{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T04:10:15Z","timestamp":1790136615273,"version":"4.0.1"},"reference-count":40,"publisher":"World Scientific Pub Co Pte Ltd","issue":"04","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J. Bioinform. Comput. Biol."],"published-print":{"date-parts":[[2026,8]]},"abstract":"<jats:p>\n                    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.\u00a0aureus Staphylococcus genomes and &gt;20,000 background bacterial genomes. Seven highly conserved, unannotated targets (SA-1\u2013SA-7) were identified, with all seven translated into qPCR primer sets (SAP-1\u2013SAP-7), among which three (SAP-1\u2013SAP-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.\u00a0aureus complex. In vitro testing using crude cell lysates confirmed specific amplification of S.\u00a0aureus DNA without non-target cross-reactivity, establishing a qualitative limit of detection (LOD) of 10\n                    <jats:sup>5<\/jats:sup>\n                    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.\n                  <\/jats:p>","DOI":"10.1142\/s0219720026500095","type":"journal-article","created":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T07:07:01Z","timestamp":1785481621000},"source":"Crossref","is-referenced-by-count":0,"title":["A scalable pan-genomic pipeline for annotation-free discovery of species-specific markers: Application to\n                    <i>Staphylococcus aureus<\/i>"],"prefix":"10.1142","volume":"24","author":[{"given":"Yuyang","family":"Zhou","sequence":"first","affiliation":[{"name":"College of Biological Science and Medical Engineering, Donghua University Shanghai 201620, P.\u00a0R.\u00a0China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiayi","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Biological 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