid genome) of the human genome:
- 3x109 base pairs (nucleotides)
How much does it cost to sequence?
- James Watson: ~$300,000
- Today: $5,000 - $100,000
- Goal: $1000 (soon < $100?)
How much time to sequence?
- First genome sequenced (2004):
. Estimated - 15 years (1990’s)
. Actual - 13 years (capillary sequencing)
. So-called ‘next generation’ sequencing
$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.
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.
1977: Fred Sanger (Cambridge, England) and Walter Gilbert (Harvard University)
Chain termination sequencing proves most versatile, robust
Sanger or di-deoxy- method of sequencing $1,000 Genome
Commercially available (1985):
Fluorescent chain termination sequencing $1,000 Genome
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 $1,000 Genome
Fluorescent chain termination sequencing: $1,000 Genome
dominates market until ~ 2005:
Next Generation (NextGen) Sequencing
First out of the blocks:
Roche 454 FLX Genome Sequencer
www.roche-applied-science.com $1,000 Genome
Genome Sequencer FLX System Customer Training Technical Overview 400 million bases/ day (5th floor, Sanger Hall)
(equal to 2 years output from cap sequencer!!)
Based on Pyrosequencing….. $1,000 Genome
Pyrosequencing – 454/Roche $1,000 Genome
Solexa/Illumina $1,000 Genome
Current Market Leader: Illumina Genome Analyzer
Reversible Terminator Chemistry $1,000 Genome
Deblock; fluor removal
free 3’ end
3’ $1,000 Genome
Cycle 1: Add sequencing reagents
First base incorporated
Remove unincorporated bases
Cycle 2-n: Add sequencing reagents and repeat
5 $1,000 Genome
T T T T T T TGT …
The identity of each base of a cluster is read off from sequential images
(sequencing genomes with the Illumina video)
New Illumina HiSeq2000: 200 X 109bases/run
- ~ 10 X 1012bases/year
- 100,000 fold increase over fluor. chain termination seq
- >3,000 human genomes!
See video….. $1,000 Genome
Best for applications where short reads are sufficient:
CHiP seq, RNA Seq…. (not de novo sequencing)
To date: still largely experimental:
Nanopore $1,000 Genome