A scalable pan-genomic pipeline for annotation-free discovery of species-specific markers: Application to Staphylococcus aureus.

Current bioinformatics approaches for bacterial diagnostic target discovery remain constrained by their reliance on gene annotations and fixed-boundary genome segmentation, which overlook unannotated intergenic regions and introduce sequence-truncation artifacts. Here, we developed an open-source, annotation-independent pan-genomic pipeline featuring an overlapping sliding-window algorithm (500-bp window, 100-bp step) and a three-tier subtractive screening funnel. Using Staphylococcus aureus as a model, the pipeline screened 1629 genomes against 852 non-S. aureus Staphylococcus genomes and >20,000 background bacterial genomes. Seven highly conserved, unannotated targets (SA-1-SA-7) were identified, with all seven translated into qPCR primer sets (SAP-1-SAP-7), among which three (SAP-1-SAP-3) were further characterized by in vitro experiments. Multi-layer in silico evaluation demonstrated 100% intraspecific sensitivity and zero cross-reactivity against background genomes, including the S. aureus complex. In vitro testing using crude cell lysates confirmed specific amplification of S. aureus DNA without non-target cross-reactivity, establishing a qualitative limit of detection (LOD) of 105 CFU/mL. Additional computational validation on draft genomes, raw sequencing reads, near-neighbor species, and a clinical truth set corroborated marker robustness under realistic conditions. This framework successfully circumvents conventional gene-centric limitations, providing a generalizable computational strategy for target discovery across other high-priority bacterial pathogens.