1. TOPICS IN (NANO) BIOTECHNOLOGY
Human Genome Project
Lecture 12
27th November, 2006
PhD Course

2. Public Consortium
Celera Genomics

5. Remember what the genome is?
Human Genome organisation
Human genome contains ~ 40,000 genes
Nuclear genome 3000 Mb
30,000 to 40,000 structural genes
24 different types of DNA duplex
22 autosomes, 2 sex chromosomes
Human Genome
Nuclear
Mitochondrial

6. Let’s define it. DEFINITION:
The entire genetic makeup of the human cell nucleus.
Includes non-coding sequences located between genes, which makes up the vast majority of the DNA in the genome (~95%)

7. What is the Human Genome Project?
DEFINITION:
The Human Genome Project is a multi-year effort to find all of the genes on every chromosome in the human body and to determine their biochemical nature.
SPECIFIC GOALS:
Identify all the genes in human DNA
Determine the sequences of the 3 billion bps
Save the information in databases
Improve tools for data analysis
Transfer related technologies to the private sector
Address the ethical, legal and social issues that may arise from the project

8. Sequencing the Human Genome

9. Find Genes Sequence Genomes Genetic Mapping, Mutation Detection
Establish
Function and
Disease Mechanism
Diagnostics/
Prognostics
Drug
Candidates
Cure
Gene Therapy

10. “Sequence the 3 billion (+) base pairs of human DNA and identify all genes contained in the human genome”
DNA Sequence ‘Reads’ - Need 60,000,000 of them!!

11. Importance and Impact Why are genome projects important?
The key to continued development of molecular biology, genetics and molecular life sciences
a catalogue containing a description of the sequence of every gene in a genome is seen as immensely valuable, even if the function is not known
aid in isolation and utilisation of new genes
stretch technology to its limits
What is the potential impact?
Improved diagnosis/therapy of disease
prokaryotic genomes: vaccine design, exploration of new microbial energy sources
plant and animal genomes: enhance agriculture

12. The primary HGP sequencing sites
The Whitehead Institute for Biomedical Research (Eric Lander, Massachusetts, USA)
The Sanger Centre (Cambridge, GB)
Baylor College of Medicine (Richard Gibbs, Houston, USA)
Washington University (Robert Wayerston, St. Louis, USA)
DoEs Joint Genome Institute, JGI (Trevor Hawkins, Walnut Creek, California, USA)
…and other genome centres worldwide...

13. The Human Genome Project - Timelines -
President announces genome working draft completed
High Resolution
Maps of Specific
Chromosomes
Announced
Celera
Genomics
Formed
Conference on HGP Feasibility
HGP Officially
Begins
1st Human Chromosome
Sequenced
E.coli Genome
Completed
1985
1989
1991
1993
1995
1997
1999
2001
1987
1986
1988
1990
1992
1994
1996
1998
2000
Low Resolution
Linkage Map of HG
Published
Fly Genome
Completed
Congress
Recommends
15 year HGP
Project
S. cerevisiae
Genome
Completed
C. elegans
Genome
Completed
Human
Genome
Published
Science (Feb. 16, 2001) - Celera
Nature (Feb. 15, 2001) - HGP

14. History of Human Genome Project
1983 Los Alamos Labs and Lawrence Livermore National Labs, both under the DOE, begin production of DNA cosmid libraries for single chromosomes
1986 DOE announces HUMAN GENOME PROJECT
1987 DOE advisory committee recommends a 15-year multi-disciplinary undertaking to map and sequence the human genome. NIH begins funding of genome projects
1988 Recognition of need for concerted effort. HUGO founded (Human Genome Organisation) to coordinate international efforts DOE and NIH sign the Memorandum of Understanding outlining plans for co-operation

15. History of Human Genome Project
1990 DOE and NIH present joint 5-year Human Genome Project to Congress. The 15 year project formally begins
1991 Genome Database (GDB) established
1992 Low resolution genetic linkage map of entire human genome published, High resolution map of Y and chromosome 21 published
1993 DOE and NIH revise 5-year goals
IMAGE consortium established to co-ordinate efficient mapping and sequencing of gene-representing cDNAs (Integrated Molecular Analysis of Genomes and their Expression)

16. History of Human Genome Project
1994 Genetic-mapping 5-year goal achieved 1 year ahead of schedule
Genetic Privacy Act proposed to regulate collection, analysis, sorage and use of DNA samples (endorsed by ELSI)
Tons of stuff happens that continues to advance the project
1998 Celera Genomics formed
New 5-year plan by DOE and NIH
1999 First chromosome completely sequenced (Chromosome 22)
2000 June 6, HGP and Celera announce they had completed ~ 97% of the human genome.
2003 April 25, HGP finally completed

