1 / 92

DNA sequencing “the technology lecture”

DNA sequencing “the technology lecture”. Part 1: Chemistry, instrumentation and data analysis Part 2: Large-scale operations, comparative sequencing. Part 3: Sequencing analysis, variation analysis. Abbrev 02/03/05, rev 10/17/05. Technology: Importance. Approaches to research

oneida
Download Presentation

DNA sequencing “the technology lecture”

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. DNA sequencing“the technology lecture” • Part 1: Chemistry, instrumentation and data analysis • Part 2: Large-scale operations, comparative sequencing. • Part 3: Sequencing analysis, variation analysis. • Abbrev 02/03/05, rev 10/17/05

  2. Technology: Importance • Approaches to research • hypothesis driven • discovery driven • technology development • Technology drives research drives technology • paradigm changing • punctuated versus gradual • Vision • personalized medicine

  3. DNA sequencing: Importance • Basic blueprint for life; Aesthetics. • Gene and protein • Function • Structure • Evolution • Genome-based diseases- “inborn errors of metabolism” • Genetic disorders • Genetic predispositions to infection • Diagnostics • Therapies

  4. Maxam-Gilbert base modification by general and specific chemicals. depurination or depyrimidination. single-strand excision. not amenable to automation Sanger DNA replication. substitution of substrate with chain-terminator chemical. more efficient automation?? DNA sequencing methodologies: ca. 1977!

  5. Maxam-Gilbert ‘chemical’ method

  6. Sanger dideoxynucleotides versus “bio” based methods

  7. DNA chemistry

  8. DNA biochemistry: replication fork

  9. O O O O O O P P P OH OH OH DNA replication: biochemistry 5’ purine or pyrimidine N HO C O purine or pyrimidine O N C O O O P OH 3’ OH

  10. O O O O O O P P P OH OH OH now, DNA “sequencing:” Sanger dideoxy method I purine or pyrimidine N HO C O dideoxyribonucleoside triphosphate (ddNTP) H

  11. O O O O O O P P P OH OH OH DNA sequencing: Sanger II purine or pyrimidine N HO C O purine or pyrimidine O chain termination method N C O O O P OH H

  12. DNA sequencing: chemistry * * * * * * * * * * * * * *

  13. DNA sequencing: in practice template + polymerase + 1 dCTP dTTP dGTP dATP ddATP primer 2 dCTP dTTP dGTP dATP ddGTP primer 3 dCTP dTTP dGTP dATP ddTTP primer 4 dCTP dTTP dGTP dATP ddCTP primer electrophoresis A•T G•C A•T T•A C•G T•A G•C G•C A•T G•C T•A T•A C•G T•A G•C A•T extension

  14. Manual radioactive sequencing

  15. Semi-automated fluorescent DNA sequencing • Fred Sanger et. al., 1977. • Walter Gilbert et. al., 1977. • Leroy Hood et. al. 1986. • Applied Biosystems, Inc. • DuPont Company

  16. DNA sequencing: upgrade, second iteration, terminator-label • Disadvantages of primer-labels: • four reactions • tedious • limited to certain regions, custom oligos or • limited to cloned inserts behind ‘universal’ priming sites. • Advantages: • Solution: • fluorescent dye terminators

  17. DNA sequencing: chemistry template + polymerase + dCTP dTTP dGTP dATP ddATP ddGTP ddTTP ddCTP electrophoresis A•T G•C A•T T•A C•G T•A G•C G•C A•T G•C T•A T•A C•G T•A G•C A•T extension

  18. DNA sequencing: photochemistry

  19. ABI series: 370, 373 and 377 • semi-automated • “best” pre- and post- • higher throughput operations. • bioinformatics limitations, ‘scuze me- “opportunities.’”

