69. One Tier Pooling of a Total Genomic BAC Library 

D.C. Torney, J.L. Longmire, D.C. Bruce, J. Fawcett, M. Campbell, J. Tesmer, M. Maltbie, B. Taggett, T. Tatum, P. Jewett, J. Meyne, N. Lenhert, Y. Valdez, S. Bailey, A. Schliep¹, L.L. Deaven, and N.A. Doggett 
Life Sciences Division and Center for Human Genome Studies, Los Alamos National Laboratory, Los Alamos, NM 87545 and ¹University of Cologne, Cologne, Germany 
doggett@gnome.lanl.gov 

We have developed a single-tier pooling approach which enables the screening of a 12X diploid human genomic BAC library of 221,184 clones, 165 kb average insert size (one-half of the total 24X RPCI-11 library, http://bacpac.med.buffalo.edu) in a single screen of 376 PCR reactions, followed by confirmatory reactions of the predicted positive BAC clones. Prior to pooling of the library, the 384 well library stock plates were translated to 96 well plates. Pooling of the library was performed in increments of a quarter at a time: each a 3x coverage containing 55,296 clones. Ninety-four pools were made from each quarter of the library including two sets of plate pools (11 pools each), two sets of row pools (12 pools each), two sets of column pools (12 pools each), and one set of left and right diagonal pools (12 pools each). Each of these eight sets of pools was made from a plate-rearranged 24x24 array of 96-well microtitre dishes. Thus, most pools contain 4,608 clones. The plate rearrangements were selected to minimize the numbers of co-incidences of pairs of clones in pools. For the row, column, and diagonal pools, the "lines" of clones from the array are combined to make the final 12 pools, with no pool containing more than one line incident on any plate. Pool construction was accomplished, straightforwardly, with manifolds which have been designed to work on the Robbins Hydra (manuscript in preparation). Prior to pooling, the performance of the single-tier design (in the presence of experimental errors) was simulated. As with real data, the Markov chain Monte Carlo procedure was used to rank the candidate positives. Results compared favorably with a random 8-sets pooling design (Knill et al., J. Comp. Biol., 3, 395-406 (1996), and Bruno et al., Genomics, 26, 21-30 (1995)). We will present results from screening the pools with STS primer pairs that demonstrate that these single-tier pools will serve as valuable resources for rapidly isolating BAC clones for mapping and sequencing. Supported by the U.S. D.O.E. Office of Biological and Environmental Research under contract W-7405-ENG-36.