Menu
September 22, 2019  |  

16S rRNA long-read sequencing of the granulation tissue from nonsmokers and smokers-severe chronic periodontitis patients

Smoking has been associated with increased risk of periodontitis. The aim of the present study was to compare the periodontal disease severity among smokers and nonsmokers which may help in better understanding of predisposition to this chronic inflammation mediated diseases. We selected deep-seated infected granulation tissue removed during periodontal flap surgery procedures for identification and differential abundance of residential bacterial species among smokers and nonsmokers through long-read sequencing technology targeting full-length 16S rRNA gene. A total of 8 phyla were identified among which Firmicutes and Bacteroidetes were most dominating. Differential abundance analysis of OTUs through PICRUST showed significant (p>0.05) abundance of Phyla-Fusobacteria (Streptobacillus moniliformis); Phyla-Firmicutes (Streptococcus equi), and Phyla Proteobacteria (Enhydrobacter aerosaccus) in nonsmokers compared to smokers. The differential abundance of oral metagenomes in smokers showed significant enrichment of host genes modulating pathways involving primary immunodeficiency, citrate cycle, streptomycin biosynthesis, vitamin B6 metabolism, butanoate metabolism, glycine, serine, and threonine metabolism pathways. While thiamine metabolism, amino acid metabolism, homologous recombination, epithelial cell signaling, aminoacyl-tRNA biosynthesis, phosphonate/phosphinate metabolism, polycyclic aromatic hydrocarbon degradation, synthesis and degradation of ketone bodies, translation factors, Ascorbate and aldarate metabolism, and DNA replication pathways were significantly enriched in nonsmokers, modulation of these pathways in oral cavities due to differential enrichment of metagenomes in smokers may lead to an increased susceptibility to infections and/or higher formation of DNA adducts, which may increase the risk of carcinogenesis.


September 22, 2019  |  

Microbial phylogenetic profiling with the Pacific Biosciences sequencing platform.

High-throughput sequencing of 16S rRNA gene amplicons has revolutionized the capacity and depth of microbial community profiling. Several sequencing platforms are available, but most phylogenetic studies are performed on the 454-pyrosequencing platform because its longer reads can give finer phylogenetic resolution. The Pacific Biosciences (PacBio) sequencing platform is significantly less expensive per run, does not rely on amplification for library generation, and generates reads that are, on average, four times longer than those from 454 (C2 chemistry), but the resulting high error rates appear to preclude its use in phylogenetic profiling. Recently, however, the PacBio platform was used to characterize four electrosynthetic microbiomes to the genus-level for less than USD 1,000 through the use of PacBio’s circular consensus sequence technology. Here, we describe in greater detail: 1) the output from successful 16S rRNA gene amplicon profiling with PacBio, 2) how the analysis was contingent upon several alterations to standard bioinformatic quality control workflows, and 3) the advantages and disadvantages of using the PacBio platform for community profiling.


September 22, 2019  |  

Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation.

Shotgun metagenomics methods enable characterization of microbial communities in human microbiome and environmental samples. Assembly of metagenome sequences does not output whole genomes, so computational binning methods have been developed to cluster sequences into genome ‘bins’. These methods exploit sequence composition, species abundance, or chromosome organization but cannot fully distinguish closely related species and strains. We present a binning method that incorporates bacterial DNA methylation signatures, which are detected using single-molecule real-time sequencing. Our method takes advantage of these endogenous epigenetic barcodes to resolve individual reads and assembled contigs into species- and strain-level bins. We validate our method using synthetic and real microbiome sequences. In addition to genome binning, we show that our method links plasmids and other mobile genetic elements to their host species in a real microbiome sample. Incorporation of DNA methylation information into shotgun metagenomics analyses will complement existing methods to enable more accurate sequence binning.


