OKC Grand Rounds 2009

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  • 1. Sean Davis, M.D., Ph.D. Genetics Branch, Center for Cancer Research National Cancer Institute National Institutes of Health Genomics for Pediatrics An Overview of Technologies for Observing the Human Genome
  • 2.  
  • 3.  
  • 4. The Human Genome Project
  • 5. The Central Dogma
  • 6. phenotype Gene Copy Number Sequence Variation Chromatin Structure and Function Gene Expression Transcriptional Regulation DNA Methylation Patient and Population Characteristics
  • 7. Overview
    • Microarrays
      • Gene expression
      • 8. Comparative genomic hybridization
      • 9. Tiling arrays and data integration
    • Next-generation sequencing
      • DNAse-Seq application
  • 10. Normal Karyotype Tumor Karyotype
  • 11. Hybridization
    • Highly robust propensity of nucleic acid polymers to form dimers with known base pairings
    • 12. Integral to life as we know it
    • 13. Can be leveraged to build systems for interrogating biologic processes
  • 14. ~30,000 genes 1 2 3 4 5
  • 15. DNA Microarrays
    • Gene Expression
      • RNA hybridized to DNA, one spot per array
  • 16. Gene Expression Microarrays Golub et al., Science 286:531-537. (1999). Spellman et al., Molecular Biology of the Cell 9, 3273-3297. (1999).
  • 17.
    • Ductal Carcinoma In Situ
    • 18. Breast cancer precursor or benign lesion?
    • 19. Histologic grading used to help define treatment
    • 20. Low grade and high grade clear-cut, but intermediate grade problematic
  • 21. DNA Microarrays
    • Gene Expression
      • RNA hybridized to DNA, one probe per gene
      • 22. RNA hybridized to DNA, multiple probes per gene
      • 23. RNA hybridized to DNA, one or more probes per exon
  • 24. DNA Microarrays
    • Gene Expression
    • 25. Comparative Genomic Hybridization (CGH)
      • Genomic DNA hybridized to DNA
      • 26. Useful for determining relative copy number of DNA across the entire genome
      • 27. Not useful (directly) for determining genome structure
  • 28. Array Comparative Genomic Hybridization (aCGH) Tumor DNA Normal DNA Hybridize (Tumor Suppressors) (Oncogenes) Normal Copy Number DNA Loss Amplification
  • 29. Tumor Chromosome Normal Chromosome
  • 30. A Genome View of Copy Number
  • 31. Frequency of Copy Number Changes Summary of copy number changes from 46 breast cancer cell lines
  • 32.
    • GLI3 mutations and deletions can lead to Greig cephalopolysyndactyly syndrome (GCPS)
    • 33. Typically requires mutation screening, FISH, and some small deletions may be missed
    • 34. Use array comparative genomic hybridization to get very high-resolution view of the region
  • 35.  
  • 36. DNA Microarrays
    • Gene Expression
    • 37. Comparative Genomic Hybridization (CGH)
    • 38. Single Nucleotide Polymorphisms
      • Arrays can be used as a genotyping platform
      • 39. Again, DNA hybridized to DNA, but designed to detect the differences in hybridization due to a SNP
      • 40. Can be used for measuring copy number, finding stretches of uniparental disomy, risk alleles for certain conditions, and in linkage and association studies
  • 41. DNA Microarrays
    • Gene Expression
    • 42. Comparative Genomic Hybridization (CGH)
    • 43. Single Nucleotide Polymorphisms
    • 44. DNA methylation
    • 45. MicroRNA expression
    • 46. “Tiling array” applications
    • 47. Others....
  • 48. Tiling Array Technology
  • 49. Simultaneous Gene Expression and Copy Number on Tiling Arrays Annotated Genes Expression Copy Number, Sample 1 Copy Number, Sample 2 Simultaneous measurement of copy number in two samples and gene expression in one sample overlayed on map of genes in the region
  • 50. Simultaneous Gene Expression and Copy Number on Tiling Arrays Annotated Genes Expression Copy Number, Sample 1 Copy Number, Sample 2 Increased expression in the small amplicon does not include all genes, giving clues as to the biologically important genes in the region
  • 51. Simultaneous Gene Expression and Copy Number on Tiling Arrays Annotated Genes Expression Copy Number, Sample 1 Copy Number, Sample 2 Spikes of expression at exons of ERBB2
  • 52. Simultaneous Expression and Copy Number Copy Number Expression Copy Number Evolutionary Conservation Expression Copy Number Opposite Strand Expression Evolutionary Conservation Expression Copy Number
  • 53. Growth in Density Over Time And these numbers are from only a single array!
