GenomicGapID: leveraging spatial distribution of conserved genomic sites for broad-spectrum microbial identification

Bacterial detection and identification methods can be broadly classified as either untargeted with expansive taxonomic coverage or targeted with narrow taxonomic focus. Untargeted approaches, such as culture and sequencing, are often time-consuming and/or costly, whereas targeted methods, such as PCR, can offer faster and more cost-effective results but require <i>a priori</i> knowledge of the likely pathogen to select the appropriate assay. GenomicGapID, a novel approach that leverages the spatial distribution of conserved genetic regions across microbial genomes, represents a significant advancement in the field of microbial identification. This technique has the potential to provide the taxonomic breadth of culture and sequencing while maintaining the speed, simplicity, and cost-effectiveness of PCR. By leveraging the conservation and relative positioning of highly conserved coding regions across different species, GenomicGapID enables the development of universal primer sets that amplify the non-conserved gaps between these regions. This creates a unique electrophoretic signature that facilitates rapid and accurate target-agnostic microbial identification. In this study, we apply the principles of GenomicGapID to the critical task of identifying clinical pathogens. We focus on expanding the coverage of a previously developed universal bacterial identification system, which initially targeted the 16s-23s internal transcribed spacer (ITS) region and was capable of discerning 45 pathogens. To enhance this system, we assembled a comprehensive database of 189 clinically relevant bacterial species. We then identified conserved primer binding sites that produce unique amplicon size signatures for each species. While we found that the use of amplicon size signatures alone would require an impractical number of universal primer sets, we demonstrate that this challenge can be effectively mitigated through concurrent melt analysis. Ultimately, we show that just three universal primer sets, guided by the GenomicGapID framework, are sufficient to cover 189 clinical bacterial pathogens, representing a majority of microbes identified in positive cultures in a clinical microbiology setting, with experimental validation of a subset of these pathogens. This study not only enhances the existing universal bacterial identification system but also establishes GenomicGapID as a versatile and powerful tool in microbial diagnostics and beyond, paving the way for new avenues of research in genomics with the potential to advance molecular biology, clinical practice, and public health.IMPORTANCERapid and accurate microbial identification is critical in both clinical and research settings. Traditional untargeted methods, such as culture and sequencing, are often time-consuming and expensive, while targeted techniques like PCR offer speed and cost-effectiveness but require pre-selection of pathogens. Our work introduces GenomicGapID, a novel bacterial identification system that provides the taxonomic breadth of untargeted methods, coupled with the speed, simplicity, and affordability of targeted PCR-based techniques. By leveraging the gap between conserved genetic regions and analyzing the associated unique electrophoretic and melt analysis signatures, GenomicGapID enables target-agnostic bacterial identification using a parsimonious set of universal primers.Our work has significant implications not only in clinical microbiology but also in genomics, environmental microbiology, and public health. We believe this manuscript aligns well with the mission of Microbiology Spectrum to publish innovative and impactful research that advances the field of microbial sciences.