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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. 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)
  2. 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
  3. 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
  4. 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
  5. The machine ‣ dNTP flow (dATPαS instead of dATP) ‣

    Substrate flow (D- luciferin, APS etc) ‣ Apyrase flow (destroys triphosphates) Margulies et al. 2005
  6. 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