Next Generations of Sequencing Technologies...Next Generations of Sequencing Technologies Jeffery A....
Transcript of Next Generations of Sequencing Technologies...Next Generations of Sequencing Technologies Jeffery A....
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Next Generations of Sequencing Technologies
Jeffery A. Schloss, Ph.D.Program Director, Technology Development Coordination
National Human Genome Research InstituteNational Institutes of Health
U.S.A.
House of Lords Inquiry on Genomic MedicineVisit to NHGRI
June 4, 2008
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Progress in Genotyping Technology
1 10 102 103 104 105 106
Nb of SNPs
Cos
tper
gen
otyp
e(C
ents
, USD
)
10
1
102
ABITaqMan
ABISNPlex
IlluminaGolden Gate
IlluminaInfinium/Sentrix
Affymetrix100K/500K
Perlegen
AffymetrixMegAllele
2001 2005
Affymetrix10K
Courtesy S. Chanock, NCI
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…feasible to assay 375,000 or more single nucleotide polymorphisms (SNPs), capturing 80% or more of the HapMap-defined genomic variation, in roughly 2,000 subjects for roughly $1.7 million per study.
cost to genotype a human genome =$850
Genotyping vs. Sequencing Costs
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Fully Integrated System
The Applied Biosystems 3730xl™ DNA Analyzer
96-capillariesSimultaneous injection and analysis of 96 samplesAutomated plate loading from a stacker that accommodates up to 16 plates (96 or 384 well)Internal barcode readerBench top unit
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Human Genome Project Sequencing Centers
Slide credit: Eric Green, NHGRI
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Slide credit: Eric Green, NHGRI
Human Genome Project Sequencing Centers
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Coordinating Committee Process
March 15, 2006
• structural variation in 48 HapMap samples (fosmid end sequencing). The genomes of any two humans are thought to differ by several hundred insertions, deletions and inversions.
• add DNA sequence to existing draft sequences of a number of primate species and add additional sequence information in regions of high biological interest for rhesus macacque, marmoset and orangutan
• low-density draft (2-fold coverage) for 8 mammals
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$0.01
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t per
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Decrease in the Cost of Finished DNA Sequencing
10 years to reduce cost by 2 orders of magnitude
technology improvementsautomationeconomies of scale
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Nature, April 2003
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Nature, April 2003
Quantum Leaps in Technology:
.….Genome sequencing at $1000 or less for a
mammalian genome…..
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NHGRI DNA Sequencing Technology Development Requests for Applications
• current technologies are able to produce the sequence of a mammalian-sized genome of the desired data quality (high-quality draft) for $10 to $50 million; the goal of this initiative is to reduce costs by at least two/four orders of magnitude.
• RFA goal is sequencing technology that produces assembled sequence at high accuracy (10-4-10-5 error rate), de novo. Ultimate goal is higher accuracy.
• applications that propose technology development for re-sequencing should explain how they will achieve the projected reduction in cost compared to technologies that can produce data of similar quality today
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• current technologies are able to produce the sequence of a mammalian-sized genome of the desired data quality (high-quality draft) for $10 to $50 million; the goal of this initiative is to reduce costs by at least two/four orders of magnitude.
• initial awards were made in 2004.• goal: technologies for 100x reduction in cost by ~2009
• goal: technologies for 10,000x reduction in cost by ~2014
http://www.genome.gov/10000368#6 – “Advanced Sequencing Technology Awards”
NHGRI DNA Sequencing Technology Development Requests for Applications
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$99 MTOTAL56$16 MRound 453$13 MRound 346$31 MRound 220$39 MRound 1
Cumulative % “$1,000”
NHGRI DNA Sequencing Technology Development $100,000 and $1,000 genome
Investment
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Sanger,
micr
ochan
nel in
tegrated
syste
m (3)
Sange
r, atom
ic for
ce m
icros
cope s
epara
tion (
1)
Sequenc
ing by
cyclic
synth
esis
(10)
Single m
olecu
le se
quenc
ing by s
ynthes
is (4)
Sequen
cing b
y liga
tion (
4)
Seque
ncing
-by-hyb
ridiza
tion (
4)
Other n
ear-fie
ld se
nsing
unmod
ified D
NA (2)
Free-ru
nning p
olymeras
e/othe
r enz
yme (
3)
Nanopo
re with
modif
ied ana
lyte (
4)
Biolog
ical n
anop
ore (3
)
Synthe
tic na
nopore
/gap (8
)
Toward the $1,000 genome
NHGRI DNA Sequencing Technology Development $100,000 and $1,000 genome portfolio
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Sanger chemistry, miniaturized and integrated
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B. Paegal, R. Blazej & R. Mathies, 2003, Curr. Opin. Biotechnol. 14, 42
“A microfabricated device for high-throughput DNA sequencing that couples clone isolation, template amplification, Sanger extension, purification, and electrophoretic analysis in a single microfluidic circuit is now attainable.”.
U of California, BerkeleyRichard A. Mathies, Ph.D.
