Abstract
IntroductionAccurate analyses of microbiota composition of low-density communities (103–104 bacteria/sample) can be challenging. Background DNA from chemicals and consumables, extraction biases as well as differences in PCR efficiency can significantly interfere with microbiota assessment. This study was aiming to establish protocols for accurate microbiota analysis at low microbial density.MethodsTo examine possible effects of bacterial density on microbiota analyses we compared microbiota profiles of serial diluted saliva and low (nares, nasopharynx) and high-density (oropharynx) upper airway communities in four healthy individuals. DNA was extracted with four different extraction methods (Epicentre Masterpure, Qiagen DNeasy, Mobio Powersoil and a phenol bead-beating protocol combined with Agowa-Mag-mini). Bacterial DNA recovery was analysed by 16S qPCR and microbiota profiles through GS-FLX-Titanium-Sequencing of 16S rRNA gene amplicons spanning the V5–V7 regions.ResultsLower template concentrations significantly impacted microbiota profiling results. With higher dilutions, low abundant species were overrepresented. In samples of <105 bacteria per ml, e.g. DNA <1 pg/µl, microbiota profiling deviated from the original sample and other dilutions showing a significant increase in the taxa Proteobacteria and decrease in Bacteroidetes. In similar low density samples, DNA extraction method determined if DNA levels were below or above 1 pg/µl and, together with lysis preferences per method, had profound impact on microbiota analyses in both relative abundance as well as representation of species.ConclusionThis study aimed to interpret microbiota analyses of low-density communities. Bacterial density seemed to interfere with microbiota analyses at < than 106 bacteria per ml or DNA <1 pg/µl. We therefore recommend this threshold for working with low density materials. This study underlines that bias reduction is crucial for adequate profiling of especially low-density bacterial communities.
Highlights
Accurate analyses of microbiota composition of low-density communities (103–104 bacteria/sample) can be challenging
For comprehensive and accurate insight in the microbiota of these low-density regions, and interindividual comparison, understanding the effect of low bacterial 16S gene template concentrations on deep sequencing analyses is relevant, especially since most studies have been focusing on bacterial habitats, where bacterial density, composition and diversity is different from these habitats e.g. gut microbiota [5,9,10,11]
Bacterial density in nasopharyngeal samples During a vaccine intervention trial, nasopharyngeal swabs were collected in 1003 infants during the first 24 months of their life [12]
Summary
Accurate analyses of microbiota composition of low-density communities (103–104 bacteria/sample) can be challenging. The upper airway is the port d’entree for infections and insight into microbial community structures in these sites could contribute to our understanding of pathogenesis of respiratory infections. Most of these niches, such as the nasopharynx, are colonized at low density. For comprehensive and accurate insight in the microbiota of these low-density regions, and interindividual comparison, understanding the effect of low bacterial 16S gene template concentrations on deep sequencing analyses is relevant, especially since most studies have been focusing on bacterial habitats, where bacterial density, composition and diversity is different from these habitats e.g. gut microbiota [5,9,10,11]
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