September 22, 2019  |  

Profiling of oral microbiota in early childhood caries using Single-Molecule Real-Time Sequencing

Background: Alterations of oral microbiota are the main cause of the progression of caries. The goal of this study was to characterize the oral microbiota in childhood caries based on single-molecule real-time sequencing. Methods: A total of 21 preschoolers, aged 3-5 years old with severe early childhood caries, and 20 age-matched, caries-free children as controls were recruited. Saliva samples were collected, followed by DNA extraction, Pacbio sequencing and phylogenetic analyses of the oral microbial communities. Results: 876 species derived from 13 known bacterial phyla and 110 genera were detected from 41 children using Pacbio sequencing. At the species level, 38 species, including Veillonella spp., Streptococcus spp., Prevotella spp. and Lactobacillus spp., showed higher abundance in the caries group compared to the caries-free group (p<0.05). The core microbiota at the genus and species levels was more stable in the caries-free micro-ecological niche. At follow-up, oral examinations 6 months after sample collection, development of new dental caries was observed in 5 children (the transitional group) among the 21 caries free children. Compared with the caries-free children, in the transitional and caries groups, 6 species, which were more abundant in the caries-free group, exhibited a relatively low abundance in both the caries group and the transitional group (p<0.05). We conclude that Abiotrophia spp., Neisseria spp. and Veillonella spp., are essential for maintaining a healthy oral microbial ecosystem. Prevotella spp., Lactobacillus spp., Dialister spp. and Filifactor spp. may be related to the pathogenesis and progression of dental caries.


September 22, 2019  |  

Comprehensive exploration of the rumen microbial ecosystem with advancements in metagenomics

Ruminant farming and its environmental impact has long remained an economic concern. Metagenomics unravel the vast structural and functional diversity of the rumen microbial community that plays a major role in animal nutrition. Hereby, we summarize rumen metagenomic studies that have enhanced the knowledge of rumen microbe dynamics subsequently leading to development of better feed strategies to improve livestock production and reduce methane emissions.


September 22, 2019  |  

Molecular characterization of eukaryotic algal communities in the tropical phyllosphere based on real-time sequencing of the 18S rDNA gene.

Foliicolous algae are a common occurrence in tropical forests. They are referable to a few simple morphotypes (unicellular, sarcinoid-like or filamentous), which makes their morphology of limited usefulness for taxonomic studies and species diversity assessments. The relationship between algal community and their host phyllosphere was not clear. In order to obtain a more accurate assessment, we used single molecule real-time sequencing of the 18S rDNA gene to characterize the eukaryotic algal community in an area of South-western China.We annotated 2922 OTUs belonging to five classes, Ulvophyceae, Trebouxiophyceae, Chlorophyceae, Dinophyceae and Eustigmatophyceae. Novel clades formed by large numbers sequences of green algae were detected in the order Trentepohliales (Ulvophyceae) and the Watanabea clade (Trebouxiophyceae), suggesting that these foliicolous communities may be substantially more diverse than so far appreciated and require further research. Species in Trentepohliales, Watanabea clade and Apatococcus clade were detected as the core members in the phyllosphere community studied. Communities from different host trees and sampling sites were not significantly different in terms of OTUs composition. However, the communities of Musa and Ravenala differed from other host plants significantly at the genus level, since they were dominated by Trebouxiophycean epiphytes.The cryptic diversity of eukaryotic algae especially Chlorophytes in tropical phyllosphere is very high. The community structure at species-level has no significant relationship either with host phyllosphere or locations. The core algal community in tropical phyllopshere is consisted of members from Trentepohliales, Watanabea clade and Apatococcus clade. Our study provided a large amount of novel 18S rDNA sequences that will be useful to unravel the cryptic diversity of phyllosphere eukaryotic algae and for comparisons with similar future studies on this type of communities.


September 22, 2019  |  

A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system.

Establishing the time since death is critical in every death investigation, yet existing techniques are susceptible to a range of errors and biases. For example, forensic entomology is widely used to assess the postmortem interval (PMI), but errors can range from days to months. Microbes may provide a novel method for estimating PMI that avoids many of these limitations. Here we show that postmortem microbial community changes are dramatic, measurable, and repeatable in a mouse model system, allowing PMI to be estimated within approximately 3 days over 48 days. Our results provide a detailed understanding of bacterial and microbial eukaryotic ecology within a decomposing corpse system and suggest that microbial community data can be developed into a forensic tool for estimating PMI. DOI:http://dx.doi.org/10.7554/eLife.01104.001.


