Physical map and gene survey of the Ochrobactrum anthropi genome using bacterial artificial chromosome contigs.

Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes research aimed at determining the genome structure of Ochrobactrum anthropi, an opportunistic human pathogen that has potential applications in biodegradation of hazardous organic compounds. A BAC library for O. anthropi was constructed that provides a 70-fold genome coverage based on an estimated genome size of 4.8 Mb. The library contains 3072 clones with an average insert size of 112 kb. High-density colony filters of the library were made, and a physical map of the genome was constructed using a hybridization without replacement strategy. In addition, 1536 BAC clones were fingerprinted with HindIII and analyzed using IMAGE and Fingerprint Contig software (FPC, Sanger Centre, U.K.). The FPC results supported the hybridization data, resulting in the formation of two major contigs representing the two major replicons of the O. anthropi genome. After determining a reduced tiling path, 138 BAC ends from the reduced tile were sequenced for a preliminary gene survey. A search of the public databases with the BLASTX algorithm resulted in 77 strong hits (E-value < 0.001), of which 89% showed similarity to a wide variety of prokaryotic genes. These results provide a contig-based physical map to assist the cloning of important genomic regions and the potential sequencing of the O. anthropi genome.

An algorithm for the assembly of robust physical maps based on a combination of multi-level hybridization data and fingerprinting data.

We have developed an algorithm which combines data obtained from restriction digestion experiments and hybridization experiments to construct robust physical maps of whole chromosomes. The algorithm has been incorporated into a program which accepts hybridization data consisting of an unordered hybridization matrix and fingerprinting data containing band coordinates for each clone. The combined data is used to produce a non-redundant, ordered matrix which can be further reduced to represent a minimum tile coverage of the chromosome. In addition, the method also takes into account multi-level hybridization events which allows for an improved treatment of the hybridization data. The program is evaluated against several other contig building programs using simulated and real data sets. Finally, it is applied to construct a physical map of the 4.1 Mb genome of Ochrobactrum anthropi based on 1387 clones and 70 probes, as well as 624 fingerprints.