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Large-Scale BAC End Sequencing to Aid Sequence-Ready Map Construction

Mark D. Adams, Steve Rounsley, Casey Field, Jenny Kelley, Steve Bass, Brook Craven, and J. Craig Venter
The Institute for Genomic Research, Rockville, MD 20850 mdadams@tigr.org

As the genome project shifts into the large-scale sequencing phase, an overwhelming technical challenge resides in developing an efficient method for producing minimum tiling paths of sequence-ready clones across the entire genome. To illustrate this challenge, consider what a sequencing center that proposes to finish 100 megabases (Mb) of DNA sequence per year must do: on average, each day 2-3 bacterial artificial chromosomes (BACs), averaging 150 kilobases (kbp) in length each, must be sequenced.

Deep representation BAC libraries are currently being developed in Dr. Mel Simon's laboratory at CalTech and in Dr. Pieter de Jong's laboratory at the Roswell Park Cancer Institute. The DNA for preparation of these libraries was prepared from volunteer donors who had given fully informed consent to use of their DNA for the human genome project. Extensive provisions for maintaining the anonymity of the donors are in place. These libraries will form the core resource used by sequencing centers in the US and around the world for sequencing the human genome.

BAC end sequencing is used to precisely mark the position of each BAC clone relatively to completely sequenced BACs. Exhaustive end-sequencing of BAC clones from 16p is currently underway as is a larger project to end-sequence up to 300,000 BAC clones to provide markers for construction of sequence-ready maps across the genome.

We are in the midst of obtaining end-sequence data from each of 300,000 clones from these new libraries. These end sequences will be invaluable for making highly efficient use of these clones to construct sequence-ready physical maps and to select clones for sequencing. At the proposed level of redundancy (about 15 genome equivalents), the end sequences will provide a sequence marker of 300-500 base pairs (bp) on average every 5,000 bp across the genome. Currently over 13,000 BAC end sequences have been produced and the sequencing rate is continuing to increase.

A 96-well mini-prep method has been developed that permits rapid purification of BAC DNA with sufficient quantity and purity for direct sequencing. Use of new BigDye terminator chemistry from ABI has made BAC end-sequencing quite robust.

Last modified: Wednesday, October 22, 2003

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