DNA Learning Center July 15, 2003

1
DNA Learning CenterJuly 15, 2003

• W. Richard McCombie
• Professor
• Cold Spring Harbor Laboratory and
• The Watson School of Biological Sciences

2
Basic points

• Genome research is advancing very rapidly
• Technologies are driving the progress
• These technologies and the data that results from
  them will have a revolutionary effect on the way
  biological research is done and in our
  understanding of biology and medicine

3
Major Topics

• What is genomics and in particular the human
  genome program
• Introduction and historical perspective on
  sequencing.
• Some information about genomes being sequenced
• Stategies to analyse genomes
• Comparative genomics
• How genomics has and will change biology and
  medicine

4
What is an organism

• At ONE LEVEL, it is the result of the execution
  of the code that is its genome
• We do not know the degree to which environment
  alters this execution
• We do know that in addition to physical
  attributes, many complex processes such as
  behavior have an influence from the code
• We now know that in mammals, this code is only
  comprised of about 30,000-40,000 genes and their
  control units

5
The Genome of an organism is

• The complete set of inherited instructions for
  that organism - Its complete DNA code
• When operating creates a set of proteins in an
  organized fashion
• These proteins act to cause growth, development
  and reproduction of the organism

6
What is genomics

• Genomics is the analysis of the complete set of
  genetic instructions of an organism
• These genetic instructions consist of genes,
  which direct the production of proteins and their
  control elements
• These genes consist of a series of DNA bases
• Previously we could only look at one or at most a
  few of these objects or parts at a time
• Technology now enables us to see them all

7
Why will genomics have such an impact

• Important biological problems such as cancer and
  learning and memory are extraordinarily complex
• Genomics lets us integrate this complex
  information in a meaningful way
• Ultimately, much of biological research will be
  driven by computational analysis

8
Sizes of some important genomes

• Virus 0.003 - 0.300 million
• Bacteria 0.8- 6 million
• Yeast 15 million
• C. elegans 100 million
• Rice 435 million
• Arabidopsis 130 million
• Fugu 800 million
• Mouse 2.5 billion
• Corn 2.5 billion
• Human 3 billion
• Wheat 16-20 billion
• Loblolly pine 20 billion

9
Genome sequencing efficiencies per person

• 1980 0.1-1 kb per year
• 1985 1-5 kb per year
• 1990 25-50 kb per year
• 1996 100-200 kb per year
• 2000 500-1000 kb per year
• 2002 10,000 - 25,000 kb per year

10
(No Transcript)
11
(No Transcript)
12
Methods to analyse a complex genome

• Mapping
• Genetic
• Physical
• Expressed gene analysis
• Genome sequence analysis
• Complete sequence
• Skimming
• Rough draft

13
Salient features of genome organization

• Higher organisms have large genomes with
  considerable amount of repeat sequences
• Genes from higher organisms are interrupted by
  non-coding regions
• Only a small portion of a genome codes for genes
• Related organisms have related genomes

14
Expressed Sequence Tags (sequencing parts of the
processed genes)

• Advantages
• Inexpensive
• Know sequence is coding
• Information about tissue or developmental stage
  expression

• Disadvantages
• Coverage is incomplete
• Position of sequence in the genome is unknown
• Only partial information about each gene
• No information about structural elements

15
Steps in genome sequencing

• Construction of a large-insert library
• Construction of a small insert subclone library
• Isolation of DNA
• Sequencing of the DNA fragments (8-10x)
• Assembly of the data into contiguous regions
• Filling the gaps in the sequence and resolving
  discrepancies
• Confirmation of the sequence
• Analysis

16
High Accuracy Genomic Sequencing (6-10x plus
resolution of problems)

• Advantages
• Normalized coverage of all genes
• Information about gene structure
• Information about regulatory elements
• Genome organization

• Disadvantages
• Cost
• Time
• Difficult to determine if a sequence codes for a
  gene

17
Rough draft

• Can be thought of as
• High coverage skimming
• Low coverage complete sequencing
• Advantages and disadvantages are intermediate
  between skimming and complete sequencing -
  dependent on the coverage