September 22, 2019  |  

Improved OTU-picking using long-read 16S rRNA gene amplicon sequencing and generic hierarchical clustering

BACKGROUND: High-throughput bacterial 16S rRNA gene sequencing followed by clustering of short sequences into operational taxonomic units (OTUs) is widely used for microbiome profiling. However, clustering of short 16S rRNA gene reads into biologically meaningful OTUs is challenging, in part because nucleotide variation along the 16S rRNA gene is only partially captured by short reads. The recent emergence of long-read platforms, such as single-molecule real-time (SMRT) sequencing from Pacific Biosciences, offers the potential for improved taxonomic and phylogenetic profiling. Here, we evaluate the performance of long- and short-read 16S rRNA gene sequencing using simulated and experimental data, followed by OTU inference using computational pipelines based on heuristic and complete-linkage hierarchical clustering. RESULTS: In simulated data, long-read sequencing was shown to improve OTU quality and decrease variance. We then profiled 40 human gut microbiome samples using a combination of Illumina MiSeq and Blautia-specific SMRT sequencing, further supporting the notion that long reads can identify additional OTUs. We implemented a complete-linkage hierarchical clustering strategy using a flexible computational pipeline, tailored specifically for PacBio circular consensus sequencing (CCS) data that outperforms heuristic methods in most settings: https://github.com/oscar-franzen/oclust/. CONCLUSION: Our data demonstrate that long reads can improve OTU inference; however, the choice of clustering algorithm and associated clustering thresholds has significant impact on performance.


September 22, 2019  |  

The bacterial microbiome of Dermacentor andersoni ticks influences pathogen susceptibility.

Ticks are of medical importance owing to their ability to transmit pathogens to humans and animals. The Rocky Mountain wood tick, Dermacentor andersoni, is a vector of a number of pathogens, including Anaplasma marginale, which is the most widespread tick-borne pathogen of livestock. Although ticks host pathogenic bacteria, they also harbor bacterial endosymbionts that have a role in tick physiology, survival, as well as pathogen acquisition and transmission. The goal of this study was to characterize the bacterial microbiome and examine the impact of microbiome disruption on pathogen susceptibility. The bacterial microbiome of two populations of D. andersoni with historically different susceptibilities to A. marginale was characterized. In this study, the microbiome was disrupted and then ticks were exposed to A. marginale or Francisella novicida to determine whether the microbiome correlated with pathogen susceptibility. Our study showed that an increase in proportion and quantity of Rickettsia bellii in the microbiome was negatively correlated to A. marginale levels in ticks. Furthermore, a decrease in Francisella endosymbionts was associated with lower F. novicida infection levels, demonstrating a positive pathogen-endosymbiont relationship. We demonstrate that endosymbionts and pathogens have varying interactions, and suggest that microbiome manipulation may provide a possible method for biocontrol by decreasing pathogen susceptibility of ticks.


September 22, 2019  |  

MetaSort untangles metagenome assembly by reducing microbial community complexity.

Most current approaches to analyse metagenomic data rely on reference genomes. Novel microbial communities extend far beyond the coverage of reference databases and de novo metagenome assembly from complex microbial communities remains a great challenge. Here we present a novel experimental and bioinformatic framework, metaSort, for effective construction of bacterial genomes from metagenomic samples. MetaSort provides a sorted mini-metagenome approach based on flow cytometry and single-cell sequencing methodologies, and employs new computational algorithms to efficiently recover high-quality genomes from the sorted mini-metagenome by the complementary of the original metagenome. Through extensive evaluations, we demonstrated that metaSort has an excellent and unbiased performance on genome recovery and assembly. Furthermore, we applied metaSort to an unexplored microflora colonized on the surface of marine kelp and successfully recovered 75 high-quality genomes at one time. This approach will greatly improve access to microbial genomes from complex or novel communities.


September 22, 2019  |  

Species-level bacterial community profiling of the healthy sinonasal microbiome using Pacific Biosciences sequencing of full-length 16S rRNA genes.

