Comparative Analysis of Gut Microbiota in Children with Obstructive Sleep Apnea: Assessing the Efficacy of 16S RRNA Gene Sequencing in Metabolic Function Prediction Based on Weight Status

Background: Analyzing bacterial microbiomes consistently using next-generation sequencing (NGS) is challenging due to the diversity of synthetic platforms for 16S rRNA genes and their analytical pipelines. This study compares the efficacy of full-length (V1-V9 hypervariable regions) and partial-length (V3-V4 hypervariable regions) sequencing of synthetic 16S rRNA genes from human gut microbiomes, with a focus on childhood obesity.

Methods: In this observational and comparative study, we explored the differences between these two sequencing methods in taxonomic categorization and weight status prediction among twelve children with obstructive sleep apnea.

Results: The full-length NGS method by Pacbio identified 118 genera and 248 species in the V1-V9 regions, all with a 0% unclassified rate. In contrast, the partial-length NGS method by Illumina detected 142 genera (with a 39% unclassified rate) and 6 species (with a 99% unclassified rate) in the V3-V4 regions. These approaches showed marked differences in gut microbiome composition and functional predictions. The full-length method distinguished between obese and non-obese children using the / ratio, a known obesity marker ( = 0.046), whereas the partial-length method was less conclusive ( = 0.075). Additionally, out of 73 metabolic pathways identified through full-length sequencing, 35 (48%) were associated with level 1 metabolism, compared to 28 of 61 pathways (46%) identified through the partial-length method. The full-length NGS also highlighted complex associations between body mass index z-score, three bacterial species (, , and ATCC 15912), and 17 metabolic pathways. Both sequencing techniques revealed relationships between gut microbiota composition and OSA-related parameters, with full-length sequencing offering more comprehensive insights into associated metabolic pathways than the V3-V4 technique.

Conclusion: These findings highlight disparities in NGS-based assessments, emphasizing the value of full-length NGS with amplicon sequence variant analysis for clinical gut microbiome research. They underscore the importance of considering methodological differences in future meta-analyses.