Assessing saliva microbiome collection and processing methods

Abigail J S Armstrong,Veenat Parmar,Martin J Blaser

doi:10.1038/s41522-021-00254-z

Abigail J S Armstrong, Veenat Parmar + Show 1 more

Open Access

https://doi.org/10.1038/s41522-021-00254-z

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Abstract

The oral microbiome has been connected with lung health and may be of significance in the progression of SARS-CoV-2 infection. Saliva-based SARS-CoV-2 tests provide the opportunity to leverage stored samples for assessing the oral microbiome. However, these collection kits have not been tested for their accuracy in measuring the oral microbiome. Saliva is highly enriched with human DNA and reducing it prior to shotgun sequencing may increase the depth of bacterial reads. We examined both the effect of saliva collection method and sequence processing on measurement of microbiome depth and diversity by 16S rRNA gene amplicon and shotgun metagenomics. We collected 56 samples from 22 subjects. Each subject provided saliva samples with and without preservative, and a subset provided a second set of samples the following day. 16S rRNA gene (V4) sequencing was performed on all samples, and shotgun metagenomics was performed on a subset of samples collected with preservative with and without human DNA depletion before sequencing. We observed that the beta diversity distances within subjects over time was smaller than between unrelated subjects, and distances within subjects were smaller in samples collected with preservative. Samples collected with preservative had higher alpha diversity measuring both richness and evenness. Human DNA depletion before extraction and shotgun sequencing yielded higher total and relative reads mapping to bacterial sequences. We conclude that collecting saliva with preservative may provide more consistent measures of the oral microbiome and depleting human DNA increases yield of bacterial sequences.

Highlights

The oral microbiome has high biomass second only to the gut[1], and has been connected with both local[2,3] and systemic illnesses[2,4]
The oral microbiome has a strong connection with the lung microbiome as oral microbes are enriched in pulmonary fluids[6,7,8], and their presence in the lung has been associated with inflammation[7]
Each subject provided two saliva samples with and without preservative; six subjects provided a second set of samples the following day. 16S rRNA gene (V4) sequencing was performed on all of the samples, and shotgun metagenomics was performed on eight of the samples collected with preservative with and without human DNA depletion before sequencing (Fig. 1)

Summary

Introduction

The oral microbiome has high biomass second only to the gut[1], and has been connected with both local[2,3] and systemic illnesses[2,4]. Our goal was to examine both the effect of saliva collection method and sequence processing on measurement of microbiome depth and diversity using both 16S rRNA genes and shotgun metagenomics. We further hypothesized that depleting human DNA preceding DNA extraction of saliva will increase the depth of the bacterial reads in shotgun metagenomic sequencing but will not have significant impact on the microbiome composition.

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Conclusion