Pan-bacterial 16S rRNA microbiome surveys performed with massively parallel DNA sequencing technologies have transformed community microbiological studies. Current 16S profiling methods, however, fail to provide sufficient taxonomic resolution and accuracy to adequately perform species-level associative studies for specific conditions. This is due to the amplification and sequencing of only short 16S rRNA gene regions, typically providing for only family- or genus-level taxonomy. Moreover, sequencing errors often inflate the number of taxa present. Pacific Biosciences’ (PacBio’s) long-read technology in particular suffers from high error rates per base. Herein, we present a microbiome analysis pipeline that takes advantage of PacBio circular consensus sequencing (CCS) technology to sequence and error correct full-length bacterial 16S rRNA genes, which provides high-fidelity species-level microbiome data.Analysis of a mock community with 20 bacterial species demonstrated 100% specificity and sensitivity with regard to taxonomic classification. Examination of a 250-plus species mock community demonstrated correct species-level classification of >?90% of taxa, and relative abundances were accurately captured. The majority of the remaining taxa were demonstrated to be multiply, incorrectly, or incompletely classified. Using this methodology, we examined the microgeographic variation present among the microbiomes of six sinonasal sites, by both swab and biopsy, from the anterior nasal cavity to the sphenoid sinus from 12 subjects undergoing trans-sphenoidal hypophysectomy. We found greater variation among subjects than among sites within a subject, although significant within-individual differences were also observed. Propiniobacterium acnes (recently renamed Cutibacterium acnes) was the predominant species throughout, but was found at distinct relative abundances by site.Our microbial composition analysis pipeline for single-molecule real-time 16S rRNA gene sequencing (MCSMRT, https://github.com/jpearl01/mcsmrt ) overcomes deficits of standard marker gene-based microbiome analyses by using CCS of entire 16S rRNA genes to provide increased taxonomic and phylogenetic resolution. Extensions of this approach to other marker genes could help refine taxonomic assignments of microbial species and improve reference databases, as well as strengthen the specificity of associations between microbial communities and dysbiotic states.


September 22, 2019  |  

CATCh, an ensemble classifier for chimera detection in 16S rRNA sequencing studies.

In ecological studies, microbial diversity is nowadays mostly assessed via the detection of phylogenetic marker genes, such as 16S rRNA. However, PCR amplification of these marker genes produces a significant amount of artificial sequences, often referred to as chimeras. Different algorithms have been developed to remove these chimeras, but efforts to combine different methodologies are limited. Therefore, two machine learning classifiers (reference-based and de novo CATCh) were developed by integrating the output of existing chimera detection tools into a new, more powerful method. When comparing our classifiers with existing tools in either the reference-based or de novo mode, a higher performance of our ensemble method was observed on a wide range of sequencing data, including simulated, 454 pyrosequencing, and Illumina MiSeq data sets. Since our algorithm combines the advantages of different individual chimera detection tools, our approach produces more robust results when challenged with chimeric sequences having a low parent divergence, short length of the chimeric range, and various numbers of parents. Additionally, it could be shown that integrating CATCh in the preprocessing pipeline has a beneficial effect on the quality of the clustering in operational taxonomic units. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


September 22, 2019  |  

Limited effects of variable-retention harvesting on fungal communities decomposing fine roots in coastal temperate rainforests.

Fine root litter is the principal source of carbon stored in forest soils and a dominant source of carbon for fungal decomposers. Differences in decomposer capacity between fungal species may be important determinants of fine-root decomposition rates. Variable-retention harvesting (VRH) provides refuge for ectomycorrhizal fungi, but its influence on fine-root decomposers is unknown, as are the effects of functional shifts in these fungal communities on carbon cycling. We compared fungal communities decomposing fine roots (in litter bags) under VRH, clear-cut, and uncut stands at two sites (6 and 13 years postharvest) and two decay stages (43 days and 1 year after burial) in Douglas fir forests in coastal British Columbia, Canada. Fungal species and guilds were identified from decomposed fine roots using high-throughput sequencing. Variable retention had short-term effects on ß-diversity; harvest treatment modified the fungal community composition at the 6-year-postharvest site, but not at the 13-year-postharvest site. Ericoid and ectomycorrhizal guilds were not more abundant under VRH, but stand age significantly structured species composition. Guild composition varied by decay stage, with ruderal species later replaced by saprotrophs and ectomycorrhizae. Ectomycorrhizal abundance on decomposing fine roots may partially explain why fine roots typically decompose more slowly than surface litter. Our results indicate that stand age structures fine-root decomposers but that decay stage is more important in structuring the fungal community than shifts caused by harvesting. The rapid postharvest recovery of fungal communities decomposing fine roots suggests resiliency within this community, at least in these young regenerating stands in coastal British Columbia.IMPORTANCE Globally, fine roots are a dominant source of carbon in forest soils, yet the fungi that decompose this material and that drive the sequestration or respiration of this carbon remain largely uncharacterized. Fungi vary in their capacity to decompose plant litter, suggesting that fungal community composition is an important determinant of decomposition rates. Variable-retention harvesting is a forestry practice that modifies fungal communities by providing refuge for ectomycorrhizal fungi. We evaluated the effects of variable retention and clear-cut harvesting on fungal communities decomposing fine roots at two sites (6 and 13 years postharvest), at two decay stages (43 days and 1 year), and in uncut stands in temperate rainforests. Harvesting impacts on fungal community composition were detected only after 6 years after harvest. We suggest that fungal community composition may be an important factor that reduces fine-root decomposition rates relative to those of above-ground plant litter, which has important consequences for forest carbon cycling. Copyright © 2018 American Society for Microbiology.


