Human Genome Lecture

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Human Genome Lecture

• Historical aspects of the HGP
• EST sequencing
• Finding new genes faster than ever
• Using 3 ESTs to generate human gene maps
• First comprehensive genome-wide human gene maps
• Sequence of human genome
• Complex genomic regions and sequence limitations

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Key pre-HGP scientific advances

• Structure of DNA determined (1953)
• Watson Crick
• Recombinant DNA created (1972)
• P. Berg Cohen and Boyer
• Methods for DNA sequencing developed (1977)
• Maxam Gilbert F. Sanger
• PCR invented (1985)
• K. Mullis
• Automated DNA sequencer developed (1986)
• L. Hood

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Obstacles to formation of the HGP

• 1) Financial/political Big biology is bad
  biology
• -departure from cottage industry culture of
  biology
• -devoid of hypothesis-driven research
• -what will it cost?
• -will it take away from other programs?
• 2) Why sequence the Junk?
• -protein coding regions make up lt1.5 of the
  genome
• -waste of time/money to sequence repetitive,
  hard-to-sequence regions
• 3) It is impossible to do
• -mid 1980s
• -primitive sequencing capabilities (500
  bp/day/lab)
• -primitive computer capabilities/bioinformatics
  resources

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Significance of the HGP

• The book of life, The grail of human biology,
  Code of codes
• The instructions to create a human being
• The genome is a product of evolution
• - molecular replicator (DNA) heritable
  variation time changing environment genome
• - record of the evolutionary history of our
  species
• Comparative genomics the genes that make us
  human
• The genome unparalled system of information
  storage
• - 70 trillion cells in human body
• - each cell stores 3 billion units of
  information

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Significance of the HGP (cont)

• Biology in the 21st century
• - equivalent of learning to read a new language
• The genome as dynamic not static
• - perspective on past/future of the species
• Implications for health and disease
• -Genetic disease gene discovery single-gene
  diseases multifactorial diseases
• -DNA-based diagnostics
• -New drug targets
• -Gene therapy implications
• -Therapeutic uses vs. enhancements
• Accumulation of a molecular parts lists of
  human physiology anatomy
• - Lander Periodic Table of the Elements
  analogy

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Genomics Timelines
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Rapid Gene Identification Mapping ESTs and
Gene-based STSs

• Single-pass sequencing of randomly selected cDNA
  clones
• Obtain sequences from 5 and 3 ends of cDNA
  inserts
• Rapidly cheaply identify human genes
• Alzheimers gene discovered by EST database
  search
• 3UT sequence ideal for STS development
  PCR-based gene mapping
• Readily scaled up for development of most
  comprehensive human gene maps (Science 1996,
  1998)

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One gene one STS

• Gene-based STSs as the basis for a human gene map
• Berry et al, Nature Genetics 1995
• ESTablishing a human transcript map
• Boguski and Schuler, Nature Genetics 1995

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Boguski Schuler, Nat. Genet. 1995
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Size and gene content of the 24 human
chromosomes. A, Size of each human chromosome,
in millions of base pairs (1 million base pairs
1 Mb). Chromosomes are ordered left to right by
size. B, Number of genes identified on each human
chromosome. Chromosomes are ordered left to right
by gene content. (Based on www.ensembl.org, v36.)
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Genomic sequencing vs EST sequencing

• EST (single pass cDNA) sequencing
• - very fast but not error-free (e.g. 99
  accuracy)
• - very rapid gene identification (reliance on
  mRNA)
• - cDNA abundance influences coverage
• some genes will be missed
• normalized cDNA libraries improve coverage
• provides a gene expression profile
• Genomic sequencing
• - pre-2001 much slower method for gene finding
• -must do gene id by computer prediction
• - will generate complete gene and genome
  information, e.g. introns, regulatory regions,
  intergenic regions, repeats, etc.
• - more expensive way to id genes
• - independent of gene expression level concerns
• - highly accurate when complete

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Significant findings arising from analysis of the
draft sequence of the human genome

