Genome sequencing

Size of human genome

 23 pairs of chromosomes
 3.1 billion bp
 If code written in NYC phone books and
stacked up would reach top of
Washington monument.
Human Genome Project
 Began as a academic effort
 Initially involved 5 research centers in
US and England.
 Soon joined by Celera, spin off
company.
Some surprises
 Initial estimate 100,000 to 150,000
genes but found to be 35,000 to
50,000. (C. elegans ~19,000 genes)
 Mass of genome that codes for protein
originally estimated as 5% but found to
be 1.5%.
Some completely sequenced
genomes
 Mycoplasma genetialium
 578,000 bp, 400 genes
 Haemophilus influenza
 1,830,138 bp, 1738 genes
 E. coli
 4,639,221 bp, 4377 genes
 S. cervisiae
 12 x 106 bp, 5885 genes
More genomes
 C. elegans
 95.5 x 106, 19,820 genes
 D. melanogaster
 1.8 x 108, 13,601 genes
 A. thaliana
 1.17 x 108, 25, 498 genes
More genomes
 M. musculus
 3 x 109, ~30,000 genes
 H. sapiens
 3.3 X 109, 30-50,000 genes
 O. sativa
 4.3 x 108, 30-63,000 genes
The beginning
 Human genome project initially
discussed at a UC-Santa Cruz meeting
in 1985.
What were the concerns?
 What will it do to biology?
 How will be pay for it?
 Is this really science?
 Why bother to sequence it all?
 all vs. just the genes (skim sequencing)
Dept. of Energy
 Initially funded project in 1987.
 $5.3 million
 Study radiation induced mutations,
repair and effect on humans.
NIH
 Joined in 1988.
 James Watson leader
 3% of research budget devoted to
examining the ethical, legal, and social
implications of gene research (ELSI)
Other genomes
 Parallel sequencing of E. coli, S.
cerevisiae, C. elegans, D. melanogaster ,
and M. musculus
 Why
 Work out the technology and methods
Watson’s vision
 Sequence it all not just genes.
 Use genetic maps and markers to help
assemble the pieces.
Academic players
 Wash U
 Baylor
 Whitehead
 Wellcome Trust
 Joint Genome Institute—DOE Center
$1 to 10 cents a finished bp
 automated processing of cloned DNA
 automated DNA sequencing
 computer system to support sequence
data
 algorithms to assess sequence fidelity,
assemble sequences, and “find” genes.
Maps
 Thomas Hunt Morgan (early 1900s)—
low resolution phenotypic markers
 1970s restriction maps
 1980s RFLPs
 1989 Maynard Olson, Leroy Hood,
Charles Cantor, and David Botstein
sequence itself is a marker! (STS)
PCR
 Polymerase Chain Reaction
 [Link]
 Techniques
 Amplifying
 Making copies of DNA
The PCR revolution
 1985
 Kary Mullis-Cetus Corporation
 No need to send clones back and forth
 Allowed automated DNA sequencing
 No need for large clone repositiory for
all human genes
 Unrestricted access to genes via public
sequence databases.
Kary Mullis talks about PCR
 [Link]
 Techniques
 Amplifying
 Interviews
 Making DNA copies
 Naming PCR
Sequencing-the old way
 Maxim and Gilbert or Sanger methods
 [Link]
 Techniques
 Sorting and Amplifying
 Early DNA sequencing
 [Link]
 Techniques
 Sorting and Amplifying
 Interviews
 Dideoxy method of sequencing
Automated Sequencing
 Automation made possible by new dye
chemistry developed by Leroy Hood and
Lloyd Smith at Cal. Inst. Tech. in 1986.
 [Link]
 Techniques
 Sorting and Amplifying
 Cycle Sequencing
Inside the automated sequencer
 Collaboration with ABI produced first
automated sequencer.
 Laser detection of each bp.
 [Link]
 Techniques
 Sorting and Amplifying
 Interviews
 Making sequencing automated

 Inside an automated sequencer

Sequencing
 Detect all 4 nucleotides in one lane so
quadrupled the output from a single
sequencing gel.
 Dupont dye terminators—allowed all
four nucleotides to be attached to
terminal nucleotide in the same
sequencing reaction.
 Capillary eliminated need to cast gels.
Sequencing the Genome an
Overview
 Show [Link] file containing
movie about sequencing the human
genome.
Two approaches to sequence the
genome
 Hierarchical Shotgun clone libraries
 Use map to pick pieces of genome in order,
break them, sequence and reassemble.
(Watson)
 Whole genome shotgun
 Break up genomic DNA randomly,
sequence several genome equivalents, and
reassemble. (Ventner)
Hierarchical Shotgun Clone
Libraries
 Top-down strategy
 Ordered library of clones based on large
scale maps.
 Subclone larger inserts into sequencing
vector.
 Reassemble sequence.
 Based on order.
ESTs
 Expressed sequence tags
 Reverse transcribe mRNA and
sequence.
 Venter used nonspecific primer to
randomly amplify 150-400 bp fragments
of genes.
Patent controversy
 NIH announced it would seed a patent
on Venter’s STS.
 Very controversial since functionally
unknown.
 More appropriate to private company.
 Watson said it was “sheer lunacy” and
resigned due to conflict with Bernardine
Healy NIH director.
More patent
 Many biotech companies arose at the
time to mine ESTs and applied for
patents on the genes for diagnostics
and pharmaceuticals.
 NIH withdrew patent application.
 ESTs must be novel to be patented.
 ESTs must be useful to be patented.
The result
 No patents granted thus far on genes
without known function.
Whole genome shotgun
 Break the genome into a bunch of pieces
often by mechanical shearing.
 Sequence pieces and reassemble.
 Weber (Marshfield Medical Research
Foundation) and Myers (U of AZ) proposed
method to speed sequencing.
 1998 Venter leaves NIH to head Celera and
promised to sequence human genome in 3
years for $300 million.
 Accelerated the public project.
 Whole genome method was tested by
sequencing 120 Mbp of Drosophila
genome.