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

Abstract

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.

Fig. 1

Boxplots representing alpha diversity indices stratified by sequencing platform and sample group (Human or Pig). A) Boxplot of Shannon diversity. The mean diversity values for each group are 1.841, 1.504, 1.526, and 1.474, with a p-value of 0.4807, indicating no statistically significant differences between groups. B) Boxplot of observed OTUs. The mean observed OTU counts for each group are 93.40, 20.75, 100.04, and 64.29, with a p-value < 0.0001, suggesting statistically significant differences between groups.

Fig. 2

Principal Coordinates Analysis (PCoA) of beta diversity distances stratified by sequencing platform and sample group (Human or Pig). (A) Bray-Curtis distances: PERMANOVA results indicate no effect of the model (Df = 3, SumOfSqs = 8.060, R² = 0.327, F = 10.373, p = 0.001), with residual variation accounting for R² = 0.673. (B) Jaccard distances: PERMANOVA also shows no significant model effect (Df = 3, SumOfSqs = 8.060, R² = 0.327, F = 10.373, p = 0.001), with residual variation accounting for R² = 0.673. Both analyses were conducted using 999 permutations.

Fig. 3

Stacked barplots displaying the five most abundant taxa in each sample, resolved to the genus level when possible, prioritizing the most specific taxonomic classification. (A) This graph represents Human samples collected from ventilator-associated pneumonia (VAP) patients, with a diverse range of pathogens identified as the causative agents of infection. The taxa composition reflects the microbial communities associated with this heterogeneous infection. (B) This graph represents an experimental VAP model in swine, inoculated with Pseudomonas aeruginosa, a common pathogen associated with VAP. The barplot highlights the microbial community dynamics in a controlled setting where P.aeruginosa is the primary pathogen.

Fig. 4

Log-Fold Changes of Taxa Across Platform and Timepoints. Differential abundance of bacterial taxa is shown with log-fold change (LFC) values displayed along the y-axis for each taxon. The x-axis represents sequencing methods, with Illumina, and Nanopore at time points 12, 18, 24, and 72 h. LFC is represented using a color gradation, where yellow indicates enrichment and blue indicates depletion. A) This plot represents Human samples collected from ventilator-associated pneumonia (VAP) patients, with a diverse range of pathogens identified as the causative agents of infection. The taxa composition on the ANCOM-BC2 reflects the heterogenicity the human samples due to the lower number of taxa presents in the analysis. B) This plot represents an experimental VAP model in swine, inoculated with Pseudomonas aeruginosa, a common pathogen associated with VAP. The taxa composition on the ANCOM-BC2 reflects the similarity and standardization of the preclinical model microbiome.