Unilag workshop complex genome analysis

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  • Figure 10.2
  • Figure 10.3
  • Figure 10.4
  • Figure 10.5
  • Figure 10.5 a, b
  • Figure 10.5 c
  • Figure 10.8
  • Figure 10.9
  • Figure 10.10
  • Figure 10.12
  • Figure 10.13
  • Figure 10.16
  • Figure 10.14
  • Fig. 10.15
  • Figure 10.20
  • Figure 10.18 (top) / Figure 10.19a (bottom)
  • Figure 10.20
  • Figure 10.21
  • Figure 10.22
  • Figure 10.24

Transcript

  • 1. COMPLEX GENOME ANALYSIS Ikhide Imumorin, PhD Assistant Professor of Animal Molecular & Quantitative Genetics Cornell University Ithaca, NY 14853 USA
  • 2. GENOMICS ANALYSIS
  • 3. The genomes of living organisms vary enormously in size.
  • 4. Genomicists look at two basic features of genomes: sequence and polymorphism.
    • Major challenges to determine sequence of each chromosome in genome and identify many polymorphisms:
      • How does one sequence a 500 Mb chromosome 600 bp at a time?
      • How accurate should a genome sequence be?
        • DNA sequencing error rate is about 1% per 600 bp.
      • How does one distinguish sequence errors from polymorphisms?
        • Rate of polymorphism in diploid human genome is about 1 in 500 bp.
      • Repeat sequences may be hard to place.
      • Unclonable DNA cannot be sequenced.
        • Up to 30% of genome is heterochromatic DNA that can not be cloned
  • 5. Divide and conquer strategy meets most challenges.
    • Chromosomes are broken into small overlapping pieces and cloned.
    • Ends of clones sequenced and reassembled into original chromosome strings
    • Each piece is sequenced multiple times to reduce error rate.
      • 10-fold sequence coverage achieves a rate of error less than 1/10,000.
  • 6. Fig. 10.2
  • 7. Techniques for mapping and cloning
    • Cloning
      • Library of DNA fragments 500 – 1,000,000 bp
      • Insert into one of a variety of vectors
    • Hybridization
      • Location of a particular DNA sequence within the library of fragments
    • PCR amplification
      • Direct amplification of a particular region of DNA ranging from 1 bp to > 20kb
    • DNA sequencing
      • Automated DNA sequencer using Sanger method determines sequences 600 bp at a time.
    • Computational tools
      • Programs for identifying matches between a particular sequence and a large population of previously sequenced fragments
      • Programs for identifying overlaps of DNA fragments
      • Programs for estimating error rates
      • Programs for identifying genes in chromosomal sequences
  • 8. Making a large scale linkage map
    • Types of DNA polymorphisms used for large-scale mapping:
      • Single nucleotide polymorphisms (SNPs) – 1/500 – 1/1000 bp across genome
      • Simple sequence repeats (SSRs) – 1/20-1/40 kb across genome
        • 2-5 nucleotides is repeated 4-50 or more times.
    • Most SNPs and SSRs have little or no effect on the organism.
    • Serve as DNA markers across the chromosomes
    • Must be able to rapidly identify and assay in populations from 100s to 1000s of individuals
    Fig. 10.3
  • 9. Genome wide identification of genetic markers
    • Initial genetic maps used SSRs which are highly polymorphic.
    • Identified by screening DNA libraries with SSR probes
    • Amplified by PCR and length differences assayed
    • SNPs – millions more recently identified by comparison of orthologous regions of cDNA clones from different individuals
  • 10.
    • Homologous – genes with enough sequence similarity to be related somewhere in evolutionary history
    • Orthologous – genes in two different species that arose from the same gene in the two species’ common ancestor
    • Paralogous – arise by duplication within same species
    • Orthologous genes are always homologous, but homologous genes are not always orthologous.
  • 11. SNPs and SSRs for genome coverage
    • Until recently, maps were constructed from about 500 SSRs evenly spaced across genome (1 SSR every 6 Mb).
    • SNPs provide more than 500,000 DNA markers across the genome.
  • 12. Genome wide typing of genetic markers
    • Two-stage assay for simple sequence repeats
      • PCR amplification
      • Size separation
    Fig. 10.4
  • 13. Long range physical maps: karyotypes and genomic libraries position markers on chromosomes.
    • Physical map
      • Overlapping DNA fragments ordered and oriented that span each of the chromosomes
      • Based on direct analysis of DNA rather than recombination on which linkage maps are based
      • Chart actual number of bp, kb, or Mb that separate a locus from its neighbors
      • Linkage vs. physical maps
        • 1 cM = 1 Mb in humans
        • 1 cM = 2 Mb in mice
  • 14. Vectors used for clone large inserts for physical mapping
    • YACs (yeast artificial chromosomes)
      • Insert size 100-1,000,000 Mb
    • BACs (bacterial artificial chromosomes)
      • Insert size 50 – 300 kb
      • More stable and easier to purify from host DNA than YACs
  • 15. How to determine order of clones across genome
    • Overlapping inserts help align cloned fragments.
