Deep sequencing analyses of low density microbial communities: working at the boundary of accurate microbiota detection

This study aimed to interpret microbiota analyses of low-density communities. Bacterial density seemed to interfere with microbiota analyses at < than 10(6) 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.

To 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.

Lower template concentrations significantly impacted microbiota profiling results. With higher dilutions, low abundant species were overrepresented. In samples of <10(5) 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.