•  The genomic landscape shows marked variation in
  the distribution of a number of features,
  including genes, transposable elements, GC
  content, CpG islands and recombination rate. This
  gives us important clues about function. For
  example, the developmentally important HOX gene
  clusters are the most repeat-poor regions of the
  human genome, probably reflecting the very
  complex coordinate regulation of the genes in the
  clusters.
•  There appear to be about 30,00040,000
  protein-coding genes in the human genomeonly
  about twice as many as in worm or fly. However,
  the genes are more complex, with more alternative
  splicing generating a larger number of protein
  products.
•  The full set of proteins (the 'proteome')
  encoded by the human genome is more complex than
  those of invertebrates. This is due in part to
  the presence of vertebrate-specific protein
  domains and motifs (an estimated 7 of the
  total), but more to the fact that vertebrates
  appear to have arranged pre-existing components
  into a richer collection of domain architectures.
•  Hundreds of human genes appear likely to have
  resulted from horizontal transfer from bacteria
  at some point in the vertebrate lineage. Dozens
  of genes appear to have been derived from
  transposable elements.
•  Although about half of the human genome derives
  from transposable elements, there has been a
  marked decline in the overall activity of such
  elements in the hominid lineage. DNA transposons
  appear to have become completely inactive and
  long-terminal repeat (LTR) retroposons may also
  have done so.
•  The pericentromeric and subtelomeric regions of
  chromosomes are filled with large recent
  segmental duplications of sequence from elsewhere
  in the genome. Segmental duplication is much more
  frequent in humans than in yeast, fly or worm.
•  Analysis of the organization of Alu elements
  explains the longstanding mystery of their
  surprising genomic distribution, and suggests
  that there may be strong selection in favour of
  preferential retention of Alu elements in GC-rich
  regions and that these 'selfish' elements may
  benefit their human hosts.
•  The mutation rate is about twice as high in
  male as in female meiosis, showing that most
  mutation occurs in males.
•  Cytogenetic analysis of the sequenced clones
  confirms suggestions that large GC-poor regions
  are strongly correlated with 'dark G-bands' in
  karyotypes.
•  Recombination rates tend to be much higher in
  distal regions (around 20 megabases (Mb)) of
  chromosomes and on shorter chromosome arms in
  general, in a pattern that promotes the
  occurrence of at least one crossover per
  chromosome arm in each meiosis.
•  More than 1.4 million single nucleotide
  polymorphisms (SNPs) in the human genome have
  been identified. This collection should allow the
  initiation of genome-wide linkage disequilibrium
  mapping of the genes in the human population.

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Patterns of intrachromosomal and interchromosomal
duplication in the human genome
Bailey, et al, Science, 2002
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Distribution of gt50 kb gaps in HapMap phase 1 -
CEU
HapMap phase 1
chromosome lengths
gt50 kb gap between SNPs
excluding centromere gaps
heterochromatin
T. Hudson
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Bailey, et al, Science, 2002
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Genome Structural Variation

• Broadest sense all changes in the genome not due
  to single base-pair substitutions
• Copy number variations (CNVs)
• CNV loci may cover 12 of genome
• Insertions/Deletions (indels)
• e.g. Repeats STRs, VNTRs
• Inversions
• Duplications and translocations

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Limitations of Genome Sequencing

• Nexgen sequencers are short read
• Repeated/duplicated sequences often cant be
  positioned
• Segmental duplications make up 5 of genome
• gt95 identity gt20kb
• Smaller-size, highly duplicated sequence families
  exist
• Complex, duplication-rich regions
• gt200 gaps (gt50kb each) in human genome
• Difficult to accurately assemble
• Linked to many human diseases
• Linked to evolutionary adaptation
• Location of missing heritability of GWAS?
• Are critical regions of the genome being
  missed/ignored?

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Limitations of next-generation genome sequence
assembly Can Alkan, Saba Sajjadian Evan E
Eichler
Nature Methods Volume 8, Pages 6165 Year
published (2011) DOI doi10.1038/nmeth.1527
Published online 21 November 2010
Abstract Abstract High-throughput sequencing
technologies promise to transform the fields of
genetics and comparative biology by delivering
tens of thousands of genomes in the near future.
Although it is feasible to construct de novo
genome assemblies in a few months, there has been
relatively little attention to what is lost by
sole application of short sequence reads. We
compared the recent de novo assemblies using the
short oligonucleotide analysis package (SOAP),
generated from the genomes of a Han Chinese
individual and a Yoruban individual, to
experimentally validated genomic features. We
found that de novo assemblies were 16.2 shorter
than the reference genome and that 420.2 megabase
pairs of common repeats and 99.1 of validated
duplicated sequences were missing from the
genome. Consequently, over 2,377 coding exons
were completely missing. We conclude that
high-quality sequencing approaches must be
considered in conjunction with high-throughput
sequencing for comparative genomics analyses and
studies of genome evolution.