Clone Pooling Methods for Physical Mapping

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DESCRIPTION: (provided by applicant) The main goal of this proposal is to develop and validate novel pooling-based methods for the rapid physical mapping of BAC libraries. The first method is Short-Tag Pooled Genomic Indexing ("ST-PGI") for comparative physical mapping of BACs of one species onto an assembled genome of another. The second method is Clone-Array Pooled Shotgun MAPping ("CAPS-MAP") method for direct (non-comparative) overlap detection between BACs. Pooled Genomic Indexing ("PGI") is a novel method for comparative physical mapping of BACs. PGI is carried out by pooling arrayed BAC clones, generating shotgun sequence pools to appropriate coverage, and by using this information to map individual BACs onto homologous sequences of a related organism. The first two steps, pooling of BAC clones and shotgun sequencing, draw on existing high-throughput methodology at the Baylor College of Medicine Human Genome Sequencing Center. The last step, deconvolution of the pools into specific BACs, is accomplished by comparison of DNA sequence reads from pools against a reference genomic sequence of a related organism. If one read from a row pool and one from a column pool match the reference sequence at a close distance, they are both assigned (deconvoluted) to the BAC at the intersection of the two pools and the BAC is mapped onto the region of the reference sequence between the two matches. When mapping closely related genomes, efficiency of PGI can be increased 4 to l2-fold by sequencing concatenamers comprising 50 to l50 bp sequence tags, a method which we refer to as Short-Tag PGI ("ST-PGI"). Clone-Arrayed Pooled Shotgun MAPping ("CAPS-MAP") is a method related to PGI. While both CAPS-MAP and PGI utilize essentially the same pooling experimental data, CAPS-MAP deconvolutes by assembling reads into contigs. A contig containing a read from a row and a column is deconvoluted to the BAC at the intersection. A contig deconvoluting to multiple BACs indicates BAC overlap. CAPS-MAP thus provides a direct, non-comparative mapping method complementary to PGI. This proposal aims to further develop ST-PGI and CAPS-MAP, and to validate experimentally and by simulation an integrated strategy for pooling-based physical mapping of multi-gigabase genomes.