BackgroundAdvances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost and benchmarking the techniques so that potential sources of bias can be better characterized.MethodsWe present a triple-index amplicon sequencing strategy to sequence large numbers of samples at significantly lower c ost and in a shorter timeframe compared to existing methods. The design employs a two-stage PCR protocol, incorpo rating three barcodes to each sample, with the possibility to add a fourth-index. It also includes heterogeneity spacers to overcome low complexity issues faced when sequencing amplicons on Illumina platforms.ResultsThe library preparation method was extensively benchmarked through analysis of a mock community in order to assess biases introduced by sample indexing, number of PCR cycles, and template concentration. We further evaluated the method through re-sequencing of a standardized environmental sample. Finally, we evaluated our protocol on a set of fecal samples from a small cohort of healthy adults, demonstrating good performance in a realistic experimental setting. Between-sample variation was mainly related to batch effects, such as DNA extraction, while sample indexing was also a significant source of bias. PCR cycle number strongly influenced chimera formation and affected relative abundance estimates of species with high GC content. Libraries were sequenced using the Illumina HiSeq and MiSeq platforms to demonstrate that this protocol is highly scalable to sequence thousands of samples at a very low cost.ConclusionsHere, we provide the most comprehensive study of performance and bias inherent to a 16S rRNA gene amplicon sequencing method to date. Triple-indexing greatly reduces the number of long custom DNA oligos required for library preparation, while the inclusion of variable length heterogeneity spacers minimizes the need for PhiX spike-in. This design results in a significant cost reduction of highly multiplexed amplicon sequencing. The biases we characterize highlight the need for highly standardized protocols. Reassuringly, we find that the biological signal is a far stronger structuring factor than the various sources of bias.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0279-1) contains supplementary material, which is available to authorized users.

BackgroundDetermining ecological roles of community members and the impact of specific taxa on overall biodiversity in the gastrointestinal (GI) microbiota is of fundamental importance. A step towards a systems-level understanding of the GI microbiota is characterization of biotic interactions. Community time series analysis, an approach based on statistical analysis of changing population abundances within a single system over time, is needed in order to say with confidence that one population is affecting the dynamics of another.ResultsHere, we characterize biotic interaction structures and define ecological roles of major bacterial groups in four healthy individuals by analysing high-resolution, long-term (>180 days) GI bacterial community time series. Actinobacteria fit the description of a keystone taxon since they are relatively rare, but have a high degree of ecological connectedness, and are positively correlated with diversity both within and between individuals. Bacteriodetes were found to be a foundation taxon in that they are numerically dominant and interact extensively, in particular through positive interactions, with other taxa. Although community structure, diversity and biotic interaction patterns were specific to each individual, we observed a strong tendency towards more intense competition within than between phyla. This is in agreement with Darwin’s limiting similarity hypothesis as well as a published biotic interaction model of the GI microbiota based on reverse ecology. Finally, we link temporal enterotype switching to a reciprocal positive interaction between two key genera.ConclusionsIn this study, we identified ecological roles of key taxa in the human GI microbiota and compared our time series analysis results with those obtained through a reverse ecology approach, providing further evidence in favour of the limiting similarity hypothesis first put forth by Darwin. Larger longitudinal studies are warranted in order to evaluate the generality of basic ecological concepts as applied to the GI microbiota, but our results provide a starting point for achieving a more profound understanding of the GI microbiota as an ecological system.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0107-4) contains supplementary material, which is available to authorized users.

The human gut microbiota plays a vital role in health and disease, and microbial colonization is a key process in infant development. Here, we analyze 2684 fecal specimens from 12 infants during their first year of life, providing detailed insights into the human gut colonization process. Maturation of the gut microbial community shows strong temporal structure and specific developmental stages. At 2–4 months of age, there is a period of accelerated convergence concurrent with a bloom of Bifidobacterium, a genus associated with metabolism of oligosaccharides found in breast milk. The end of this period coincides with the introduction of solid food, a reduction in the relative abundance of Bifidobacterium, and an increase in several groups of Firmicutes. Our findings highlight the dynamic nature and individuality of the gut colonization process, and the need for high-frequency sampling over an extended period when designing and interpreting infant microbiome studies.

BackgroundThe gelada monkey (Theropithecus gelada), endemic to the Ethiopian highlands, is the only graminivorous primate, i.e., it feeds mainly on grasses and sedges. In spite of known dental, manual, and locomotor adaptations, the intestinal anatomy of geladas is similar to that of other primates. We currently lack a clear understanding of the adaptations in digestive physiology necessary for this species to subsist on a graminoid-based diet, but digestion in other graminivores, such as ruminants, relies heavily on the microbial community residing in the gastrointestinal (GI) system. Furthermore, geladas form complex, multilevel societies, making them a suitable system for investigating links between sociality and the GI microbiota.ResultsHere, we explore the gastrointestinal microbiota of gelada monkeys inhabiting an intact ecosystem and document how factors like multilevel social structure and seasonal changes in diet shape the GI microbiota. We compare the gelada GI microbiota to those of other primate species, reporting a gradient from geladas to herbivorous specialist monkeys to dietary generalist monkeys and lastly humans, the ultimate ecological generalists. We also compare the microbiotas of the gelada GI tract and the sheep rumen, finding that geladas are highly enriched for cellulolytic bacteria associated with ruminant digestion, relative to other primates.ConclusionsThis study represents the first analysis of the gelada GI microbiota, providing insights into the adaptations underlying graminivory in a primate. Our results also highlight the role of social organization in structuring the GI microbiota within a society of wild animals.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0468-6) contains supplementary material, which is available to authorized users.

The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I-related molecule known to protect IgG and albumin from catabolism and transport IgG across polarized epithelial cells in a bidirectional manner. Previous studies have shown species-specific differences in ligand binding, IgG transport direction, and steady-state membrane distribution when expressed in polarized epithelial cells. We hypothesized that these differences may be due to the additional N-glycans expressed on the rat Mutant human FcRn clones that contained additional N-glycan side-chain modifications, including that which was fully rodentized, still exhibited specificity for human IgG and failed to bind to mouse IgG. At steady state, the mutant human FcRn with additional N-glycans redistributed to the apical cell surface similar to that of rat FcRn. Furthermore, the rodentized human FcRn exhibited a reversal of IgG transport with predominant transcytosis from an apical-to-basolateral direction, which resembled that of the rat FcRn isoform. These studies show that the N-glycans in FcRn contribute significantly to the steady-state membrane distribution and direction of IgG transport in polarized epithelia. The neonatal Fc receptor (FcRn)2 was originally proposed by Brambell and colleagues to be the Fc receptor responsible for transfer of IgG through neonatal rodent intestinal epithelium and yolk sac of pregnant rabbits more than three decades ago (1). These functional properties were confirmed to be mediated by FcRn with the subsequent isolation from neonatal rat intestinal brush borders followed by the cloning of the rat homologue identified as a 50-kDa major histocompatibility complex class I-related heavy chain in non-covalent association with a 12-kDa subunit consistent with