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RG Blazej, P Kumaresan & RA Mathies, 2006, PNAS USA 103:7240
Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing
Richard A. Mathies, Ph.D. U of Calif., Berkeley
Annelise Barron, Ph.D. Northwestern University
Microchip Biotechnologies, Inc.Stevan Jovanovich, Ph.D.
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RG Blazej, P Kumaresan & RA Mathies, 2006, PNAS USA 103:7240
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Integrated affinity capture, purification & inline injection capillary electrophoresis for attomole-scale Sanger DNA sequencing
Mathies lab:Blazej, et al., 2007, Anal. Chem., 79 (12), 4499 -4506Toriello, et al., 2007, Anal. Chem., ASAP Article 10.1021/ac0712547 S0003-2700(07)01254-1
Copyright © 2007 American Chemical Society
~150 000 captured fluorescent molecules
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Sequencing by cyclic synthesis
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Sequencing by cyclic synthesis (SBS)5’
G
T
C
A
G
T
C
A
G
T
C
A
GT
3’
5’
C
A
G
TC
A
T
C
A
C
C
TAG
CG
TA
First base incorporated
Cycle 1: Add sequencing reagents
Remove unincorporated bases
Detect signal
Cycle 2-n: Add sequencing reagents and repeat
Do this on a microarray with hundreds of thousands or millionsof different molecules on a surface!
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A CuraGen Corporation 03/30/04
Process OverviewProcess Overview
1) Prepare adapter ligated ssDNA library2) Capture DNA fragments on excess of capture beads
3) Clonal Amplification on 28 µ beads
5) Perform sequencing by synthesison the 454 instrument
4) Load beads and enzymes in PicoTiter Plate™
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A CuraGen Corporation 03/30/04
454 Technology 454 Technology -- Sequencing InstrumentSequencing Instrument
M Margulies, et al., 2005, Nature advance online publication 31 July
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Clonal Single Molecule Arrays™
~1000 molecules per ~ 1 um cluster ~1000 clusters per 100 um square~40 million clusters per experiment
Prepare DNA fragments
Ligate adapters Attach single molecules to surfaceAmplify to form clusters
Random array of clusters
100um
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100 microns20 microns
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SBS system using Reversible Photocleavable FluorescentNucleotide Terminators
Jingyue JuColumbia University
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Jingyue Ju, Columbia University
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J Schendure, et al. Sciencexpress 4 August 2005Science, 2005, 309:1728-1732
Harvard Medical SchoolGeorge M. Church
Single molecules generated from a cell-free, mate-paired library of E. coli genomic DNA, were amplified in parallel and attached to 1 micron beads, by emulsionpolymerase chain reaction. Millions of beads were immobilized and subjected to automated cycles of sequencing by ligation and four-color imaging. Off-the shelf reagents and an inexpensive epifluorescence microscope were used. Sequence accuracy is sufficient for re-sequencing applications.
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Braslavsky, et al., 2003, PNAS 100:3960-4
Stanford UniversitySteve Quake
Single molecules sequencing by synthesis.
Helicos BiosciencesTim Harris
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Roche Applied Science -- Genome Sequencer FLX System (454)
First commercial sequencer available 2005; FLX released 2007
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Illumina Genome Analyzer (Solexa)
First commercial sequencer available 2006
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Applied Biosystems SOLiD™ System 2.0 (Agencourt)
First commercial sequencer available 2007
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Helicos HeliScope™ Single Molecule Sequencer
First commercial sequencer available 2008
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Dover Systems The Polonator G.007
First commercial sequencer available 2008
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Free-running polymerase
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Pacific Biosciences
DNA Polymerase As a Sequence Reader
DNA polymerase
Primer
Template
Nucleotides
•Fast: 750 bps•Frugal•Faithful (1 in 105 )•Small
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Pacific Biosciences
Solution: Zero Mode Waveguide with Polymerase….
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Nanopore sequencing with electronic detection
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Single-stranded nucleic acid molecules passing through a nanometer-sized pore modulate the ionic conductance across the membrane. This observation may one day lead to a device for single molecule DNA sequencing.
U of California, Santa CruzDavid W. Deamer, Ph.DMark Akeson, Ph.D.Harvard UniversityDaniel Branton, Ph.D.Jene Golovchenko, Ph.D
Nature Biotechnology 2001 19:248-252
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Nature 2001 412:166-169
Solid state fabrication methods were developed to create a pore small enough for single-stranded DNA analysis. A beam of massive argon ions closes a pre-made hole. Size control is achieved by monitoring ion flux through the pore. The result is a “robust electronic detector consisting of a single nanopore in a Si3N4 membrane, capable of registering single DNA molecules in aqueous solution.”
Harvard UniversityJene Golovchenko, Ph.D Daniel Branton, Ph.D.
