S. MacMillan¹, C. Hott¹, D. Anderson¹, E. C. Rouchka², B. D. Dunford-Shore, B. Brownstein¹, R. Mazzarella², V. Nowotny², and D. J. States² 
Washington University in St. Louis, St. Louis, Missouri 
states@ibc.wustl.edu 

The human genome is polymorphic at all scales ranging from single nucleotide polymorphism to cytogenetically visible translocation spanning tens of megabases, but it remains difficult to characterize variation between these scales. We have proposed a method for screening for the presence of large-scale structural variants in the human genome. To demonstrate the feasibility of our strategy, STS markers derived from regions of finished genomic sequence are used to screen BAC and PAC libraries derived from 9 individuals with coverage in excess of 20 fold using a hierarchical multiplex hybridization and PCR approach. Recovered clones are subjected to both end-sequence analysis and four-enzyme restriction (RE) digest fingerprinting. End-sequence reads are aligned with the reference genomic sequence and their separation is compared with the molecular size of the clone as determined by the sum of the RE fragments sizes. The set of restriction digests predicted from the region spanned by the end-sequence alignments is compared with the experimental digests. Our method is validated by applying them towards verification of three BAC sequencing projects from the Chen laboratory, demonstrating a fingerprint sizing accuracy of better than 1% for bands with molecular weight between 1.2 and 15 kb. Successful fingerprints and end-sequence were generated for all clones. No false positive or false negative calls were identified in 302 bands scored for comparison. To date, 61 markers spanning 2 megabase of sequence (color vision, BRCA2, and TCR beta) have been screened retrieving 249 clones. 15 sites have been identified where multiple clone demonstrate a consistent pattern of deviation from the predicted digest pattern, including the presence of novel bands as well as the absence of predicted bands. To validate variations, a second tier of RE has been implement to further characterize these variants and PCR assays are being developed to test for the presence of these variants in uncloned genomic DNA.