454

An overview of 454 technology (2021)

Transcript

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2. The first NGS
   ‣ Pyrosequencing
   ‣ 454 Process
   ‣ Multiplexing
   ‣ Paired end sequencing
   

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3. Nucleotide chemistry
   

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4. Polymerization
   

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5. Ligation
   

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6. Nick translation
   

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7. Three ATP-like derivatives
   ATP
   dATPαS
   A 5' phosphosulfate
   (APS)
   

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8. Pål Nyrén
   

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9. The 4 enzymes of
   pyrosequencing
   

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10. Pyrosequencing process
    

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11. Output
    

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12. 454
    Library preparation
    Emulsion
    PCR
    Deposition
    sequencing reaction
    

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14. Jonathan Rothberg
    

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15. Library preparation
    dsDNA
    fragments
    P
    A B
    Ligatio
    n
    Fill
    in
    Capture on SA-Beads &
    Wash
    Alkaline Elution
    A B
    P Bio
    Bio
    Bio
    Bio
    Bio
    Primer A Key Library fragment Primer B
    #bases: 40 4
    Standard
    Library
    Seq. primer Read
    

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16. Emulsion PCR
    UCSC Sequencing Center
    Mix DNA Library
    & capture beads
    (limited dilution)
    “Break micro-reactors”
    Isolate DNA containing beads
    •  Generation of millions of clonally amplified sequencing templates on each bead
    •  No cloning and colony picking
    Create
    “Water-in-oil”
    emulsion
    + PCR Reagents
    + Emulsion Oil
    Perform emulsion PCR
    Adapter carrying
    library DNA
    A
    B
    Micro-reactors
    

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17. Deposition
    UCSC Sequencing Center
    Centrifuge Step
    Load Enzyme
    Beads
    44 µm
    Load beads into
    PicoTiter™Plate
    

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18. Sequencing
    UCSC Sequencing Center
    DNA Capture Bead
    Containing Millions of
    Copies of a Single
    Clonal Fragment
    A A T C G G C A T G C T A A A A G T C A
    Anneal Primer
      Simultaneous sequencing of the entire genome in hundreds of
    thousands of picoliter-size wells
      Pyrophosphate signal generation
    T
    PP
    i
    ATP
    Light + oxy luciferin
    Sulfurylase
    Luciferase
    APS
    luciferin
    

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19. The machine
    ‣ dNTP flow (dATPαS
    instead of dATP)
    ‣ Substrate flow (D-
    luciferin, APS etc)
    ‣ Apyrase flow
    (destroys
    triphosphates)
    Margulies et al. 2005
    

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20. Output
    Margulies et al. 2005
    

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21. Margulies et al. 2005
    

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22. Margulies et al. 2005
    

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23. January 2013
    

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24. Multiplexing
    UCSC Sequencing Center
    Primer A MID 1
    Key Library fragment Primer B
    Seq. primer Read
    #bases: 15 4 10
    Primer A Key Library fragment Primer B
    #bases: 40 4
    MID
    Library
    Standard
    Library
    Seq. primer Read
    Primer A MID 2
    Key Library fragment Primer B
    Primer A MID n
    Key Library fragment Primer B
    

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25. Mate-pair with 454
    UCSC Sequencing Center
    

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26. Target enrichment
    CSB2008 August 2008 UCSC Sequencing Center
    gDNA
    Exon 1 Exon 2 Exon 3 Exon 4 Exon 5
    Fragment and hybridize to
    NimbleGen capture array
    HT-Sequencing
    Analyze
    Exon
    Sequences
    Elute
    

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