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Harvard University U of California, Santa CruzJene Golovchenko David W. DeamerDaniel Branton Mark Akeson
http://www.mcb.harvard.edu/branton/
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DNA Sequencing Through Nanopore Sensors
Recent progress:• further demonstrations of single-base
discrimination, though not yet sequencing• fabricating pores/sensors • understanding the physics of DNA transport
through pores of the same diameter as the molecule
• analyzing the potential to distinguish between the four bases as the molecule passes the sensor
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Identification of Deoxyribonucleoside 5'-Monophosphates by Using an Engineered Protein Nanopore Equipped with a Molecular Adapter
Yann Astier, Orit Braha, and Hagan Bayley 2006,J. Am. Chem. Soc.,128 (5), 1705 -1710 Copyright © 2006 American Chemical Society
University of OxfordJ Hagan P Bayley
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A Single-Molecule Nanopore Device Detects DNA Polymerase Activity with Single-Nucleotide Resolution
Cockroft, et al. 2008 J. Am. Chem. Soc. 130:818-820
Scripps Research InstituteM Reza Ghadiri
Copyright © 2008 American Chemical Society
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For the sensor configuration shown at left, with electrodes in the walls of a nanopore, modeling shows that the distributions of current values for each nucleotide will be sufficiently different to allow for rapid sequencing.
M. Di Ventra, 2006, Nano Lett. 6:779-782
University of North Carolina Chapel Hill J. Michael Ramsey
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Measurement (lower left) of electrical signal difference between “poly” and “Si” electrodes (shown above) as DNA molecule passes through a 7 nm pore through the sensor. Computer simulation (lower right) of helical polyC20 through the device produces “movie” frames in which DNA charge centers are seen moving through the sensor. Peaks in the simulated trace (lower right) show that electrical signals should be strong enough to detect individual bases. Narrower pores are needed to reduce noise.
J-P Leburton, 2006, Nanotechnology 17:622-633
University of Illinois UC Gregory Timp
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• bases tilt with ACX -field in pore• induced potential changes due to switching
polarity of the applied bias• predict: that nucleotides can be discriminated
υp
υs
υm
υp
υs
υm
Sequencing Using an ACSequencing Using an AC--field in a Porefield in a Pore(MD simulations of 1nm pore in MOS cap)
dC25 d(C15AC9)
• predict: sequence can be determined with single nucleotide precision by gradually increasing bias
G Timp & A AksimentievNano Letters 2008 8,56
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“…nanochannels would confine the DNA over its entire length rather than at a single point, and…one would expect to see a straight DNA molecule moving by without any kinks.”RH Austin (2003) Nature Materials 2, 567-568
W. Reisner, et al., 2005, Phys. Rev. Lett. 94, 196101
Princeton University Cornell UniversityEdward C. Cox Harold G. CraigheadRobert Austin North Carolina State University
Robert Riehn
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Throughput of DNA Sequencing Technologies
Baseline:
Human Genome “Reference” Sequence
$300 million~8 years
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Throughput of DNA Sequencing Technologies
Capillary Array Electrophoresis96 channels x 24 runs/day x 800 bp per run ≈ 1.8 Mb/day6x coverage of 3 Gb genome takes 26 years with 1 machine,~ 3 months with 100 machines
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Throughput of DNA Sequencing Technologies
Capillary Array Electrophoresis96 channels x 24 runs/day x 800 bp per run ≈ 1.8 Mb/day6x coverage of 3 Gb genome takes 26 years with 1 machine,~ 3 months with 100 machines
Sequencing by synthesis on array1 Gb/run, 2.5 days/run, 20x coverage of 6 Gb genome takes 1 yearthese are still early days for this collection of emerging
technologies e.g., 4-6x improvement over next year~2 months with one machine
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Throughput of DNA Sequencing Technologies
Capillary Array Electrophoresis96 channels x 24 runs/day x 800 bp per run ≈ 1.8 Mb/day6x coverage of 3 Gb genome takes 26 years with 1 machine,~ 3 months with 100 machines
Sequencing by synthesis on array1 Gb/run, 2.5 days/run, 20x coverage of 6 Gb genome takes 1 yearthese are still early days for this collection of emerging
technologies e.g., 4-6x improvement over next year~2 months with one machine
Nanosensor1 msec per base10x coverage of 6 Gb genome takes
~ 2 years with single nanopore; < 1 day with 1000 nanopore array
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• sequence variation (SNP, indel, and larger) – rare variants, not just the common ones
• haplotypes
• rearrangements
• expression analysis -- COUNTING
• allele-specific expression analysis
• alternative splicing
• microRNAs
• rare samples (e.g., in mixtures) – dynamic range
• genomes – re-sequencing, de novo?
• targeted regions (some)
• methylation status
Applications
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Next-next generation genome sequencing?
• Cost• MUCH longer reads
– Assembly, haplotypes, microbiomes…• Read all 5 bases• Read RNA (directly?), protein• Re-read same template – impact on data
quality• …• Medical care for individual patients
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Single-Cell Analysisto get at the ‘unculturables’
Requires ability to read a LOT of genomes!
Marcy et al., 2007, PNAS 104:11889
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