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

Abstract

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.

Furbellow (Brown Algae) Extract Increases Lifespan in Drosophila by Interfering with TOR-Signaling

“…DNA was eluded in 100 µL sterile elution buffer and stored at −20 • C until sequencing. The bacterial variable regions 1 and 2 of 16S rRNA genes were amplified as described by Rausch et al [27,28]. The amplicons were sequenced using the Illumina MiSeq with 2 × 300 bp paired-end sequencing.…”

Advancing Our Functional Understanding of Host–Microbiota Interactions: A Need for New Types of Studies

“…Second, application of a neutral model to the microbiota composition in different host organisms revealed that microbiota community structure is generally consistent with neutral expectations and suggested that transient deviations from neutrality play a role in various diseases . Third, a large scale metagenomics study revealed that the transition from aquatic to a terrestrial habitat marked a major event in the evolution of host‐associated microbiota . Finally, using basal metazoans as model systems enabled the recent elucidation of important conceptual and molecular advances for host–microbiota interactions.…”

The distal intestinal microbiome of hybrids of Hainan black goats and Saanen goats

“…The V3-V4 regions of the 16S rRNA genes were amplified using PCR assays [25,26]. A set of 6-nucleotide barcodes was added to the universal forward primer 338F (5 0 -ACTCCTACGG GAGGCAGCA-3 0 ) and the reverse primer 806R (5 0 -GGACTACHVGGGTWTCTAAT-3 0 ), which was targeted at domain bacteria [24].…”