18
Cost of various types of sequencing (per base)

• Base perfect (uncomplicated) 0.3
• 8x shotgun - no finishing 0.1
• 4x shotgun - no finishing 0.05
• 3x shotgun - no finishing 0.04
• 1x shotgun - no finishing 0.01

19
The Human Genome Project

• Human genome consists of three billion base pairs
  Adenine, Cytosine, Guanine, Thymine
• Printing out the A,C,G,T would fill over 150,000
  telephone book pages
• Disease is often caused by a single variation in
  the three billion bases - one different letter in
  150,000 pages

20
The human genome project

• A concerted effort to build resources to unravel
  the human control code
• To develop map resources to link genetic elements
  (such as disease genes) to a physical
  representation of the genome
• To determine the sequence of all of the DNA that
  combines to make the human control code

21
2-15-01
22
Genome sequencing assignments
I
II
III
IV
V
CSHSC
Kazusa
TIGR
SPP
ESSA
Kazusa
Genoscope
23
(No Transcript)
24
Gene Families
25
(No Transcript)
26
(No Transcript)
27
Cytogenetic map of chromosome 4S
Paul Fransz
28
Complete genomic sequencing reduces the genetics
of an organism to a closed, finite system
29
FRUITFULL Gene Function
The AGL8 gene was renamed FRUITFULL (ful1)
30
Genetic Redundancy
ap1 cal ful triple mutants have flowers replaced
by shoots

• apetala1 cauliflower double mutants have
  proliferating floral meristems ressembling
  cauliflowers

31
The state of Arabidopsis research200??

• Complete annotated sequence available
• Time to clone a gene has decreased from months to
  years to weeks in some cases
• People are beginning to look at global features
  of Arabidopsis
• Gene trap insertion in every gene
• Insertion site sequences known, linked to
  physical and genetic map

32
Analysis of not the first, or the second, but
subsequent genomes

• The information from the first few genomes will
  enable huge cost and time savings
• A major emphasis will be to determine the
  function of genes

33
What are the genes and what do they do???

• Computational analysis
• Functional analysis
• Microarrays
• Transposons
• Various other methods
• Comparative analysis

34
Comparative Genomics
35
What can we learn from comparative analysis

• Evolutionary relationships
• Better annotation of genes, particularly of
  beginning and ends of genes
• Detection of conserved regulatory regions
• Functional evidence

36
Benefits of having a model genome reference
sequence with conserved local gene order to your
plant of interest

• Requirements for sequence accuracy decrease for
  most of the genome
• you can fill in with high accuracy where needed
• The reference genome can be used as a scaffold
  allowing the anchoring of clones (allowing
  partial sequence coverage to infer complete clone
  coverage)

37
Co-linearity among cereal genomes
38
What type of comparisons are useful?

• Arabidopsis to very closely related species
• Annotate the Arabidopsis sequence
• Arabidopsis to related crop plants (soybean,
  tomato, Medicago truncatula)
• Determine the degree of locally conserved gene
  order between these crops and Arabidopsis
• Determine how the Arabidopsis sequence can be
  used in the analysis of these species
• Arabidopsis to distant plants (rice for instance)
• Gene discovery
• Systems analysis
• Gene order conservation???
• Arabidopsis to animals
• How plants and animals differ in carrying out
  basic biological processes
• How plant and animals organize and manage gene
  expression

39
Mammalian Comparative Genomics

• Canine vs. Human Genome
• Sequence canine ESTs
• In collaboration with Elaine Ostrander (FHCRC)
  map to the dog genome
• Map computationally to the human genome
• Use to better annotate the human sequence
• Starting material for microarrays
• Use in gene discovery (behavior and cancer)

40
(No Transcript)
41
myosin, light polypeptide 4, alkali
42
How will genomics effect the way we do biological
research
43
Rate at which genes can be identified

• Cloning - weeks to years
• Database searches - seconds to minutes

44
What are the areas where genome technology will
impact us

• Diagnostics
• Forensics
• Understanding of diseases such as cancer at the
  molecular level
• Treatments for diseases customized to the
  individual

