The saliva microbiome profiles are minimally affected by collection method or DNA extraction protocols

Abstract

Saliva has attracted attention as a diagnostic fluid due to the association of oral microbiota with systemic diseases. However, the lack of standardised methods for saliva collection has led to the slow uptake of saliva in microbiome research. The aim of this study was to systematically evaluate the potential effects on salivary microbiome profiles using different methods of saliva collection, storage and gDNA extraction. Three types of saliva fractions were collected from healthy individuals with or without the gDNA stabilising buffer. Subsequently, three types of gDNA extraction methods were evaluated to determine the gDNA extraction efficiencies from saliva samples. The purity of total bacterial gDNA was evaluated using the ratio of human β-globin to bacterial 16S rRNA PCR while 16S rRNA gene amplicon sequencing was carried out to identify the bacterial profiles present in these samples. The quantity and quality of extracted gDNA were similar among all three gDNA extraction methods and there were no statistically significant differences in the bacterial profiles among different saliva fractions at the genus-level of taxonomic classification. In conclusion, saliva sampling, processing and gDNA preparation do not have major influence on microbiome profiles.

Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines

“…Saliva samples with long storage time and multiple freezing-thawing cycles seem unsuitable for microbiota research. DNA yield from included saliva samples was lower than described in the literature (35,36) (Fig. S1).…”

The Human Oral Microbiome in Health and Disease: From Sequences to Ecosystems

“…Whatever the technique being employed, it is important that researchers come to a consensus on the exact procedure for collection and sequencing to ensure reproducibility. While some of these referenced studies have shown that the microbiome profile of a sample is not heavily influenced by the collection technique [78,79], these are focused on large-scale differences. However, as sequencing technologies become more efficient and microbiome-associated databases become more complete, researchers will continue to compare samples at finer scales, so minor technical variability stemming from different techniques of swabbing collection sites or using a different solution for oral rinse collections could potentially impact results.…”

“…Since these niches tend to have very similar overall microbiome compositions at all but the lowest taxonomic levels, many researchers choose to treat the oral cavity as an individual habitat and analyze global compositions and processes therein [10,12,112]. A few studies have shown that this is a viable and efficient method for sample collection to investigate the oral microbiome [78,79], and standardized procedures have been proposed [113,114]. Essentially, an individual would refrain from eating, drinking, brushing, or smoking (anything that might temporarily shift the typical microbial composition) for at least 30 min prior to collection.…”

Toward standards in clinical microbiome studies: comparison of three DNA extraction methods and two bioinformatic pipelines

“…DNA yield from saliva samples was lower as compared to literature (43, 44) (Fig S1). Only a single DNA extraction had a concentration of slightly above one ng/μl (1.18; Table S4), while all other extractions had concentrations between 0.04 and 0.68 ng/μl.…”