Interpreting 16S metagenomic data without clustering to achieve sub-OTU resolution

The standard approach to analyzing 16S tag sequence data, which relies on clustering reads by sequence similarity into Operational Taxonomic Units (OTUs), underexploits the accuracy of modern sequencing technology. We present a clustering-free approach to multi-sample Illumina datasets that can identify independent bacterial subpopulations regardless of the similarity of their 16S tag sequences. Using published data from a longitudinal time-series study of human tongue microbiota, we are able to resolve within standard 97% similarity OTUs up to 20 distinct subpopulations, all ecologically distinct but with 16S tags differing by as little as 1 nucleotide (99.2% similarity). A comparative analysis of oral communities of two cohabiting individuals reveals that most such subpopulations are shared between the two communities at 100% sequence identity, and that dynamical similarity between subpopulations in one host is strongly predictive of dynamical similarity between the same subpopulations in the other host. Our method can also be applied to samples collected in cross-sectional studies and can be used with the 454 sequencing platform. We discuss how the sub-OTU resolution of our approach can provide new insight into factors shaping community assembly.Comment: Updated to match the published version. 12 pages, 5 figures + supplement. Significantly revised for clarity, references added, results not change

Combinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage Priming

Epiblast cells in the early post-implantation stage mammalian embryo undergo a transition described as lineage priming before cell fate allocation, but signaling pathways acting upstream remain ill defined. Genetic studies demonstrate that Smad2/3 double-mutant mouse embryos die shortly after implantation. To learn more about the molecular disturbances underlying this abrupt failure, here we characterized Smad2/3-deficient embryonic stem cells (ESCs). We found that Smad2/3 double-knockout ESCs induced to form epiblast-like cells (EpiLCs) display changes in naive and primed pluripotency marker gene expression, associated with the disruption of Oct4-bound distal regulatory elements. In the absence of Smad2/3, we observed enhanced Bmp target gene expression and de-repression of extra-embryonic gene expression. Cell fate allocation into all three embryonic germ layers is disrupted. Collectively, these experiments demonstrate that combinatorial Smad2/3 functional activities are required to maintain distinct embryonic and/or extra-embryonic cell identity during lineage priming in the epiblast before gastrulation. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved

Metabolic flexibility as a major predictor of spatial distribution in microbial communities

A better understand the ecology of microbes and their role in the global ecosystem could be achieved if traditional ecological theories can be applied to microbes. In ecology organisms are defined as specialists or generalists according to the breadth of their niche. Spatial distribution is often used as a proxy measure of niche breadth; generalists have broad niches and a wide spatial distribution and specialists a narrow niche and spatial distribution. Previous studies suggest that microbial distribution patterns are contrary to this idea; a microbial generalist genus (Desulfobulbus) has a limited spatial distribution while a specialist genus (Methanosaeta) has a cosmopolitan distribution. Therefore, we hypothesise that this counter-intuitive distribution within generalist and specialist microbial genera is a common microbial characteristic. Using molecular fingerprinting the distribution of four microbial genera, two generalists, Desulfobulbus and the methanogenic archaea Methanosarcina, and two specialists, Methanosaeta and the sulfate-reducing bacteria Desulfobacter were analysed in sediment samples from along a UK estuary. Detected genotypes of both generalist genera showed a distinct spatial distribution, significantly correlated with geographic distance between sites. Genotypes of both specialist genera showed no significant differential spatial distribution. These data support the hypothesis that the spatial distribution of specialist and generalist microbes does not match that seen with specialist and generalist large organisms. It may be that generalist microbes, while having a wider potential niche, are constrained, possibly by intrageneric competition, to exploit only a small part of that potential niche while specialists, with far fewer constraints to their niche, are more capable of filling their potential niche more effectively, perhaps by avoiding intrageneric competition. We suggest that these counter-intuitive distribution patterns may be a common feature of microbes in general and represent a distinct microbial principle in ecology, which is a real challenge if we are to develop a truly inclusive ecology

A Core Human Microbiome as Viewed through 16S rRNA Sequence Clusters

We explore the microbiota of 18 body sites in over 200 individuals using sequences amplified V1–V3 and the V3–V5 small subunit ribosomal RNA (16S) hypervariable regions as part of the NIH Common Fund Human Microbiome Project. The body sites with the greatest number of core OTUs, defined as OTUs shared amongst 95% or more of the individuals, were the oral sites (saliva, tongue, cheek, gums, and throat) followed by the nose, stool, and skin, while the vaginal sites had the fewest number of OTUs shared across subjects. We found that commonalities between samples based on taxonomy could sometimes belie variability at the sub-genus OTU level. This was particularly apparent in the mouth where a given genus can be present in many different oral sites, but the sub-genus OTUs show very distinct site selection, and in the vaginal sites, which are consistently dominated by the Lactobacillus genus but have distinctly different sub-genus V1–V3 OTU populations across subjects. Different body sites show approximately a ten-fold difference in estimated microbial richness, with stool samples having the highest estimated richness, followed by the mouth, throat and gums, then by the skin, nasal and vaginal sites. Richness as measured by the V1–V3 primers was consistently higher than richness measured by V3–V5. We also show that when such a large cohort is analyzed at the genus level, most subjects fit the stool “enterotype” profile, but other subjects are intermediate, blurring the distinction between the enterotypes. When analyzed at the finer-scale, OTU level, there was little or no segregation into stool enterotypes, but in the vagina distinct biotypes were apparent. Finally, we note that even OTUs present in nearly every subject, or that dominate in some samples, showed orders of magnitude variation in relative abundance emphasizing the highly variable nature across individuals

