scholarly journals CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems

2021 ◽  
Vol 9 (4) ◽  
pp. 816
Author(s):  
Matthew G. Links ◽  
Tim J. Dumonceaux ◽  
E. Luke McCarthy ◽  
Sean M. Hemmingsen ◽  
Edward Topp ◽  
...  
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Dna Sequences ◽  
Pcr Amplification ◽  
Shotgun Sequencing ◽  
Natural Ecosystems ◽  
Gene Markers ◽  
Microbial Profile ◽  
Microbial Ecosystems ◽  
Pcr Targeting

Background. The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with “universal” PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes. However, both amplicon-based and shotgun sequencing approaches have shortcomings that limit the ability to study microbiome dynamics. Methods. We present a novel, amplicon-free, hybridization-based method (CaptureSeq) for profiling complex microbial communities using probes based on the chaperonin-60 gene. Molecular profiles of a commercially available synthetic microbial community standard were compared using CaptureSeq, whole metagenome sequencing, and 16S universal target amplification. Profiles were also generated for natural ecosystems including antibiotic-amended soils, manure storage tanks, and an agricultural reservoir. Results. The CaptureSeq method generated a microbial profile that encompassed all of the bacteria and eukaryotes in the panel with greater reproducibility and more accurate representation of high G/C content microorganisms compared to 16S amplification. In the natural ecosystems, CaptureSeq provided a much greater depth of coverage and sensitivity of detection compared to shotgun sequencing without prior selection. The resulting community profiles provided quantitatively reliable information about all three domains of life (Bacteria, Archaea, and Eukarya) in the different ecosystems. The applications of CaptureSeq will facilitate accurate studies of host-microbiome interactions for environmental, crop, animal and human health. Conclusions: cpn60-based hybridization enriched for taxonomically informative DNA sequences from complex mixtures. In synthetic and natural microbial ecosystems, CaptureSeq provided sequences from prokaryotes and eukaryotes simultaneously, with quantitatively reliable read abundances. CaptureSeq provides an alternative to PCR amplification of taxonomic markers with deep community coverage while minimizing amplification biases.

scholarly journals CaptureSeq: Hybridization-based enrichment of cpn60 gene fragments reveals the community structures of synthetic and natural microbial ecosystems

2018 ◽  
Author(s):  
Matthew G. Links ◽  
Tim J. Dumonceaux ◽  
Luke McCarthy ◽  
Sean M. Hemmingsen ◽  
Edward Topp ◽  
...  
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Shotgun Sequencing ◽  
Natural Ecosystems ◽  
Gene Markers ◽  
Microbiome Composition ◽  
Microbial Profile ◽  
Domains Of Life ◽  
Metagenome Sequencing ◽  
Pcr Targeting

AbstractBackgroundMolecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with universal PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes. However, both amplicon-based and shotgun sequencing approaches have shortcomings that limit the ability to study microbiome dynamics.MethodsWe present a novel, amplicon-free, hybridization-based method (CaptureSeq) for profiling complex microbial communities using probes based on the chaperonin-60 gene. Molecular profiles of a commercially available synthetic microbial community standard were compared using CaptureSeq, whole metagenome sequencing, and 16S universal target amplification. Profiles were also generated for natural ecosystems including antibiotic-amended soils, manure storage tanks, and an agricultural reservoir.ResultsThe CaptureSeq method generated a microbial profile that encompassed all of the bacteria and eukaryotes in the panel with greater reproducibility and more accurate representation of high G/C content microorganisms compared to 16S amplification. In the natural ecosystems, CaptureSeq provided a much greater depth of coverage and sensitivity of detection compared to shotgun sequencing without prior selection. The resulting community profiles provided quantitatively reliable information about all three Domains of life (Bacteria, Archaea, and Eukarya) in the different ecosystems. The applications of CaptureSeq will facilitate accurate studies of host-microbiome interactions for environmental, crop, animal and human health.

scholarly journals A step-by-step sequence-based analysis of virome enrichment protocol for freshwater and sediment samples

2020 ◽  
Author(s):  
Federica Pinto ◽  
Moreno Zolfo ◽  
Francesco Beghini ◽  
Federica Armanini ◽  
Francesco Asnicar ◽  
...  
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Microbial Community Composition ◽  
Genetic Material ◽  
Amplicon Sequencing ◽  
Sample Type ◽  
Natural Ecosystems ◽  
Sediment Samples ◽  
Water And Sediment ◽  
Water And Sediment Samples

AbstractCultivation-free metagenomic analysis afforded unprecedented details on the diversity, structure and potential functions of microbial communities in different environments. When employed to study the viral fraction of the community that is recalcitrant to cultivation, metagenomics can shed light into the diversity of viruses and their role in natural ecosystems. However, despite the increasing interest in virome metagenomics, methodological issues still hinder the proper interpretation and comparison of results across studies. Virome enrichment experimental protocols are key multi-step processes needed for separating and concentrating the viral fraction from the whole microbial community prior to sequencing. However, there is little information on their efficiency and their potential biases. To fill this gap, we used metagenomic and amplicon sequencing to examine the microbial community composition through the serial filtration and concentration steps commonly used to produce viral-enriched metagenomes. The analyses were performed on water and sediment samples from an Alpine lake. We found that, although the diversity of the retained microbial communities declined progressively during the serial filtration, the final viral fraction contained a large proportion (from 10% to 40%) of non-viral taxa, and that the efficacy of filtration showed biases based on taxonomy. Our results quantified the amount of bacterial genetic material in viromes and highlighted the influence of sample type on the enrichment efficacy. Moreover, since viral-enriched samples contained a significant portion of microbial taxa, computational sequence analysis should account for such biases in the downstream interpretation pipeline.ImportanceFiltration is a commonly used method to enrich viral particles in environmental samples. However, there is little information on its efficiency and potential biases on the final result. Using a sequence-based analysis on water and sediment samples, we found that filtration efficacy is dependent on sample type and that the final virome contained a large proportion of non-viral taxa. Our finding stressed the importance of downstream analysis to avoid biased interpretation of data.

