71. Systematic Conversion of a YAC/STS Map into a Sequence Ready BAC Map 

C. Han and N.A. Doggett 
Joint Genome Institute, Center for Human Genome Studies, Los Alamos National Laboratory, Los Alamos, NM 87545 
chan@telomere.lanl.gov 

We are starting with a previously constructed integrated physical map of human chromosome 16 (Doggett et al., Nature 377:Suppl:335-365, 1995) and converting this to a new sequence-ready BAC of this chromosome. The YAC/STS component of the integrated map consists of 900 CEPH megaYACs, and 300 flow-sorted 16-specific miniYACs that are localized to and ordered within somatic cell hybrid breakpoint intervals with 1150 STSs. This YAC/STS map provides nearly complete coverage of the euchromatic arms of the chromosome and provides STS markers on average every 78 kb. The integrated map also includes 470 genes/ESTs/exons, 400 genetic markers, and 530 cosmid contigs (110 kb average size, and covering 60% of the chromosome). To create large sequenceable targets of this chromosome we are using a systematic approach to screen high density BAC filters with evenly spaced probes. Probes are either pooled overlapping oligonucleotides (overgos, method developed by John McPherson, Wash U.). In order to select evenly spaced probes we first identified all available sequences in the integrated map. These include sequences from genes, ESTs, STSs, and cosmid end sequences. Since the integrated map was constructed on a physical scale we are able to select for sequences at a spacing of 50 kb - 100 kb when these were available. We then used BLAST to identify 36 bp unique fragments of DNA for overgo probes. Up to 236 overgos have been pooled in a single hybridization against a 12X coverage human BAC library (RPCI-11). Positive BACs that are identified from the pooled overgos are rearrayed on membranes and hybridized with either two-dimensional subpools of overgos to determine which BAC clones are positive for individual overgos. Probe-content BAC contigs are constructed in this manner. BAC contigs are then restriction mapped to select the optimal tiling sets for sequencing. Thus far we have identified over 6000 BACs from the chromosome 16 long arm and from an 11 Mb region of the short arm by the hybridization of 1,00 3 overgos. 35 Mb of BAC probe-content maps (by completion of the 2-dimensional hybridizations) and 10 Mb of sequence ready restriction maps have been constructed from these targets. Supported by the US DOE, OBER under contract W-7405-ENG-36.