45
Genomic Information allows us to look at the
entire gene content of an organism simultaneously
46
gt 9 of the 10 Leading Causes of Mortality Have
Genetic Components

• 1. Heart disease (29.5 of deaths in 00)
• 2. Cancer (22.9)
• 3. Cerebrovascular diseases (6.9)
• 4. Chronic lower respiratory dis. (5.1)
• 5. Injury (3.9)
• 6. Diabetes (2.9)
• 7. Pneumonia/Influenza (2.8)
• 8. Alzheimer disease (2.0)
• 9. Kidney disease (1.6)
• 10. Septicemia (1.3)

47
Genomic Health Care

• About conditions partly
• Caused by mutation(s) in gene(s)
• e.g., breast cancer, colon cancer, autism,
  atherosclerosis, inflammatory bowel disease,
  diabetes, Alzheimer disease, mood disorders,
  etc., etc.
• Prevented by mutation(s) in gene(s)
• e.g., HIV (CCR5), ?atherosclerosis, ?cancers,
  ?diabetes , etc., etc.

48
Genomic Health Care

• Will change health care by...
• Creating a fundamental understanding of the
  biology of many diseases (and disabilities), even
  many non-genetic ones
• Helping to redefine illnesses by etiology rather
  than by symptomatology

49
Genomic Health Care

• Knowledge of individual genetic predispositions
  will allow
• Individualized screening
• Individualized behavior changes
• Presymptomatic medical therapies, e.g.,
  antihypertensive agents before hypertension
  develops, anti-mood disorder agents before mood
  disorder occurs

50
Crystal Ball - 2010

• Predictive genetic tests for 10 - 25 conditions
• Intervention to reduce risk for many of them
• Gene therapy for a few conditions
• Primary care providers begin to practice genetic
  medicine
• Preimplantation diagnosis widely available,
  limits fiercely debated
• Effective legislative solutions to genetic
  discrimination privacy in place in US
• Access remains inequitable, especially in
  developing world

51
Crystal Ball - 2020

• Gene-based designer drugs for diabetes,
  hypertension, etc. coming on the market
• Cancer therapy precisely targets molecular
  fingerprint of tumor
• Pharmacogenomic approach is standard approach for
  many drugs
• Mental illness diagnosis transformed, new
  therapies arriving, societal views shifting
• Homologous recombination technology suggests
  germline gene therapy could be safe

52
Crystal Ball - 2030

• Genes involved in aging fully cataloged
• Clinical trials underway to extend life span
• Full computer model of human cells replaces many
  laboratory experiments
• Complete genomic sequencing of an individual is
  routine, costs less than 100
• Major anti-technology movements active in US,
  elsewhere
• Worldwide inequities remain

53
Genomics

• May also change society
• Genetic stratification, e.g., in employment or
  marriage
• Genetic engineering against (and for) diseases
  and characteristics
• Cloning
• Increased opportunity for private eugenics

54
Genomics

• If we are all mutants, what is the definition of
  normal?

55
Conclusions

• Genomics will be the knowledge base or
  infrastructure for virtually all biology and
  medicine of the 21st century
• In silico biology will be a driving force in
  research and medicine
• Treatments for diseases will be radically
  improved by our understanding of complex diseases

56
Collaborators and Funding
Rob Martienssen Pablo Rabinowicz Lincoln
Stein Susan McCouch Steve Tanksley Rick
Wilson Marco Marra Elaine Mardis John
McPherson Bob Waterston The WUGSC Special thanks
to NHGRI for some of the slides used
Rod Wing and the CUGI Group Doug Cook Mike
Bevan Our ESSA-MIPS Collaborators Daphne
Preuss The AGI NSF, USDA, DOE NIH (NHGRI) and
NCI Monsanto, Westvaco, David Luke III
57
(No Transcript)
58

• It is now conceivable that our children's
  children will know the term cancer only as a
  constellation of stars.
• President Clinton at the White House, June 26,
  2000 announcing completion of the human genome
  draft sequence