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Pre-genomic era: finding your own clones

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Imagine initial cDNA or PCR fragment probe hybridizes to clones 3, 9, 16, 22

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GENOME

CHROMOSOMES

YACsor

BACs (map)

PlasmidSub-clones

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Gridded (arrayed/ordered) library

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18,000 BACs for Drosophila

300,000 BACs for Humans

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STS- Sequence Tagged Site Mapping

STS Probe orPCR product

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Minimal Tiling Path

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Sequence >700ntfrom each end

Plasmids

Sub-clone into smaller segments and mapOr use primer walking- NOT EFFICIENT

Instead use shotgun sequencing

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Virtual DNA(sequence)Assembly

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2 or 3 libraries ofDifferent size fragments

ShotgunSequencing

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Mates: read-pairs

2kb library of clones

10kb library of clones

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* * **

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123 23

GENOME

CHROMOSOMES

YACsor

BACs (map)

PlasmidSub-clonesDon’t map Sequence ends

Assemble sequence of BAC

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Mates: read-pairs

2kb library of clones

10kb library of clones

150kb library of clones

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C. Elegans 100Mb

Drosophila 120Mb

Human 3, 200Mb

Cosmids, YACs ordered

BACs ordered- STS mapping+ fingerprinting

BACs ordered- fingerprinting

Whole Genome shotgun

Whole Genome shotgun

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1.454 sequencing

Amplify singleDNA molecules on single beads

Sequence eachDNA/bead bystepwise Incorporation ofA, G,C or T in mini-wells

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Aqueous microsphere

bead

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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-attachedTemplates from one original template molecule

Anneal primer for sequencing and loadDNA polymerase and SSB after enrichingFor template-loaded beads

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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 = beads, amplification, emulsion, magnetics = cloning DNA molecules via PCR on beads

No template

No bead

No template or bead

Had one template

Had another template

Aqueous microspheres

Remove oil

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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

30APS = adenosine phosphosulfate

Destroy old nucleoside triphosphate substrate before adding new one

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33Red, green, blue, pink

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2005

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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)

2. Solexa/Illumina sequencing

Intelligent Bio-Systems (Jue, Turro… Columbia)

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Solexa-Illumina

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3. Applied Biosystems SOLiD sequencing

Shendure, Church et al.

Polony (polymerase colony) by emulsion PCRor similar on beads (BEAMing)Attach beads to glass slide for sequencing

Sequence by ligation!

Webinar:http://appliedbiosystems.cnpg.com/lsca/webinar/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.

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ATTACGGC

AACCGGTT

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5 primer roundsIn total

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