454 sequencing

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1. 454 sequencing
Amplify single DNA molecules on single beads
Sequence each DNA/bead by stepwise Incorporation
of A, G,C or T in mini-wells
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bead
Aqueous microsphere
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BEAMing beads, amplification, emulsion,
magnetics cloning DNA molecules via PCR on
beads (Dressman et al., Vogelstein lab. PNAS
2003.)
No template and no bead
Aqueous microspheres
Had one template
Had another template
No template
No bead
Remove oil
E.g., hybridization to a complementray probe
Or other ways to read out (e.g., CCD camera,
laser scanner)
 Dressman, D., Yan, H., Traverso, G., Kinzler,
K.W., and Vogelstein, B. 2003. Transforming
single DNA molecules into fluorescent magnetic
particles for detection and enumeration of
genetic variations. Proc Natl Acad Sci U S A 100
8817-8822.
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Attached oligomers were pre-labeld red or green,
then mixed and emulsified. See single beads in
aqueous microspheres in oil.
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BEAMing PCR on beads compartmentalized in a
water-oil emulsion.
Millions of primers attached to each
bead, Producing millions of copies of
bead-attached Templates from one original
template molecule
Anneal primer for sequencing and load DNA
polymerase and SSB after enriching for
template-loaded beads
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Big beads- Template, primer, DNA
polymerase
Small beads- ATP sulfurylase, Luciferase
Solution- One dNTP Luciferin, APS
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Pyrosequencing
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Destroy old nucleoside triphosphate substrate
before adding new one
APS adenosine phosphosulfate
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Red, green, blue, pink
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Advantage relatively long read lengths
(400-500) (Sanger sequencing 800)
Problem long homopolymer runs (gt8?)
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2. Solexa/Illumina sequencing Intelligent
Bio-Systems (Jue, Turro Columbia)
Amplification in situ on glass surface of flow
cell (PCR that keeps different DNAs separate-
micro-cloning Sequencing with reversible
fluorescent terminator dNTPs (one nucleotide at a
time)
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Solexa-Illumina
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Jingyue Ju lab, Columbia U.
Sequencing by synthesis
http//genome4.cpmc.columbia.edu/researcher/ju.htm
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http//genome4.cpmc.columbia.edu/researcher/ju.htm
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Jingyue Ju, Columbia U.
Sequencing by synthesis
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Record image
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3. Applied Biosystems SOLiD sequencing Shendure,
Church et al.
Webinar http//appliedbiosystems.cnpg.com/lsca/we
binar/rhodes/chemistry/20070618/ Shendure, J.,
Porreca, G.J., Reppas, N.B., Lin, X., McCutcheon,
J.P., Rosenbaum, A.M., Wang, M.D., Zhang, K.,
Mitra, R.D., and Church, G.M. 2005. Accurate
multiplex polony sequencing of an evolved
bacterial genome. Science 309 1728-1732.
Polony (polymerase colony) by emulsion PCR or
similar on beads (BEAMing) Attach beads to glass
slide for sequencing Sequence by ligation!
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8-mers, z universal hybridizers n degenerate
(all 64 possibilites) All with TT labeled blue,
etc.
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Ligase
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5
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8 cycles cover 35 nt)
AT TA CG GC
AA CC GG TT
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5 primer rounds in total
Know n-1 base is, say, A because it is part of
the primer Then overlapping colors define a
unique sequence
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http//www.helicosbio.com/Technology/TrueSingleMol
eculeSequencing/tabid/64/Default.aspx
Like Illumina, but immobilized templates are SS
DNA moelcule (200 nt) Each cycle adds one
base,records, and then cleaves the fluorescent
group and washes it away. Several billion
single molecule spots per slide.
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Helicos virtual terminator Inhibits DNA Pol once
incorporated Cleavable via the S-S bond (reduce
it)
Free 3 OH never blocked
dU-3P,5P
dUTP
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Quanti?cation of the yeast transcriptome by
single-moleculesequencing Lipson et al. VOL.27
NUMBER7 JULY2009 NATURE BIOTECHNOLOGY
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http//www.pacificbiosciences.com
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