Comparative analysis of amplicon and metagenomic sequencing methods reveals key features in the evolution of animal metaorganisms

Background The interplay between hosts and their associated microbiome is now recognized as a fundamental basis of the ecology, evolution, and development of both players. These interdependencies inspired a new view of multicellular organisms as “metaorganisms.” The goal of the Collaborative Research Center “Origin and Function of Metaorganisms” is to understand why and how microbial communities form long-term associations with hosts from diverse taxonomic groups, ranging from sponges to humans in addition to plants. Methods In order to optimize the choice of analysis procedures, which may differ according to the host organism and question at hand, we systematically compared the two main technical approaches for profiling microbial communities, 16S rRNA gene amplicon and metagenomic shotgun sequencing across our panel of ten host taxa. This includes two commonly used 16S rRNA gene regions and two amplification procedures, thus totaling five different microbial profiles per host sample. Conclusion While 16S rRNA gene-based analyses are subject to much skepticism, we demonstrate that many aspects of bacterial community characterization are consistent across methods. The resulting insight facilitates the selection of appropriate methods across a wide range of host taxa. Overall, we recommend single- over multi-step amplification procedures, and although exceptions and trade-offs exist, the V3 V4 over the V1 V2 region of the 16S rRNA gene. Finally, by contrasting taxonomic and functional profiles and performing phylogenetic analysis, we provide important and novel insight into broad evolutionary patterns among metaorganisms, whereby the transition of animals from an aquatic to a terrestrial habitat marks a major event in the evolution of host-associated microbial composition

Composition of Microbial Oral Biofilms during Maturation in Young Healthy Adults

<div><p>In the present study we aimed to analyze the bacterial community structure of oral biofilms at different maturation stages in young healthy adults. Oral biofilms established on membrane filters were collected from 32 human subjects after 5 different maturation intervals (1, 3, 5, 9 and 14 days) and the respective phylogenetic diversity was analyzed by 16S rDNA amplicon sequencing. Our analyses revealed highly diverse entire colonization profiles, spread into 8 phyla/candidate divisions and in 15 different bacterial classes. A large inter-individual difference in the subjects’ microbiota was observed, comprising 35% of the total variance, but lacking conspicuous general temporal trends in both alpha and beta diversity. We further obtained strong evidence that subjects can be categorized into three clusters based on three differently occurring and mutually exclusive species clusters.</p></div

Species and subject clusters.

<p>Combined consensus clustering and ordination (PCA) of robust species and human subjects. (A) First (PC1) and second (PC2) axis and (B) first (PC1) and third axis (PC3) of the ordination space of individual subject samples are shown. Species data points (small spheres) are projected into the ordination space as weighted averages and grouped into three clusters according to species consensus clustering: “<i>Prevotella</i> cluster” (magenta), “<i>Streptococcus</i> cluster” (orange), “Proteobacteria cluster” (green). Large spheres represent the centroids of individual sample points for each human subject, color-coded according to the result of subject consensus clustering. (C) Relative species abundances in subject clusters. Color coding of species clusters is analogous to (A) and (B).</p