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Next Generation Sequencing revolution (February 2010 - PhD retreat)

F4ae403ab821b6c55bed3a7efd4c6aa1?s=47 Andrea Telatin
November 03, 2010

Next Generation Sequencing revolution (February 2010 - PhD retreat)

Introduction to the sequencing revolution referred to as "Next Generation Sequencing", namely describing 454 and SOLiD.

F4ae403ab821b6c55bed3a7efd4c6aa1?s=128

Andrea Telatin

November 03, 2010
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  1. University of Padua CRIBI Biotechnology Center Andrea Telatin (PhD candidate)

    Genomics Group — Giorgio Valle The DNA sequencing revolution …and the magic behind it San Martino di Castrozza - February 2010
  2. DNA SEQUENCING Why is Sanger no longer suitable? 1977 –

    Sanger method 1980 – PCR 1986 – Applied release first sequencer 1990 – HGP begins (estimated 13 billion $) 1996 – Yeast genome + pyrosequencing 2006 – 1000$ genome (Science)
  3. DNA SEQUENCING Why is Sanger no longer suitable? 1977 –

    Sanger method 1980 – PCR 1986 – Applied’s first fluorescent sequencer 1990 – HGP begins (estimated 13 billion $) 1996 – Yeast genome + pyrosequencing 2006 – 1000$ genome race
  4. SANGER METHOD: LIMITS • Clonal amplification • Sequencing reaction •

    Gel electrophoresis DNA polymerase and fluorescent di-deoxy NTPs Fragment genome, then clone fragments
  5. NEXT GEN SEQUENCERS Roche 454 (2005) Illumina GA (2007) AB

    SOLiD (2008) Higher throughput, shorter reads How did they do?
  6. NEXT GEN SEQUENCERS • Roche 454 • Cloning à Emulsion

    PCR • Pyrosequencing reaction • Gel à Real time imaging • AB SOLiD • Cloning à Emulsion PCR • Sequencing by ligation • Gel à Real time imaging • Illumina G.A. • Cloning à Bridge amplification • Reversible terminators (~Sanger) • Gel à Real time imaging Single read is ~500 bp 0.5 Gb per run (10 hours) Longest reads, expensive Single read is 50bp 60 Gb per run (7 days) Best dollars/Gb ratio Single read is max 100bp About 15 Gb per run (10d) Best compromise
  7. 454: LIBRARY PREPARATION • Shear DNA • Attach adaptors A-B

    • Discard A-A and B-B molecules with avidin coated beads and some magic • Emulsion PCR • Mix PrimerA-coated beads, single strand template, PCR mix and oil • Adding proper ratio of DNA/beads maximize the case of droplets with 1 DNA molecule inside. • Million different templates in a single tube A single step instead of cloning into bacteria!
  8. 454: LIBRARY PREPARATION • Shear DNA • Attach adaptors A-B

    • Discard A-A and B-B molecules with avidin coated beads and some magic • Emulsion PCR • Mix PrimerA-coated beads, single strand template, PCR mix and oil • Adding proper ratio of DNA/beads maximize the case of droplets with 1 DNA molecule inside. • Million different templates in a single tube A single step instead of cloning into bacteria!
  9. 454: PYROSEQUENCING • Bead deposition • 1 bead fits 1

    well • Pyrosequencing • Polymerase extends the primer but 1 base at time is added • PPi release is coupled with flash light • An issue with long homopolymers • Hi resolution CCD camera records images • Computer then rebuild sequences
  10. SOLID: CHEMISTRY • Library preparation similar with 454 • Bead

    deposition on a glass slide… • …then sequence by ligation!!! 16 set of probes, 4 colours Primer “n”
  11. SOLID: CHEMISTRY • Library preparation similar with 454 • Bead

    deposition on a glass slide… • …then sequence by ligation!!! 16 set of probes, 4 colours Primer “n”
  12. SOLID: COLOR SPACE • We don’t detect bases, but degenerated

    dinucleotides… • …but each base is called twice • SNP ≠ sequencing error • Determine sequence? • Here how it looks like: Do you like to think in a colourful way?
  13. SOLID: COLOR SPACE • We don’t detect bases, but degenerated

    dinucleotides… • …but each base is called twice • SNP ≠ sequencing error • Determine sequence? • Here how it looks like:
  14. MATE PAIRS LIBRARIES Repeated region Repeated region

  15. MATE PAIRS LIBRARIES Repeated region

  16. MOST COMMON APPLICATIONS • Roche 454 is suitable for •

    Genome de novo sequencing / resequencing • Amplicon analysis • cDNA sequencing • SOLiD better for • Gene expression (RNA-Seq) • Chromatin immunoprecitation (ChIP-Seq) • Genome Resequencing • SNP detection (?) Single cell transcriptom e! Metagenomics, epigenetics… Virus evolution (amplicons)
  17. FUTURE PERSPECTIVES • Next-next generation • Completely eliminate amplification à

    Single molecule seq. • Either longer reads (Visigen claims to reach 10kbp!) or higher througput (SOLiD will do 100Gbp!) • Could you sequence DNA with a pore? • Sanger’s revenge • Microfluidic based Sanger method. Feasible? What whould you do with a 1000$ human genome?
  18. THANK YOU for your kind attention

  19. Sequencing by ligation (1)

  20. Sequencing by ligation (2)

  21. SOLiD throughput

  22. SNP in color space