Sequencing the Whole Human Genome -
Classical Strategies and New Ones

Introduction (Brian Fox)

1. Molecular biology tools
2. Sequence Tagged Sites - STS
3. Small scale sequencing reactions
4. Putting it all together
5. Progress

The BAC-end strategy (Nitin Sharma)

1. Problems with the classical approach
2. Description
3. Efficiency and cost analysis

The papers:

Venter, J. C., Smith, H. O., Hood, L. (1996) "A New Strategy for Genome Sequencing." Nature 381, 364-367.

Karp, R. M., Shamir, R. (1998) "Optimizing the BAC-End Strategy for Sequencing the Human Genome." In preparation.

Useful web resources:

Human Genome Project Information http://www.ornl.gov/hgmis/

Tutorial describing genome project: http://www.genome.washington.edu/UWGC/tutorial/

Web page for Natl. Human Genome Research Inst. http://www.nhgri.nih.gov/

Database of STSs http://www.ncbi.nlm.nih.gov/dbSTS/

Intro: Molecular biology tools

"Cloning" The process of taking a particular piece of DNA and inserting into another organism (bacteria, virus or yeast), which can faithfully replicate the foreign DNA.

The piece of foreign DNA can be in many sizes, depending on the biological system used.

"Library" - A collection of clones (virus, bacteria, or yeast) which each has a different DNA sequence in their vector. A genomic library has all the human DNA (3 Gb) represented in all of the clones of the collection. A library can be cataloged/sorted/copied/distributed …

Intro: Sequence Tagged Sites

Definition: A unique DNA sequence (200-500 bp) which has been localized to one location in the genome.

Described as a "common language for physical mapping of the human genome." Olsen, et. al. Science 245 (1989) 1434.

Can use a series of STSs to identify and order clones from a library.

Intro: Small scale sequencing reactions

• DNA sequencing techniques need: (1) pure, (2) homogenous, (3) single-stranded DNA with a (4) known start sequence, and a length (5) between 500 and 1000 bases.

• Solution: Use M13 phage as the vector. When a bacteria has M13 DNA inside, it will secrete virus particles into the media which have the single stranded DNA of interest inside the capsid.

• After removing the bacterial cells from the media (centrifugation), one can break down the protein coat of the virus, and purify the ssDNA for a sequencing rxn.

Intro: Putting it all together

Overall strategy:

• Make a YAC and cosmid genomic library.
• Low resolution map (ordering of YACs, 30,000 STS, every 100kb).
• High resolution map (ordering of cosmids).
• Shotgun sequencing of each minimally overlapping cosmid.
• Reconstruction of order of M13 clones for each cosmid.

Progress:

First 5-year plan established in 1990, revised in 1993.

• estimated 15 years to complete
• cost over $3 billion
• planned on new technologies to greatly increase efficiency of sequencing strategies

Current Plan, 1998:

• STS mapping completed.
• Sequence 500 Mb/year at $0.25/base.
• Finish sequencing by 2003 (50 years after Watson, Crick discovered structure of DNA).