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454

2e0188eb9315b0ecb64537ccfe70eab8?s=47 Anton Nekrutenko
February 18, 2021

 454

An overview of 454 technology (2021)

2e0188eb9315b0ecb64537ccfe70eab8?s=128

Anton Nekrutenko

February 18, 2021
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Transcript

  1. 454

  2. The first NGS ‣ Pyrosequencing ‣ 454 Process ‣ Multiplexing

    ‣ Paired end sequencing
  3. Nucleotide chemistry

  4. Polymerization

  5. Ligation

  6. Nick translation

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

  8. Pål Nyrén

  9. The 4 enzymes of pyrosequencing

  10. Pyrosequencing process

  11. Output

  12. 454 Library preparation Emulsion PCR Deposition sequencing reaction

  13. None
  14. Jonathan Rothberg

  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
  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
  17. Deposition UCSC Sequencing Center Centrifuge Step Load Enzyme Beads 44

    µm Load beads into PicoTiter™Plate
  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
  19. The machine ‣ dNTP flow (dATPαS instead of dATP) ‣

    Substrate flow (D- luciferin, APS etc) ‣ Apyrase flow (destroys triphosphates) Margulies et al. 2005
  20. Output Margulies et al. 2005

  21. Margulies et al. 2005

  22. Margulies et al. 2005

  23. January 2013

  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
  25. Mate-pair with 454 UCSC Sequencing Center

  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