ASSESSMENT OF ILLUMINA AND OXFORD NANOPORE SEQUENCING APPROACHES FOR 16S RRNA-BASED CHARACTERIZATION OF RESPIRATORY MICROBIAL COMMUNITIES

The respiratory microbiome is central to health and disease, requiring accurate sequencing for characterization. We compared Illumina NextSeq and Oxford Nanopore Technologies (ONT) for 16S rRNA profiling. Illumina produces short, highly accurate reads (∼300 bp) suited for genus-level classification but limited at species resolution. ONT generates full-length reads (∼1,500 bp), enabling higher resolution but with historically lower accuracy. We sequenced 34 respiratory samples from ventilator-associated pneumonia (VAP) patients (n=20) and a swine model (n=14) using Illumina (V3–V4) and ONT (whole gene). Data were processed with nf-core/ampliseq or epi2me-labs/wf-16s using the Silva 138.1 database. Diversity metrics and taxonomic profiles were compared. Alpha diversity showed greater species richness with Illumina, while evenness was comparable. Beta diversity differences were significant in pigs but not humans, suggesting stronger platform effects in complex microbiomes. Illumina detected a broader taxonomic range, while ONT resolved dominant species. ANCOM-BC2 revealed platform-specific biases: ONT overrepresented Enterococcus and Klebsiella but underrepresented Prevotella and Bacteroides. Illumina is suited for broad surveys, while ONT excels in species-level resolution and real-time use. Hybrid approaches may best leverage both technologies in clinical and preclinical studies.