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Lesson: Sequencing by synthesis

Understand the Central Dogma, DNA sequencing methods, Next-Generation Sequencing technology, and sequencing by synthesis process. Explore the revolutionary applications and advancements in genomics. Engage in a hands-on sequencing modeling activity to grasp the concept intricacies.

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Lesson: Sequencing by synthesis

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  1. Lesson: Sequencing by synthesis Goals: Review central dogma and limits of DNA Understand history and recent advances in sequencing Understand the process (sequencing by synthesis) used to generate data in this module

  2. What is DNA? 10010110 to (coding) as LIFE 5’GATTACA 3’ to (DNA)

  3. The Central Dogma DNA goes to protein Responsible for most of the structure and function of an organism 5’GATTACA 3’

  4. Different ways to represent DNA 3D structure 2D chemical structure Sequence 5’-ATTAGCTAGAC-3’ DNA sequencing simply means reading the sequence of the DNA

  5. Very small yet very big DNA is tiny Genomic information is massive 3 BILLION letters in the human genome • Each letter in the DNA sequence is less than one nanometer LIFE

  6. So what? What can we do with DNA sequencing? Are the following scenarios “Sci-Fi” or “Reality”?

  7. Identify whether someone is more or less likely to commit a crime Sci-fi.

  8. Figure out the identity of ancient human remains Reality.

  9. Use preserved DNA to re-create extinct plants and animals Sci-fi.

  10. Use human DNA to create a clone with the same personality Sci-fi.

  11. Track disease by monitoring toilet waste from airplanes Reality.

  12. Next-Generation Sequencing makes these advances possible 1990-2003: Human Genome Project “Sanger sequencing” technology Today “Next-generation sequencing” technology 1 human genome in 13 years ~40 sequencing institutions $3,000,000,000 per genome 16 human genomes in 3 days 1 sequencing system $1,000 per genome

  13. DNA Sequencing by Synthesis Bioinformatics Inquiry through Sequencing

  14. DNA synthesis ATGAGCTTAGCTA • Template strand • DNA polymerase • Primer • Nucleotides TACTCG T T C A T A C G G

  15. DNA synthesis ATGAGCTTAGCTA T T C A TACTCG T A C G G

  16. DNA synthesis ATGAGCTTAGCTA TACTCGAATCGAT

  17. Sequencing By Synthesis Sequence DNA by observing the synthesis of a complimentary strand MiSeqsequencer

  18. DNA is attached to the surface of a flow cell • DNA is fixed in place while various chemicals wash over it • The camera takes pictures of DNA synthesis while it happens OUTLET INLET

  19. Make identical copies • Because nucleotides are so small, they are difficult to see, even when attached to fluorescent dyes • The sequencer copies the sample sequence to form a large group of identical sequences. • This group is called a cluster • The fluorescent signal from a cluster is much greater than the fluorescent signal from a single strand

  20. DNA is fixed to the surface of a flow cell INLET OUTLET

  21. Fluorescent Dye G A T C • When excited by a laser, fluorescent dyes emit brightly colored light • Each nucleotide is attached to a unique fluorescent dye • Blue  A • Red  T • Green G • Yellow C

  22. Sequencing By Synthesis K. Voelkerding, et al, Clinical Chemistry, 2009

  23. Fluorescent Dye • Laser excites fluorophore.Camera captures color • Each color indicates a specific base (support.Illumina.com)

  24. Tracking colors • The sequencer uses a camera to identify the color of each nucleotide as it gets added • Write the first letter of the color you see: • Red • Blue • Yellow or • Green DNA synthesis happens fast and uncontrollable

  25. Blocking group controls speed 3’-ATGC-5’ 5’-TA x ?? C Deblocker

  26. Speed can now be controlled Without blocking groups With blocking groups

  27. Sequencing by Synthesis

  28. Primer and polymerase attach

  29. 1. Add nucleotide

  30. 2. Image fluorescence

  31. 3. Remove dye

  32. Sequencing by synthesis

  33. Sequencing by synthesis

  34. Sequencing by synthesis

  35. Sequencing by synthesis

  36. Sequencing by synthesis

  37. Sequencing by synthesis

  38. Complete sequence

  39. What the camera sees: Image 1 Image 2 Image 3 Image 4 Image 5 G G A C T • Blue  A • Red  T • Green  G • Yellow C

  40. What the camera sees: Image 1 Image 2 Image 3 Image 4 Image 5 G T C G A • Blue  A • Red  T • Green  G • Yellow C

  41. Modeling Activity • Everyone gets a role: • DNA Polymerase • Primer • Sequence complementary of primer • Laser • Camera • Nucleotides of the original sequence • Deblocker • Nucleotides

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