454

An overview of 454 technology (2021)

Transcript

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
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