17. People of Human Genome Project
James Watson Original Head of HGP
Francis Collins
Craig Venter

18. DNA sequencing The Sanger dideoxy termination method (remember?)
Nucleotide analogs (ddNTP) are incorporated into DNA during its synthesis together with normal nucleotides (dNTP) - when a ddNTP is inserted, the reaction stops = chain termination
Radioactively labeled ddNTPs
four different reactions are performed, each reaction contains ddA, ddG, ddC, ddT
Autoradiography enable analysis of different fragment lengths which correspond to different termination points
Fluorescently labeled ddNTPS
one reaction carried out, all four ddNTPs are incorporated but each ddNTP is labelled with a different fluourescent dye
automated DNA sequencers interfaced with computers determine the order of the dyes and hence the DNA sequence

19. Mapping the Human Genome:
Low Resolution Mapping
The Gene Linkage Map
Identifies position of genes by locating marker base sequences associated with RFLPs
Based on how close together two genes are
the closer together two genes are, the less likely they are to separate during meiotic recombination in germ cells
the frequency of recombination between two genes can help to decipher the distance between them on a gene linkage map
genes separated by more than 50cM (50 million bps) are not considered linked
Studies of families affected by genetic disease have proven useful for genetic linkage analysis

20. Mapping the Human Genome:
High Resolution Mapping
The Physical Map
Provides the actual distances in bps between genes on a given chromosome
Prepared by aligning the sequences of adjacent DNA fragments from small overlapping clones to form a contiguous map (a contig map)
Sequence tag sites (STS) mark sites on chromosomes and help to locate adjacent segments of DNA
if two DNA fragments share an STS they overlap and are contiguous

21. Mapping the Human Genome:
High Resolution Mapping
Sequence Tagged Sites (STS)
Sequences occurring only once in the human genome
Help to map locations
52,000 STS in Humans
~ 1 every 62,000 bases

22. Hierarchical (Clone-based) Approach
Know location of 30,000 – 100,000 bp region
Break into bp fragments
Sequence Fragments
Assemble based on similarity
~8-10x coverage
Current Price: $0.09 / base

23. Hierarchical (clone-based) approach
generate overlapping set of clones
select a minimum tiling path
shotgun sequence each clone

24. Hierarchical (clone-based) approach
DISADVs
map generation requires resources, time and money
Some regions not cloned
ADVs
easier to assemble smaller pieces
less chance for assembly error

25. Determining genome sequences
The aim, obviously, is to determine the entire genome sequence
A sequence has to be constructed from a series of shorter fragments
Shotgun technique
break molecule into smaller fragments
determine sequence of each one
use a computer to search for overlaps and build a master sequence

26. Shotgun Sequencing Approach
Developed 1991 TIGR
Craig Venter, Hamilton Smith
Break genome into millions of pieces
Sequence each piece
Reassemble into full genomes

27. Whole Genome Shotgun Approach
reads generated directly from a whole-genome library
assemble the genome all at once

28. Whole Genome Shotgun Approach
DISADVs
more prone to assembly error
computationally intensive
cannot effectively handle repeats
ADVs
Less overhead time up front

29. Chromosome walking
Analysis of DNA sequences of chromosomes by extending the sequenced region a little bit further each time until the tips of the chromosome are reached
The next round of sequencing is based on the results of the previous round by synthesising appropriate DNA primers to extend further

30. Base calling and Assembly Software
PHRED and PHRAP Developed (1988)
PHRED: Base calling software
PHRAP: Assists in assembly of sequenced data

31. Available Assemblers SEQAID (Peltola et al., 1984) CAP (Huang, 1992)
PHRAP (Green, 1994)
TIGR Assembler (Sutton et al., 1995)
AMASS (Kim et al., 1999)
CAP3 (Huang and Madan, 1999)
Celera Assembler (Myers et al., 2000)
EULER (Pevzner et al., 2001)
ARACHNE (Batzoglou et al., 2002)

32. Growth of GenBank 1982: 600,000 Bases 2002: 28.5 Billion Bases
Image source:

33. Results of Human Genome Project
The International Human Genome Sequencing Consortium published their results in Nature, 409(6822): , 2001
Initial Sequencing and Analysis of the Human Genome
Celera Genomics published their results in Science, 291(5507), , 2001
The Sequence of the Human Genome

34. Results of Human Genome Project
The Human genome contains million bases
The average gene size is 3,000 bases
Total number of genes is between 30-40,000
The order of 99.9% of the nucleotides is the same in all people
Of the discovered genes, the function for more than half is unknown
> 30 genes have already been associated with human disease (e.g. Cancer, blindness)