      • Bottom-up approach – overlapping sequences of tens of thousands of clones determined by restriction site analysis or sequence tag sites (STSs)
      • Top-down approach – insert is hybridized against karyotype of entire genome.
  • 16. Identifying and isolating a set of overlapping fragments from a library
    • Two approaches:
      • Linkage maps used to derive a physical map
        • Set of markers less than 1 cM apart
        • Use markers to retrieve fragments from library by hybridization.
        • Construct contigs – two or more partially overlapping cloned fragments.
        • Chromosome walk by using ends of unconnected contigs to probe library for fragments in unmapped regions
      • Physical mapping techniques:
        • Direct analysis of DNA
        • Overlapping clones aligned by restriction mapping
        • Sequence tag segments (STSs)
  • 17. Physical mapping by analysis of STSs Bottom-up approach Each STS represents a unique segment of the genome amplified by PCR. Fig. 10.5
  • 18. Human Karyotype
    • (a) Complete set of human chromosomes stained with Giemsa dye shows bands.
    • (b) Ideograms show idealized banding pattern.
    Fig. 10.6 a, b
  • 19. Chromosome 7 at three levels of resolution Fig. 10. 6 c
  • 20. FISH protocol for top-down approach Fig. 10.8
  • 21. Sequence maps show order of nucleotides in cloned piece of DNA.
    • Two strategies for sequence human genome:
      • Hierarchical shotgun approach
      • Whole-genome shotgun approach
    • Shotgun – randomly generated overlapping insert fragments:
      • Fragments from BACs
      • Fragments from shearing whole genome
        • Shearing DNA with sonication
        • Partial digestion with restriction enzymes
  • 22. Hierarchical shotgun strategy Used in publicly funded effort to sequence human genome
    • Shear 200 kb BAC clone into ~2 kb fragments
    • Sequence ends 10 times
    • Need about 1700 plasmid inserts per BAC and about 20,000 BACs to cover genome
    • Data form linkage and physical maps used to assemble sequence maps of chromosomes
    • Significant work to create libraries of each BAC and physically map BAC clones
    Fig. 10.9
  • 23. Whole-genome shotgun sequencing Used by Celera Genomics to sequence whole human genome.
    • Whole genome randomly sheared three times
      • Plasmid library constructed with ~ 2kb inserts
      • Plasmid library with ~10 kb inserts
      • BAC library with ~ 200 kb inserts
    • Computer program assembles sequences into chromosomes.
    • No physical map construction
    • Only one BAC library
    • Overcomes problems of repeat sequences
    Fig. 10.10
  • 24. Limitations of whole genome sequencing
    • Some DNA can not be cloned.
      • e.g., heterochromatin
    • Some sequences rearrange or sustain deletions when cloned.
    • Future large genome sequencing will use both shotgun approaches.
  • 25. Sequencing of the human genome
    • Most of draft took place during last year of project.
      • Instrument improvements – 500,000,000 bp/day
      • Automated factory-like production line generated sufficient DNA to supply sequencers on a daily basis.
      • Large sequencing centers with 100-300 instruments – 150,000,000 bp/day
  • 26. Integration of linkage, physical, and sequence maps
    • Provides check on the correct order of each map against other two
    • SSR and SNP DNA linkage markers readily integrated into physical map by PCR analysis across insert clones in physical map
    • SSR, SNP (linkage maps), and STS markers (physical maps) have unique sequences 20 bp or more, allowing placement on sequence map.
  • 27. Changes in biology, genetics and genomics from human genome sequence
    • Genetics parts list
    • Speeds gene-finding and gene-function analysis
      • Sequence identification in second organism through homology
      • Gene function in one organism helps understand function in another for orthologous and paralogous genes
      • Genes often encode one or more protein domains
        • Allows guess at function of new protein by comparison of protein sequence in databases of all known domains
      • Ready access to identification of known human polymorphism
      • Speeds mapping of new organisms by comparison
        • e.g., mouse and human have high similarity in gene content and order
  • 28. Major insights from human and model organism sequences
    • Approximately 25,000 human genes
    • Genes encode noncoding RNA or proteins.
    • Repeat sequences are > 50% of genome.
    • Distinct types of gene organization:
      • Gene families
      • Gene rich regions
      • Gene desert
    • Combinatorial strategies amplify genetic information and increase diversity.
    • Evolution by lateral transfer of genes from one organism to another
    • Males have twofold higher mutation rate than females.
    • Human races have very few unique distinguishing genes.
    • All living organisms evolve from a common ancestor.
  • 29. Conserved segments of syntenic blocks in human and mouse genomes Fig. 10.12
  • 30. Noncoding RNA genes
    • Transfer RNAs (tRNAs) – adaptors that translate triplet code of RNA into amino acid sequence of proteins
    • Ribosomal RNAs (rRNAs) – components of ribosome
    • Small nucleolar RNAs (snoRNAs) – RNA processing and base modification in nucleolus
    • Small nuclear RNAs (sncRNAs) - spliceosomes
  • 31. Protein coding genes generate the proteome.
