Abstract
Next Generation sequencing has greatly progressed the exploration of the oral microbiome’s role in dental diseases, however, there has been little focus on the effect of sample storage conditions and their interaction with DNA extraction method. Dental plaque samples collected from 20 healthy participants were pooled and stored in either 75% ethanol or Bead solution for up to 6-months at −80 °C, prior to DNA extraction with either QIAamp (non-bead beating) or PowerSoil (bead-beating) kit, followed by Illumina sequencing of 16S rRNA gene. We found that storage media and not extraction method had the biggest influence on the diversity and abundance of the oral microbiota recovered. Samples stored in Bead solution, independent of the extraction kit, retrieved higher diversity (PowerSoil p = 1.64E-07, QIAamp p = 0.0085) and had dissimilar overall ecologies as indicated by lower level of shared diversity (PowerSoil p = 0.0000237, QIAamp p = 0.0088). Comparatively, samples stored in Bead solution and extracted with PowerSoil recovered a higher abundance of Streptococcus species. These data indicate that Bead solution can preserve the oral microbiome in dental plaque reliably, for periods of up to 6-months at −80 °C, and is compatible, with either a bead-beating or non-bead beating DNA extraction method.
Highlights
Generation sequencing has greatly progressed the exploration of the oral microbiome’s role in dental diseases, there has been little focus on the effect of sample storage conditions and their interaction with DNA extraction method
We found a total of 280 bacterial species or Amplicon Sequence Variants (ASVs), with seven dominant species occurring above 2.0% (Fig. 2, Supplementary Table 1)
A differential abundance test, DESeq[222,23], was used to compare the abundance of ASVs present in dental plaque samples stored in Bead solution versus ethanol, and extracted with the PowerSoil versus the QIAamp kit, while controlling for length of time stored at −80 °C
Summary
A differential abundance test, DESeq[222,23], was used to compare the abundance of ASVs present in dental plaque samples stored in Bead solution versus ethanol, and extracted with the PowerSoil versus the QIAamp kit, while controlling for length of time stored at −80 °C. Three species were more abundant in Bead solution than ethanol-stored samples, independent of if the sample was extracted with PowerSoil (Fig. 5a) or QIAamp (Fig. 5b) These enriched species in Bead Solution included Streptococcus sp, Haemophilus sp. For the ethanol-stored samples, those extracted with the PowerSoil compared to the QIAamp kit (Fig. 5c), recovered a higher abundance of Streptococcus parasanguinis_II (p = 6.09E-19), Haemophilus sp. The differential abundance test indicated that Bead solution stored and PowerSoil extracted samples recovered a higher abundance, of streptococcus species, compared to ethanol-stored samples
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