35. Results of Human Genome Project
About 2% of the genome encodes instructions for the synthesis of proteins
Repeated sequences make up 50% of the genome
There are urban centres that are gene rich: stretches of C and G bases repeats (CpG islands) occur adjacent to gene rich areas
Chromosome 1 has 2,968 genes; the Y has 231
Humans:
only twice number of genes of the fly
3 times as many proteins as fly or worm
share the same gene families as fly or worm

36. Completed genomes Microbial genomes Insect genomes
Haemophilus influenzae
Escherichia coli
Bacillus subtilus
Helicobacter pylori
Streptococcus pneumonaie
Saacharomyces cerevisiae
Archaeglobus fulgidus
Methanbacterium thermoautotropicum
Methanococcus jannaschil
Mycobacterium tubercolosis
Staphylococcus aureus
and more…..
Insect genomes
Arabidopsis thaliana
Drosophilia melanogaster
Mus musculus

37. Results of Human Genome Project
Organism
Genome Size (Bases)
Estimated Genes
Human (Homo sapiens)
3 billion
30,000
Laboratory mouse (M. musculus)
2.6 billion
Mustard weed (A. thaliana)
100 million
25,000
Roundworm (C. elegans)
97 million
19,000
Fruit fly (D. melanogaster)
137 million
13,000
Yeast (S. cerevisiae)
12.1 million
6,000
Bacterium (E. coli)
4.6 million
3,200
Human immunodeficiency virus (HIV)
9700 9
Video_1

38. Ethical, legal and societal issues
The DOE and the NIH spend between 3-5% of their annual HGP budgets toward studying the ELSI associated with availability of genetic information
This budget is the world’s largest bioethics program, and has become a worldwide model
Examples of ELSI are:
privacy legislation
gene testing
patenting
forensics
behavioural genetics
genetics in the courtroom

39. Societal Concerns Who should have access to this information?
Employers
Insurers
Schools
Courts
Adoption agencies
Military
Philosophical Implications
Human responsibility
Free will versus genetic determinism
Who owns and controls genetic information?
How is privacy and confidentiality managed?
Psychological impact and stigmatisation
Effects on the individual
Effects on society’s perceptions and expectations of the individual

40. Clinical Issues Clinical Issues Genetic Counselling
Growing demand to educate health care workers
Public needs to gain scientific literary and understand the capabilities, limitations and risks
Standards need to be established including quality controls to ensure accuracy and reliability
Regulations?
Genetic Counselling
Informed consent for complex procedures
Counseling about risks, limitations and reliability of genetic screening techniques
Reproductive decision making based on genetic information
Reproductive rights
Multifactorial diseases and environmental factors
Genetic predispositions do not mandate disease development
Caution must be exercised when correlating genetic tests with predictions

41. Commercialisation and patents
Who owns genes and DNA sequences?
The person (or company) who discovered it, or the person whose body it came from
Should genetic information be the property of humanity?
Is it ethical to charge someone for access to a database of genetic information?
Is it time to raise the bar concerning patents?
Will patent protection slow the advance of research and be detrimental to society as a whole in the long run

42. Microarray Technology
Benefits of Human Genome Project
Agriculture & Bioremediation Industries
Diagnostic & therapeutic applications
Gene therapy applications
Medicine & pharmaceutical industries
Preventative measures
Biotechnology
Medicine
Bioinformatics
Proteomics
Microarray Technology
DNA chip technology
Developmental Biology
Evolutionary & Comparative Biologists
Pharmacogenomics

43. Single nucleotide polymorphisms
These occur when a single nucleotide in the genome sequence is altered (1 bp difference)
66% of SNPs involve a C to T change and they occur every bases in either coding or non-coding regions
Evolutionary stable, there are between 2 and 3 million SNPs in the human genome
Many SNPs have no effect on cell function, but:
some SNPs could be responsible for variations in how many humans respond to disease, environmental factors, drugs and other therapies
SNPs may help identify multiple genes involved in complex diseases

44. Single nucleotide polymorphisms
SNPs are NOT the same things as alleles (or so we believe so far)
Researchers have found that most SNPs are not responsible for a disease state
They serve as markers for pinpointing a disease on the human genome map, being located near a gene found to be associated with a certain disease
Occasionally, SNPs may actually cause a disease and can to be used to search for and isolate the disease-causing gene
SNPs travel together - i.e. Variations in DNA are linked
To date, Celera & Orchid Biosciences have largest databases

45. Single nucleotide polymorphisms
Goals:
Develop large scale technologies
Identify common variants in the coding regions
Create a SNP of at least 100,000 markers
Develop the intellectual foundation for studies of sequence variation
Create public resources of DNA samples and cell lines
SNP Consortium:
Ten large pharmaceutical companies and the UK Wellcome Trust
Headed by Arthur Holden
Find and map 300,000 common SNPs
Generate a widely accepted, high-quality, publically available map