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

The exploration of oral microbiome has been increasing due to its relatedness with various systemic diseases, but standardization of saliva sampling for microbiome analysis has not been established, contributing to the lack of data comparability. Here, we evaluated the factors that influence the microbiome data. Saliva samples were collected by the two collection methods (passive drooling and mouthwash) using three saliva-preservation methods (OMNIgene, DNA/RNA shield, and simple collection). A total of 18 samples were sequenced by both Illumina short-read and Nanopore long-read next-generation sequencing (NGS). The component of the oral microbiome in each sample was compared with alpha and beta diversity and the taxonomic abundances, to find out the effects of factors on oral microbiome data. The alpha diversity indices of the mouthwash sample were significantly higher than that of the drooling group with both short-read and long-read NGS, while no significant differences in microbial diversities were found between the three saliva-preservation methods. Our study shows mouthwash and simple collection are not inferior to other sample collection and saliva-preservation methods, respectively. This result is promising since the convenience and cost-effectiveness of mouthwash and simple collection can simplify the saliva sample preparation, which would greatly help clinical operators and lab workers.

Automated summary

The exploration of oral microbiome has increased due to its association with systemic diseases, but there is a lack of standardization in saliva sampling for microbiome analysis, leading to incomparable data. This study evaluated factors influencing microbiome data by comparing saliva samples collected through two methods (passive drooling and mouthwash) and preserved using three methods (OMNIgene, DNA/RNA shield, and simple collection). The alpha diversity of the mouthwash samples was significantly higher than the drooling group, while there were no significant differences between the saliva-preservation methods. The study suggests that mouthwash and simple collection are comparable to other sample collection and preservation methods, offering convenience and cost-effectiveness for saliva sample preparation.