September 22, 2019  |  

Analysis of the duodenal microbiotas of weaned piglet fed with epidermal growth factor-expressed Saccharomyces cerevisiae.

The bacterial community of the small intestine is a key factor that has strong influence on the health of gastrointestinal tract (GIT) in mammals during and shortly after weaning. The aim of this study was to analyze the effects of the diets of supplemented with epidermal growth factor (EGF)-expressed Saccharomyces cerevisiae (S. cerevisiae) on the duodenal microbiotas of weaned piglets.Revealed in this study, at day 7, 14 and 21, respectively, the compositional sequencing analysis of the 16S rRNA in the duodenum had no marked difference in microbial diversity from the phylum to species levels between the INVSc1(EV) and other recombinant strains encompassing INVSc1-EE(+), INVSc1-TE(-), and INVSc1-IE(+). Furthermore, the populations of potentially enterobacteria (e.g., Clostridium and Prevotella) and probiotic (e.g., Lactobacilli and Lactococcus) also remained unchanged among recombinant S. cerevisiae groups (P?>?0.05). However, the compositional sequencing analysis of the 16S rRNA in the duodenum revealed significant difference in microbial diversity from phylum to species levels between the control group and recombinant S. cerevisiae groups. In terms of the control group (the lack of S. cerevisiae), these data confirmed that dietary exogenous S. cerevisiae had the feasibility to be used as a supplement for enhancing potentially probiotic (e.g., Lactobacilli and Lactococcus) (P?


September 22, 2019  |  

Lack of thinning effects over inter-annual changes in soil fungal community and diversity in a Mediterranean pine forest

Predicted changes in global climate might negatively affect the soil microbiome and associated ecosystem processes in Mediterranean forests. Forest treatments, such as forest thinning, have been suggested to mitigate climate change impacts on vegetation by reducing competition between trees, thus increasing water availability. Studies addressing the combined effects of climate and forest thinning on belowground fungal communities are still scarce, being fundamental to elaborate adaptive strategies to global warming. The aim of this study was to evaluate the tree density reduction effects on soil fungal communities and their response to inter-annual changes in weather conditions. The temporal dynamics of soil fungal communities in relation to these two drivers (i.e., forest management and weather conditions) were studied from 2009 until 2014 in a set of 12 pairs of thinned and un-thinned plots dominated by Pinus pinaster Ait. Thinning (from 30% up to 70% reduction in stand basal area) was conducted in 2009 and soil fungal community composition was studied during 4?years. Here, we used autumn precipitation and temperature to describe the impact of inter-annual weather changes. We used Pacific Biosciences sequencing of fungal ITS2 amplicons to study fungal communities in soil samples. Forest thinning did not significantly affect fungal community composition nor fungal species richness and diversity, indicating that the soil fungal community is resistant to forest thinning regardless of its intensity. However, fungal species composition changed progressively across years, both at the species level and with regards to functional guilds. These changes in community composition were partly driven by inter-annual variation in precipitation and temperature, with free-living fungi increasing in abundance under wetter conditions, and symbiotic fungi being more prominent under drier and colder conditions. The results indicate that mycorrhizal communities in Mediterranean forest ecosystems can resist forest thinning, if enough trees and functional roots from thinned trees are retained.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.