Comprehensive DNA signature discovery and validation

Abstract

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. Our approach, whereby the entire genomic complement of organisms are compared to identify probe targets, is a promising method for diagnostic assay development, and it provides assay designers with the flexibility to choose probes from the most relevant genes or genomic regions. The Insignia system is freely accessible via a Web interface and has been released as open source software at: http://insignia.cbcb.umd.edu.

Figure 1. Inclusive TaqMan Assay Displaying Increased Fluorescence due to Target Amplification for All 46 V. cholerae Strains Tested, and No Fluorescent Activity among the E. coli Negative Controls

Relative florescence intensity for 40 PCR cycles is shown.

Figure 2. Exclusive TaqMan Assay Displaying Increased Fluorescent Activity for the Reference Strain of V. cholerae and No Fluorescent Activity among the 23 Non-Cholera Strains

Relative florescence intensity for 50 PCR cycles is shown.

Figure 3. TaqMan Validation Results for the 50 Assay Designs Tested on 46 V. cholerae, 22 Near Neighbors, and One E. coli Control

Organisms are grouped vertically, and assays are sorted horizontally by effectiveness. Each colored box represents the Ct value for one of the 3,450 validation experiments. For example, assays 1–5 show strong amplification for all V. cholerae strains and heavily delayed or failed amplification for all other organisms.

Figure 4. A Match Cover (M_tb) Constructed from the Exact Matches between a Target (t) and Background (b) Genome

M_tb intervals (red boxes) represent regions of the target with a contiguous match to the background (gray boxes).

Figure 5. Shared k-mers (Red Lines) Obtained from an Intersection (I_r) of Three Match Covers M_rs, M_rt, M_ru

I_r intervals (red boxes) represent regions of the reference r shared with all other target genomes s, t, u as derived from the match covers between the reference and each target (gray boxes). k-mers not contained by I_r are not shared by all targets (dotted gray lines).

Figure 6. Unique k-mers (Red Lines) Obtained from a Union (U_r) of Three Match Covers M_ra, M_rb, M_rc

U_r intervals (red boxes) represent regions of the reference r matching some background genome a, b, c as derived from the match covers between the reference and each background (gray boxes). k-mers contained by U_r match the background and are not unique (dotted gray lines).