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NGS Technologies: 454

NGS Technologies: 454

Slides describing the principle behind 454 technology

Anton Nekrutenko

January 18, 2017
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  1. 454
    RIP: 2005 - 2013

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  2. The test of time
    454
    Illumina
    PacBio
    SOLiD
    Ion Torrent
    ONP
    100
    1,000
    10,000
    100,000
    1,000,000
    10,000,000
    1132
    46559
    23063
    30288
    3318279
    265436
    Number of SRA entries (Jan 2018)

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

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  4. Nucleotide chemistry

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  5. Polymerization

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  6. Ligation

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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. Feb 2014

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  23. 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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  24. Mate-pair with 454
    UCSC Sequencing Center

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  25. Flow value separation
    Balzer et al. 2010

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  26. Flow value separation
    Balzer et al. 2010

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  27. Errors in 454 data

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  28. Errors in 454 data
    ‣ incomplete homopolymer extension
    ‣ carry forward incomplete extension
    (CAFIE)

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  29. Base/Read qualities
    Huse et al. 2007

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  30. Length versus Error rate
    Huse et al. 2007

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