      And these numbers are from only a single array!
      Excel doesn't work!
    2,000 spots, 1997 8,000 spots, 2000 36,000 spots, 2003 85,000 to 390,000 spots, 2004 10,000,000 beads, 2005
  • 54. Why did the chicken cross the road? Darwin1: It was the logical next step after coming down from the trees. Darwin2: The fittest chickens cross the road.
  • 55. Sequencing
    • Why use hybridization, which is just a measure of sequences, correct?
      • Sequencing is costly, time- and labor-intensive, and inefficient
    • Next-generation sequencing technology changes the equation such that sequencing can be more efficient, cheaper, and less time- and labor-intensive than hybridization-based methods like microarrays
  • 56.  
  • 57.  
  • 58.  
  • 59. Next-generation Sequencing
  • 60.  
  • 61. Chromatin
    • Chromatin is the complex of protein and DNA that make up the chromosomes. It is not a static structure.
    • The nucleosomes are the basic building blocks of chromatin structure. Their positioning on the genome and the regulation of their placement is not well described.
  • 62.
    • DNAse is an enzyme that cuts DNA at locations where DNA is accessible
    • 63. These “accessible” regions have been associated with open chromatin
    • 64. Regions of open chromatin are necessary for transcriptional and regulatory machinery to have access to gene neighborhoods and facilitate transcription
  • 65. DNAse Hypersensitivity
    • Method for finding regions of “open” chromatin
    • 66. In data published with the ENCODE consortium, DNAse hypersensitive (HS) were shown to be correlated with:
      • Histone modification
      • 67. Transcription start sites
      • 68. Early replicating regions
      • 69. Transcription factor binding sites (experimentally determined by ChIP/chip, etc.)
    Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. The ENCODE Consortium. Nature , 2007.
  • 70. DNAse-Seq Method Crawford, G.E., Davis, S., Scacheri, P.C., Renaud, G., Halawi, M.J., Erdos, M.R., Green, R., Meltzer, P.S., Wolfsberg, T.G., and Collins, F.S. Nat Methods , 2006
  • 71.  
  • 72.  
  • 73.  
  • 74.
    • Distances between sequences in non-DNAse HS regions have an oscillating pattern with frequency that corresponds to a single turn of the double-helix
    • 75. DNAse is known to cut preferentially in the minor groove, which is exposed every 10.4 bases when wrapped around a nucleosome
    • 76. A nucleosome is wrapped by 147 base pairs when complexed with DNA
    • 77. Implication: Nucleosomes are positioned in a highly organized, precise manner
    Nucleosome Positioning
  • 78.  
  • 79. Phenotype Gene Copy Number Sequence Variation Chromatin Modification Gene Expression Transcriptional Regulation DNA Methylation
  • 80. Public Data
    • NCBI Gene Expression Omnibus (GEO)
      • 250,000 microarray experiments already done !
    • NCBI Short Read Archive (SRA)
      • Compendium of sequencing experiments utilizing next-generation sequencing technologies
    • GWAS databases
    • 81. Databases of gene and protein function and interactions
  • 82. Final Thoughts
    • Genomic technologies are rapidly evolving and constantly improving
    • 83. These technologies will see increasing use in clinical medicine, but such use must be tempered by sound clinical hypotheses and testing
    • 84. The data generated by many of these technologies necessitates improved clinical, translational, and research information systems
  • 85.  
  • 86. Challenges
    • Most of these technologies are still quite expensive and do not adapt well to clinical laboratory settings
    • 87. Designing studies that evaluate the operating characteristics of new testing methods is costly and requires the appropriate patient populations
    • 88. There are many ethical concerns associated with the enormous amounts of personal information that might be gleaned from genomic technologies applied in the clinical setting
  • 89. The Biggest Challenge? How do we integrate all the disparate pieces of information, collected longitudinally and by many sources, to improve the health of the individual?
  • 90. Thanks
    • NCI
    • University of Sydney
      • Lucy Webster
      • 94. Rosemary Balleine
    • Duke
      • Greg Crawford
      • 95. Terry Furey
    • NHGRI
      • Julie Johnston
      • 96. Les Biesecker
  • 97. One day the zoo-keeper noticed that the orangutan was reading two books - the Bible and Darwin's The Origin of Species. In surprise he asked the ape, "Why are you reading both those books"? "Well," said the orangutan, "I just wanted to know if I was my brother's keeper or my keeper's brother." [email_address]