  20. ABI 370s-series screen dump

  21. Bioinformatics part one: pixel refinement

  22. ABI 377 envelope: 96 lanes

  23. genome sequencing strategies • Shotgun • Directed primer walks • Modified directed primer walks

  24. Sequencing strategies Whole genome Also on a smaller scale: 1. “Island walking” and 2. Primer walking.

  25. Rapid re-sequencing of human Ad1: Time trial. Have sequence of Ad 1. In theory, have a minimally tiled set of PCR primers to cover entire 36,001 base genome. In theory, have a minimally tiled set of sequencing primers as well. Want draft sequence in a minimal time, including primer delivery from a vendor. In practice design two parallel sets of minimally tiled PCR primers and amplify two sets. In practice, assume 750 base reads--> 48 primers, one direction. Compare with consensus: Determine accuracy, timing and evaluate operation. 1 36,001 115 7,315 7,300 14,500 14,400 21,600 21,500 28,700 28,600 35,885

  26. Custom primer walks and “island” hopping • Have scaffold of generic genome: related or compiled. • Have archived “islands of sequences” (lg, med, sm)- from other research interests. • Generate “in-bound” primers to re-sequence equivalents and known features, e.g., 3’-ITR. • Use custom “out-bound” primers to walk across “inter-island” sequences (PCR and sequencing. • Collect “1st +” draft genomic sequence as round 1. • Iterative walks to complete “2+1” consensus, with error rate 1/10,000 bases.

  27. Target: HAdV4 • For 36,000 bases, need 90 primers for 1x coverage (1st draft) and 270 primers for 3x coverage (finished). • Have from GenBank: 10 “islands” @ 30%= 10,883 bases, • calling for 27x2= 54 primers for complementing coverage. • Theory (if continuous sequence): 36,000-10,883= 25,117 bases. • At 400 bases per read, need 63 primers for 1x coverage, or 126 for complementing coverage. • Practice: 10 “islands” @ 30%= 10,883 bases, 80 primers. • Example: “Island 1” is 149 bases. • 1 fragment at 400 bases/read. • 2 primers for 1x coverage. • “Terminal island,” need only 1 “outbound” primer. • Total of (1x2)+1= 3 primers. • Example: “Island 2” is 2042 bases. • 5 fragments at 400 bases/read. • “Internal island,” need 2 “outbound” primers. • Total of (5x2)+2= 12 primers.

  28. Definition of tiled set of PCR primers: Data. C A B D G E F H PCR fragments “B” “C” “D” “E”

  29. Input from sequencer peak intensities Output to user DNA sequence DNA sequencing: Computation • normalize intensities • apply mobility corrections • predict bands • call bases

  30. DNA sequencing: Computation

  31. DNA sequencing: Computation

  32. Sequence assembly: “Sequencher”

  33. Applications DNA sequencing • Whole genome analysis • Comparative genomics • Applications to subfields

  34. DNA sequencing • Higher throughput

  35. DNA sequencing: Equipment

  36. DNA sequencing technology • Manual. • ABI 370s series. • DuPont “Genesis.” • Capillary array: Hitachi, ABI, Amersham... • Ultrathin horizontal: GeneSys Tech. (MJResearch), Whitehead Inst., E. Yeung. • Thin channel. • “ABI” 310, 3100, 3710……. (2002)

  37. Capillary electrophoresis

  38. Multi-capillary array

  39. Cap array screen dump

  40. Shimadzu, Ltd. • NEW ORLEANS, March 19, 2002. PittCon. • Faster and more economical DNA Sequencer. • 10 times faster and 90 percent cheaper to run than current state-of-the-art. • GenoMEMS, MA spinoff that has developed a microfabrication technology, based on Whitehead Inst. technology. • Microelectromechanical system, or MEMS, technology:microfabricated electrical and mechanical components • Five million bases per day. • Readlengths of 800 bases. • Target 2003. • TODAY (2005) Solexa, 454 etc. $1,000 genome- $100,000 genome

  41. Other considerations: automation

  42. Bioinformatics issues in comparative DNA sequencing

More Related