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454

Anton Nekrutenko
February 18, 2021

 454

An overview of 454 technology (2021)

Anton Nekrutenko

February 18, 2021
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  1. The first NGS
    ‣ Pyrosequencing
    ‣ 454 Process
    ‣ Multiplexing
    ‣ Paired end sequencing

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  2. Nucleotide chemistry

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  3. Polymerization

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  4. Nick translation

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

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  6. The 4 enzymes of
    pyrosequencing

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  7. Pyrosequencing process

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  8. 454
    Library preparation
    Emulsion
    PCR
    Deposition
    sequencing reaction

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  9. Jonathan Rothberg

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  10. 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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  11. 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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  12. Deposition
    UCSC Sequencing Center
    Centrifuge Step
    Load Enzyme
    Beads
    44 µm
    Load beads into
    PicoTiter™Plate

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  13. 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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  14. 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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  15. Output
    Margulies et al. 2005

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  16. Margulies et al. 2005

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  17. Margulies et al. 2005

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  18. January 2013

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  19. 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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  20. Mate-pair with 454
    UCSC Sequencing Center

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  21. 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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