DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

152. Optical Mapping: A Complete System For Whole Genome Shotgun Mapping

Anantharaman, T, Apodaca, J., Aston, C., Clarke, V., Gebauer, D., Delobette, S., Dimalanta, E., Edington, J., Giacalone, J., Gibaja, V., Huff, E., Jing, J., Lai, Z., Lin, J., Limm, A., Mishra, B., Ni, L., Paxia, S., Qi, R., Ramanathan, A., Skiadis, Y., Vafai, J., Wang, W., Schwartz, D.C.

University of Wisconsin - Madison

Optical Mapping is a single molecule approach for the rapid production of ordered restriction maps from single DNA molecules. Fluorescence microscopy is used to image individual DNA molecules bound to derivatized glass surfaces, and cleaved by restriction enzymes. Fragments retain their original order, and cut sites are flagged by small, visible gaps. The system has advanced in several critical areas for mapping both clones and entire genomes (D. radiodurans and P. falciparum). We mapped these entire microbial genomes using megabased-sized genomic DNA molecules. Because large fragments of randomly sheared DNA are mapped with high cutting efficiency, many overlapping restriction site landmarks allow contigs to be assembled and a shotgun mapping strategy can be employed. High resolution whole genome maps can therefore be assembled without library construction and associated cloning artifacts. Because ensembles of single molecules are analyzed, small amounts of starting material are required enabling mapping of microorganisms which are problematic to culture. Whole genome maps firstly, enable the size of the genome to be accurately determined, an important prelude to any sequencing endeavor and secondly, provide an in situ picture of the architecture of the entire genome, revealing the number of chromosomes, extrachromosomal elements etc. Populations can be potentially be characterized by comparing maps from different strains. Recent efforts have been to create maps of E. coli O157:H7 (5.4 mgb) as a scaffold for facilitated sequence assembly and verification (Collaborator: F. Blattner, U. Wisconsin). We will compare maps generated from the sequence of E. coli K12 (4.6 mgb) to identify regions unique to O157 which could be targetted for sequencing. Notably, we have constructed a map of the whole human genome at a coverage of 0.6X showing feasibility of complete mapping of the human genome. To map megabase-sized molecules, we created a system to tile overlapping microscope images, with proper pixel registration. "Gentig" then automatically generates contigs from optical mapping data by repeatedly combining the two islands that produce the greatest increase in probability density, excluding any contigs whose false positive overlap probability is unacceptable. The standard deviation, digestion rate, false cut rate and false match possibility can be altered to change the number of molecules that "Gentig" contigs together. Visualization of such information-rich data; whole chromosome maps composed of many restriction sites and deep contigs, presents a challenge. "ConVEx" (Contig Visualizer and Expander) creates contigs from maps and uses a scalable viewer to visualize assemblies for editing. "ConVEx" is a zoomable interface which allows annotation and integration of other related information such as STS markers, sequence contigs and even sequence reads. "ConVex" is built on top of PAD++, which can be run on all major operating systems.