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Finishing the Human Genome http://biochem158.stanford.edu/

Finishing the Human Genome http://biochem158.stanford.edu/. Genomics, Bioinformatics & Medicine. Doug Brutlag Professor Emeritus of Biochemistry & Medicine Stanford University School of Medicine. Chromosome 21: Public vs Celera Assemblies. Chromosome 8: Public vs. Celera.

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Finishing the Human Genome http://biochem158.stanford.edu/

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  1. Finishing the Human Genomehttp://biochem158.stanford.edu/ Genomics, Bioinformatics & Medicine Doug Brutlag Professor Emeritus of Biochemistry & Medicine Stanford University School of Medicine

  2. Chromosome 21:Public vs Celera Assemblies

  3. Chromosome 8:Public vs. Celera

  4. FinishingStrategyfor the PublicGenomeProject

  5. Polymerase Chain Reaction Overview: Exponential Amplification of DNA

  6. After Cycle 1 After Cycle 2 After Cycle 3 The First Three Cycles Original DNA After N cycles, amount of target DNA is 2N-2N

  7. PCR Requirements DNA • Need to know at least the beginning and end of DNA sequence • These flanking regions have to be unique to strand interested in amplifying • Region of interest can be present in as little as one copy • Enough DNA in 0.1 microliter of human saliva to use PCR DNA Polymerase Enzyme • DNA polymerase from Thermus aquaticus--Yellowstone • Alternatives: Thermococcus litoralis, Pyrococcus furiosus Thermocycler

  8. Temperature Cycling TAQ polymerase optimum at 72° C

  9. PCR on a Chip Uses: Reaction complete in 2-20 minutes Extremely portable

  10. Fluidigm PCR Arrayshttp://www.fluidigm.com/access-array-system.html

  11. Real-Time PCR • Uses: •Portable means to diagnose bacteria: epidemics • Bioterrorism detection • Military, medical, and municipal applications • Fast: Results in less than seven minutes

  12. Quantitative PCR

  13. QuantaLifehttp://www.quantalife.com/

  14. PCR Applications • Forensics • assessment/reassessment of crimes • Archaeology • determine gene sequences of ancient organisms • rethinking the past, human origins • Molecular Biology • Cloning genes • Sequencing genes • Finishing genome sequences • Amplification of DNA or RNA • Medicine • Diagnostics for inherited disease • Diagnostics for gene expression • Diagnostics for gene methylation

  15. FinishingStrategyfor the PublicGenomeProject

  16. Finished Sequence in 2004 (Build 35)

  17. Comparison of Chromosome 7Draft versus Finished Sequence

  18. Substitutions in BAC Overlaps withBACs from Same or Different Libraries

  19. Gaps in BAC Overlaps withBACs from Same or Different Libraries

  20. Duplications and Deletionsin the Human Genome

  21. Percentage of Chromosomes Duplicated

  22. Duplications near Centromeres

  23. Duplications near Telomeres

  24. Deletions and Duplications can Arise from Unequal Crossing Over in Repeated Regions • Crossing over between maternal and paternal chromosomes • Unequal crossing over between maternal and paternal chromosomes Maternal Paternal Offspring Offspring Maternal Paternal Offspring Offspring

  25. The Diploid Sequence of anIndividual Human (HuRef)

  26. Karyotype of J.Craig Venter Giemsa Stain FISH Stain

  27. Comparing NCBI Assembly to HuRef Assembly

  28. SNPs & InDels in HuRef Autosomes

  29. Illumina Solexa Sequencing Technology

  30. Illumina Solexa Sequencing Technology

  31. Illumina Solexa Sequencing Technology

  32. Illumina Solexa Sequencing Technology

  33. Illumina Solexa Sequencing Technology

  34. Illumina Solexa Sequencing Technology

  35. Illumina Solexa Sequencing Technology

  36. Illumina Solexa Sequencing Technology

  37. Illumina Solexa Sequencing Technology

  38. Illumina Solexa Sequencing Technology

  39. Illumina Solexa Sequencing Technology

  40. Illumina Solexa Sequencing Technology

  41. 1) Prepare Adapter Ligated ssDNA Library 2) Clonal Amplification on 28 µ beads 3) Load beads and enzymes in PicoTiter Plate™ 4) Perform Sequencing by synthesis on the 454 Instrument Life Sciences 454 Process Overview

  42. Emulsion Based Clonal Amplification Single test tube generation of millions of clonally amplified sequencing templates No cloning and colony picking A + PCR Reagents + Emulsion Oil B Anneal DNA template to capture beads Micro-reactors “Water-in-oil” emulsion Adapter carrying library DNA Break micro-reactors Isolate DNA containing beads Perform emulsion PCR

  43. Depositing DNA Beads into thePicoTiter™Plate Load Enzyme Beads 44 μm Load beads into PicoTiter™Plate Centrifuge Step • 70x75mm array of fused optical fibers with etched wells • 1.6 million wells monitored optically through fiber

  44. Simultaneous sequencing of the entire genome in hundreds of thousands of picoliter-size wells • Pyrophosphate signal generation Sequencing By Synthesis Sequencing-By-Synthesis

  45. 4-mer 3-mer 2-mer 1-mer Flowgrams and BaseCalling Flow Order TACG

  46. Pacific Biosciences SMRT Sequencing

  47. Pacific Biosciences SMRT Sequencing

  48. Phospholinked Fluorophores

  49. Processive Synthesis

  50. Synthesis of Long Duplex DNA

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