Bartolomeu, C. | Search bioRxiv

bioRxiv2026

ASPIRE: the Amplicon Sequencing Profiler for Investigating Respiratory Ecosystems

Microbial communities inhabiting the respiratory tract contribute to health status through interactions with host physiology, immune function, and local environmental conditions. Advances in small subunit ribosomal RNA (SSU or 16S rRNA) gene amplicon sequencing enable culture-independent profiling of microbial communities as amplicon sequence variants (ASVs), revealing links between microbial dysbiosis and respiratory diseases, and the use of mass spectrometry to measure volatile organic compounds (VOCs) in exhaled breath shows emerging promise for biomarker discovery. Here we present ASPIRE, the Amplicon Sequencing Profiler for Investigating Respiratory Ecosystems, an accessible Nextflow workflow for processing, analyzing, and interpreting linked ASV-VOC data from respiratory microbiome studies. ASPIRE is designed to support scalable comparative analysis across respiratory sample types while preserving intermediate file outputs for inspection and reuse within a standardized file structure.

bioinformatics↗

bioRxiv2026

Methodological Impacts on Microbiome Structure and Indicator Status in the Human Lower Respiratory Tract

RationaleLung cancer is the leading cause of cancer-related death globally, and rising incidence among traditionally low-risk individuals intensifies the need for improved early-detection methods that the lower-airway microbiome may inform. ObjectivesTo evaluate how respiratory tract sampling method shapes inferred microbiome structure, and whether bronchial brushing recovers a microbial community ecologically distinct from BAL and oral rinse. MethodsProspective cohort of 33 participants (8 lung cancer, 25 non-cancer controls) underwent oral rinse, bilateral bronchoalveolar lavage (BAL), and bronchial brushing. Microbiome structure was characterized by small subunit ribosomal rRNA gene amplicon sequence variant (ASV) profiling, with indicator-species analysis and SPIEC-EASI correlation-network mapping used to identify ASVs associated with sample type or cancer status. Measurements and Main ResultsSampling method was the dominant axis of variation. BAL communities closely resembled oral rinse (65 jointly indicative ASVs; none shared with brushing), whereas bronchial brushing yielded a distinct but low-biomass signal. After excluding host-derived sequences, two brush-specific indicator ASVs affiliated with Sphingomonadaceae and an uncultured Steroidobacteraceae were identified that co-localized within a single co-occurrence module. Cancer-status effects were not detectable in this pilot cohort, consistent with limited statistical power. ConclusionsSampling method is the primary determinant of inferred respiratory microbiome structure. Bronchial brushing recovers a distinct but low-biomass signal that is obscured when BAL is used in isolation. However, overlap between this low-biomass signal with host and contaminant sequences indicates that confidently resolving a discrete lower-airway community will require deeper sequencing and dedicated contamination controls. These methodological findings directly inform the design of future cancer-and disease-association studies.

microbiology↗