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NGS Technologies: 454

NGS Technologies: 454

Slides describing the principle behind 454 technology

2e0188eb9315b0ecb64537ccfe70eab8?s=128

Anton Nekrutenko

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

  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)
  3. The first NGS ‣ Pyrosequencing ‣ 454 Process ‣ Multiplexing

    ‣ Paired end sequencing
  4. Nucleotide chemistry

  5. Polymerization

  6. Ligation

  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. Feb 2014

  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
  24. Mate-pair with 454 UCSC Sequencing Center

  25. Flow value separation Balzer et al. 2010

  26. Flow value separation Balzer et al. 2010

  27. Errors in 454 data

  28. Errors in 454 data ‣ incomplete homopolymer extension ‣ carry

    forward incomplete extension (CAFIE)
  29. Base/Read qualities Huse et al. 2007

  30. Length versus Error rate Huse et al. 2007

  31. None
  32. None