Compositions and methods for rapid and highly efficient characterization of genetic diversity in organisms are provided. The methods involve rapid sequencing and characterization of extrachromosomal DNA, particularly plasmids, to identify useful nucleotide sequences. The method involves generating a library of extrachromosomal DNA clones, sequencing a portion of the clones, comparing the sequences against a database of existing DNA sequences, using an algorithm to select novel nucleotide sequences based on the presence or absence of the sequence in a database, and identification of at least one novel nucleotide sequence. The DNA sequence can also be translated in all six frames and the resulting amino acid sequences compared against a database of protein sequences. Organisms of particular interest include, but are not limited to bacteria, fungi, algae, and the like. Compositions comprise a mini-cosmid vector comprising a stuffer fragment and at least one cos site.