Whole-genome and pan-genome analyses reveal genomic differences among nontypeable

Background: Nontypeable Methods: We performed whole-genome sequencing and comparative genomic analysis of 27 NTHi isolates, including strains derived from patients with bronchiectasis (n = 10), community-acquired pneumonia (CAP; n = 6), and chronic obstructive pulmonary disease (COPD; n = 11). Among these, three isolates (one from each disease group) were newly sequenced using a hybrid Oxford Nanopore-Illumina approach, while the remaining 24 genomes were retrieved from public databases. Pan-genome analysis, multilocus sequence typing (MLST), core genome phylogenetic analysis, accessory genome based discriminant analysis, and pan-genome-wide association analysis (pan-GWAS) were performed to characterize genomic diversity and variation in gene content across isolates. Results: MLST and core genome phylogenetic analyses revealed substantial genetic diversity, with isolates from bronchiectasis, CAP, and COPD distributed across multiple lineages without clear disease-specific clustering. Accessory genome-based analyses indicated heterogeneous differences in gene content among isolates from different clinical backgrounds, although overlap between groups remained evident. Pan-genome-wide association analysis did not identify any accessory genes that remained statistically significant after Benjamini-Hochberg false discovery rate (FDR) correction. Under a relaxed exploratory threshold (empirical P-value < 0.35 and odds ratio > 1), a subset of accessory genes showing differential distribution patterns among disease groups was identified and reported as exploratory candidates. Comparatively greater accessory genome divergence was observed between bronchiectasis and COPD isolates. Functional annotation indicated that these candidate genes spanned multiple categories, including recombination, membrane-associated processes, transport, and nutrient utilization. Conclusions: Clinical heterogeneity among NTHi isolates was not reflected in core genome phylogeny. Differences in accessory gene content were observed across isolates from different clinical sources; however, these patterns were not supported by statistically robust associations after multiple testing correction. The identified candidate genes should therefore be regarded as exploratory, and the findings interpreted as descriptive of genomic diversity rather than evidence of disease-associated functional differentiation.