Oral Microbiome in Oral Cancer Research from Sampling to Analysis: Strategies, Challenges, and Recommendations

Area of Science:

• Microbiology
• Oncology
• Genomics

Background:

• The oral microbiome's role in oral cancer is increasingly recognized.
• Limited consensus exists on standardized methodologies for oral microbiome research in oncology.

Purpose of the Study:

• To critically evaluate current strategies for sampling, preservation, DNA extraction, sequencing, and data analysis in oral microbiome research for oral cancer.
• To provide a framework for improving study design and enabling clinical translation.

Main Methods:

• Narrative review of sampling methods (saliva, rinse, swab, brush, biopsy), preservation techniques, DNA extraction, 16S rRNA and shotgun metagenomics sequencing, and data analysis pipelines.
• Comparison of microbial yield, site specificity, and biological relevance across different sampling approaches.

Main Results:

• Sampling methods impact microbial yield and site specificity; lesion-directed methods offer greater relevance.
• Preservation and DNA extraction influence microbial stability and host DNA removal.
• 16S rRNA sequencing is common, while shotgun metagenomics provides higher resolution but faces clinical applicability challenges.
• Inconsistent data processing contributes to variable microbial profiles.

Conclusions:

• Methodological consistency across all workflow stages is essential for reproducible, high-quality data in oral microbiome research.
• Standardized approaches are vital for translating findings into clinically applicable biomarkers for oral cancer detection and risk assessment.