Deep Sequencing Analyses of Low Density Microbial Communities: Working at the Boundary of Accurate Microbiota Detection

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 5 bacteria per ml, e.g. DNA ConclusionThis study aimed to interpret microbiota analyses of low-density communities. Bacterial density seemed to interfere with microbiota analyses at 6 bacteria per ml or DNA