Patent Human Infections with the Whipworm, Trichuris trichiura, Are Not Associated with Alterations in the Faecal Microbiota

Background: The soil-transmitted helminth (STH), Trichuris trichiura colonises the human large intestine where it may modify inflammatory responses, an effect possibly mediated through alterations in the intestinal microbiota. We hypothesised that patent T. trichiura infections would be associated with altered faecal microbiota and that anthelmintic treatment would induce a microbiota resembling more closely that observed in uninfected individuals. Materials and Methods: School children in Ecuador were screened for STH infections and allocated to 3 groups: uninfected, T. trichiura only, and mixed infections with T. trichiura and Ascaris lumbricoides. A sample of uninfected children and those with T. trichiura infections only were given anthelmintic treatment. Bacterial community profiles in faecal samples were studied by 454 pyrosequencing of 16 S rRNA genes. Results: Microbiota analyses of faeces were done for 97 children: 30 were uninfected, 17 were infected with T. trichiura, and 50 with T. trichiura and A. lumbricoides. Post-treatment samples were analyzed for 14 children initially infected with T. trichiura alone and for 21 uninfected children. Treatment resulted in 100% cure of STH infections. Comparisons of the microbiota at different taxonomic levels showed no statistically significant differences in composition between uninfected children and those with T. trichiura infections. We observed a decreased proportional abundance of a few bacterial genera from the Clostridia class of Firmicutes and a reduced bacterial diversity among children with mixed infections compared to the other two groups, indicating a possible specific effect of A. lumbricoides infection. Anthelmintic treatment of children with T. trichiura did not alter faecal microbiota composition. Discussion: Our data indicate that patent human infections with T. trichiura may have no effect on faecal microbiota but that A. lumbricoides colonisation might be associated with a disturbed microbiota. Our results also catalogue the microbiota of rural Ecuadorians and indicate differences with individuals from more urban industrialised societies

Environmental DNA sequencing primers for eutardigrades and bdelloid rotifers

<p>Abstract</p> <p>Background</p> <p>The time it takes to isolate individuals from environmental samples and then extract DNA from each individual is one of the problems with generating molecular data from meiofauna such as eutardigrades and bdelloid rotifers. The lack of consistent morphological information and the extreme abundance of these classes makes morphological identification of rare, or even common cryptic taxa a large and unwieldy task. This limits the ability to perform large-scale surveys of the diversity of these organisms.</p> <p>Here we demonstrate a culture-independent molecular survey approach that enables the generation of large amounts of eutardigrade and bdelloid rotifer sequence data directly from soil. Our PCR primers, specific to the 18s small-subunit rRNA gene, were developed for both eutardigrades and bdelloid rotifers.</p> <p>Results</p> <p>The developed primers successfully amplified DNA of their target organism from various soil DNA extracts. This was confirmed by both the BLAST similarity searches and phylogenetic analyses. Tardigrades showed much better phylogenetic resolution than bdelloids. Both groups of organisms exhibited varying levels of endemism.</p> <p>Conclusion</p> <p>The development of clade-specific primers for characterizing eutardigrades and bdelloid rotifers from environmental samples should greatly increase our ability to characterize the composition of these taxa in environmental samples. Environmental sequencing as shown here differs from other molecular survey methods in that there is no need to pre-isolate the organisms of interest from soil in order to amplify their DNA. The DNA sequences obtained from methods that do not require culturing can be identified post-hoc and placed phylogenetically as additional closely related sequences are obtained from morphologically identified conspecifics. Our non-cultured environmental sequence based approach will be able to provide a rapid and large-scale screening of the presence, absence and diversity of Bdelloidea and Eutardigrada in a variety of soils.</p

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing

Peer reviewedPublisher PD

Characterization of Bacteria in Biopsies of Colon and Stools by High Throughput Sequencing of the V2 Region of Bacterial 16S rRNA Gene in Human

BACKGROUND: The characterization of the human intestinal microflora and their interactions with the host have been identified as key components in the study of intestinal disorders such as inflammatory bowel diseases. High-throughput sequencing has enabled culture-independent studies to deeply analyze bacteria in the gut. It is possible with this technology to systematically analyze links between microbes and the genetic constitution of the host, such as DNA polymorphisms and methylation, and gene expression. METHODS AND FINDINGS: In this study the V2 region of the bacterial 16S ribosomal RNA (rRNA) gene using 454 pyrosequencing from seven anatomic regions of human colon and two types of stool specimens were analyzed. The study examined the number of reads needed to ascertain differences between samples, the effect of DNA extraction procedures and PCR reproducibility, and differences between biopsies and stools in order to design a large scale systematic analysis of gut microbes. It was shown (1) that sequence coverage lower than 1,000 reads influenced quantitative and qualitative differences between samples measured by UniFrac distances. Distances between samples became stable after 1,000 reads. (2) Difference of extracted bacteria was observed between the two DNA extraction methods. In particular, Firmicutes Bacilli were not extracted well by one method. (3) Quantitative and qualitative difference in bacteria from ileum to rectum colon were not observed, but there was a significant positive trend between distances within colon and quantitative differences. Between sample type, biopsies or stools, quantitative and qualitative differences were observed. CONCLUSIONS: Results of human colonic bacteria analyzed using high-throughput sequencing were highly dependent on the experimental design, especially the number of sequence reads, DNA extraction method, and sample type