    • Proteome – collective translation of 30,000 protein coding genes into proteins
    • Complexity of proteome increase from yeast to humans.
      • More genes
      • Shuffling, increase, or decrease of functional modules
      • More paralogs
      • Alternative RNA splicing – humans exhibit significantly more
      • Chemical modification of proteins is higher in humans.
  • 32. Protein coding genes generate the proteome How transcription factor protein domains have expanded in specific lineages Fig. 10.11
  • 33. Examples of domain accretions in chromatin proteins Fig. 10.13
  • 34. Number of distinct domain architectures in four eukaryotic genomes Fig. 10.14
  • 35. Repeat sequences fall into five classes.
    • Transposon-derived repeats
    • Processed pseudogenes
    • SSRs
    • Segmental duplications of 10-300 kb
    • Blocks of repeated sequences at centromere, telomeres and other chromosomal features
  • 36. Repeat sequences constitute more than 50% of the genome. Fig. 10.15
  • 37. Gene Organization of Genome
    • Gene families
      • Closely related genes clustered or dispersed
    • Gene-rich regions
      • Functional or chance events?
    • Gene deserts
      • Span 144 Mb or 3% of genome
      • Contain regions difficult to identify?
        • e.g., big genes – nuclear transcript spans 500 kb or more with very large introns (exons < 1% of DNA)
  • 38. Genome has a distinct organization. Gene family – olfactory receptor gene family
  • 39. Class II region of human major histocompatibility complex contains 60 genes in 700 kb Fig. 10.17
  • 40. Combinatorial strategies
    • At DNA level – T-cell receptor genes are encoded by a multiplicity of gene segments.
    • At RNA level – splicing of exons in different orders
    Fig. 10.19a Fig. 10.18
  • 41. Lateral transfer of genes
    • > 200 human genes may arise by transfer from organisms such as bacteria.
    • Lateral transfer is direct transfer of genes from one species into the germ line of another.
  • 42. Twofold higher mutation rate in males
    • Comparison of X and Y chromosomes
    • Same may be true for autosomes, but difficult to measure.
    • Majority of human mutations arise in males.
    • Males give rise to more defects, but also more diversity.
  • 43. Human races have similar genes.
    • Genome sequence centers have sequenced significant portions of at least three races.
    • Range of polymorphisms within a race can be much greater than the range of differences between any two individuals of different races.
    • Very few genes are race specific.
    • Genetically, humans are a single race.
  • 44. All living organisms are a single race.
    • All living organisms have remarkably similar genetic components.
    • Life evolved once and we are descendents of that event.
    • Analysis of appropriate biological systems in model organisms provides fundamental insight into corresponding human systems.
  • 45. In the future, other features of chromosomes will become increasingly important.
    • Chemical modification of bases
      • Understand DNA methylation now
      • Others may be discovered
    • Interaction of various proteins with chromosome
    • Three dimensional structure of proteins in nucleus
      • May determine interactions of chromosomal regions with regions of nuclear envelope
    • More effective tools need to be developed to examine chromosome features.
  • 46.  
  • 47. High-throughput instruments DNA sequencer Fig. 10.20
  • 48. High-throughput instruments e.g, microarrays Fig. 10.21
  • 49. Two color - DNA microarray Fig. 10.22
  • 50. Analysis of genomic and RNA sequences
    • Quantitative analysis of mRNA levels
      • Serial analysis of gene expression (SAGE)
        • Small cDNA tags of 15 bp from 3’ ends of mRNA are linked and sequenced.
      • Massively parallel signature sequence (MPSS)
        • Transcriptome – population of mRNAs expressed in a single cell or cell type
        • MPSS allows identification of most of cell’s rarely expressed mRNAs
  • 51. Lynx therapeutics sequencing strategy of MPSS Fig. 10.24
  • 52. Systems Biology – the global study of multiple components of biological systems and their interactions
    • New approach to studying biological systems has made possible:
      • Sequencing genomes
      • High-throughput platform development
      • Development of powerful computational tools
      • The use of model organisms
      • Comparative genomics
  • 53. Human Genome Project has changed the potential for predictive/preventive medicine.
    • Provided access to DNA polymorphisms underlying human variability
      • Makes possible identification of genes predisposing to disease
      • Understanding of defective genes in context of biological systems
      • Circumvent limitations of defective genes
        • Novel drugs
        • Environmental controls
        • Approaches such as stem-cell transplants or gene therapy
  • 54. Social, ethical, and legal issues
    • Privacy of genetic information
    • Limitations on genetic testing
    • Patenting of DNA sequences
    • Society’s view of older people
    • Training of physicians
    • Human genetic engineering
      • Somatic gene therapy – inserting replacement genes
      • Germ-line therapy – modifications of human germ line