Factors influencing oral microbiome analysis: from saliva sampling methods to next-generation sequencing platforms

Area of Science:

• Microbiology
• Genomics

Background:

• The oral microbiome's link to systemic diseases is increasingly recognized.
• Lack of standardized saliva sampling hinders comparable microbiome data.
• Variability in collection and preservation methods affects oral microbiome analysis.

Purpose of the Study:

• To evaluate factors influencing oral microbiome data.
• To compare saliva collection (passive drooling vs. mouthwash) and preservation methods (OMNIgene, DNA/RNA shield, simple collection).
• To assess the impact of these factors on microbiome composition using next-generation sequencing (NGS).

Main Methods:

• Saliva samples collected via passive drooling and mouthwash.
• Samples preserved using OMNIgene, DNA/RNA shield, or simple collection.
• Sequencing performed using Illumina short-read and Nanopore long-read NGS.
• Analysis of alpha and beta diversity and taxonomic abundances.

Main Results:

• Mouthwash samples showed significantly higher alpha diversity than drooling samples with both NGS types.
• No significant differences in microbial diversity were observed between the three preservation methods.
• Mouthwash collection and simple preservation are effective alternatives.

Conclusions:

• Mouthwash collection and simple saliva preservation are viable, non-inferior methods.
• These convenient and cost-effective approaches can simplify saliva sample preparation.
• Standardization using these methods could benefit clinical and laboratory settings.