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Comprehensive DNA Signature Discovery and Validation 1* 2 1 1 3 3 Adam M. Phillippy , Jacquline A. Mason , Kunmi Ayanbule , Daniel D. Sommer , Elisa Taviani , Anwar Huq , Rita R. Colwell1,2, Ivor T. Knight1,2, Steven L. Salzberg1 1 Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, United States of America, 2 Canon U.S. Life Sciences, Rockville, Maryland, United States of America, 3 Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland, United States of America DNA signatures are nucleotide sequences that can be used to detect the presence of an organism and to distinguish that organism from all other species. Here we describe Insignia, a new, comprehensive system for the rapid identification of signatures in the genomes of bacteria and viruses. With the availability of hundreds of complete bacterial and viral genome sequences, it is now possible to use computational methods to identify signature sequences in all of these species, and to use these signatures as the basis for diagnostic assays to detect and genotype microbes in both environmental and clinical samples. The success of such assays critically depends on the methods used to identify signatures that properly differentiate between the target genomes and the sample background. We have used Insignia to compute accurate signatures for most bacterial genomes and made them available through our Web site. A sample of these signatures has been successfully tested on a set of 46 Vibrio cholerae strains, and the results indicate that the signatures are highly sensitive for detection as well as specific for discrimination between these strains and their near relatives. O