Comparative analysis of illumina and oxford nanopore sequencing platforms for 16S rRNA profiling of respiratory microbial communities

The respiratory microbiome plays a crucial role in health and disease, necessitating accurate characterization through high-throughput sequencing technologies. This study provides a comparative analysis of Illumina NextSeq and Oxford Nanopore Technologies (ONT) sequencing platforms for 16 S rRNA profiling of respiratory microbial communities. Illumina sequencing, known for its high accuracy and short-read lengths (~ 300 bp), is widely used for genus-level microbial classification but struggles with species-level resolution due to its limited read length. In contrast, ONT generates full-length 16 S rRNA reads (~ 1,500 bp), enabling higher taxonomic resolution but historically exhibiting higher error rates (5-15%). Analysis of alpha and beta diversity indicated that Illumina captured greater species richness, while community evenness remained comparable between platforms. Beta diversity differences were significant in pig samples but not in human samples, suggesting that sequencing platform effects are more pronounced in complex microbiomes. Taxonomic profiling revealed that Illumina detected a broader range of taxa, while ONT exhibited improved resolution for dominant bacterial species. ANCOM-BC2 differential abundance analysis highlighted platform-specific biases, with ONT overrepresenting certain taxa (e.g., Enterococcus, Klebsiella) while underrepresenting others (e.g., Prevotella, Bacteroides). These findings emphasize that platform selection should align with study Objective: Illumina is ideal for broad microbial surveys, whereas ONT excels in species-level resolution and real-time applications. Future research should explore hybrid sequencing approaches to leverage the strengths of both technologies, thereby improving microbiome characterization in both clinical and preclinical settings.