scholarly journals Microbial community diversity in the soil of Barrientos Island estimated by RAPD and Biolog Ecoplate methods

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Learn-Han Lee ◽  
Vengadesh Letchumanan ◽  
Nurul-Syakima Ab Mutalib ◽  
Yoke Kqueen Cheah
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Soil Microbial Communities ◽  
Community Diversity ◽  
Soil Microbial Diversity ◽  
Microbial Community Diversity ◽  
Soil Microbial ◽  
Biolog Ecoplate

The diversity of soil microbial communities at Barrientos Island with  differents soil characteristics were evaluated using PCR-based method random amplified polymorphic DNA (RAPD) and community level physiological profiles (CLPP) of Biolog Ecoplate. The soils were selected from 17 different locations around Barrientos Island inhabited by different breeders. Shannon-Weaver index and multivariate analysis were performed to characterize variations of soil microbial communities. Both RAPD and CLPP methods exhibited that most soils with different type of rookery and characteristics could possibly affect the DNA sequence diversity and soil microbial diversity. The abandoned type of rookery had the highest Shannon-Weaver index as exhibited by soil sample 445 (3.4 for RAPD) and 450 (3.09 for CLPP). Higher coefficients of DNA sequence similarity were found in soil samples colonized by similar breeders, like soil 442 and 446 (both were active Chinstrap rookery) shared highest similarity in DNA sequences (73.53). The cluster analysis of RAPD profiles by UPGMA and principle component analysis (PCA) of Biolog Ecoplate exhibited similar influence of type of rookery and soil condition towards soil microbial community diversity. The results may suggest that the change in microbial community DNA composition is accompanied with the change in microbial functional properties.

scholarly journals A conceptual framework for the phylogenetically constrained assembly of microbial communities

Microbiome ◽  
2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Daniel Aguirre de Cárcer
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Community Assembly ◽  
Phylogenetic Signal ◽  
A Priori ◽  
Amplicon Sequencing ◽  
Bioinformatic Analysis ◽  
Microbial Ecosystems ◽  
And Function ◽  
Microbial Community Assembly

Abstract Microbial communities play essential and preponderant roles in all ecosystems. Understanding the rules that govern microbial community assembly will have a major impact on our ability to manage microbial ecosystems, positively impacting, for instance, human health and agriculture. Here, I present a phylogenetically constrained community assembly principle grounded on the well-supported facts that deterministic processes have a significant impact on microbial community assembly, that microbial communities show significant phylogenetic signal, and that microbial traits and ecological coherence are, to some extent, phylogenetically conserved. From these facts, I derive a few predictions which form the basis of the framework. Chief among them is the existence, within most microbial ecosystems, of phylogenetic core groups (PCGs), defined as discrete portions of the phylogeny of varying depth present in all instances of the given ecosystem, and related to specific niches whose occupancy requires a specific phylogenetically conserved set of traits. The predictions are supported by the recent literature, as well as by dedicated analyses. Integrating the effect of ecosystem patchiness, microbial social interactions, and scale sampling pitfalls takes us to a comprehensive community assembly model that recapitulates the characteristics most commonly observed in microbial communities. PCGs’ identification is relatively straightforward using high-throughput 16S amplicon sequencing, and subsequent bioinformatic analysis of their phylogeny, estimated core pan-genome, and intra-group co-occurrence should provide valuable information on their ecophysiology and niche characteristics. Such a priori information for a significant portion of the community could be used to prime complementing analyses, boosting their usefulness. Thus, the use of the proposed framework could represent a leap forward in our understanding of microbial community assembly and function.

scholarly journals Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods

Microbiology ◽  
2004 ◽  
Vol 150 (8) ◽  
pp. 2565-2573 ◽  
Author(s):  
Xia Zhou ◽  
Stephen J. Bent ◽  
Maria G. Schneider ◽  
Catherine C. Davis ◽  
Mohammed R. Islam ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Dna Sequences ◽  
Phylogenetic Analyses ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Single Species ◽  
Rrna Gene ◽  
Caucasian Women ◽  
Tract Infections

The normal microbial flora of the vagina plays an important role in preventing genital and urinary tract infections in women. Thus an accurate understanding of the composition and ecology of the ecosystem is important to understanding the aetiology of these diseases. Common wisdom is that lactobacilli dominate the normal vaginal microflora of post-pubertal women. However, this conclusion is based on methods that require cultivation of microbial populations; an approach that is known to yield a biased and incomplete assessment of microbial community structure. In this study cultivation-independent methods were used to analyse samples collected from the mid-vagina of five normal healthy Caucasian women between the ages of 28 and 44. Total microbial community DNA was isolated following resuspension of microbial cells from vaginal swabs. To identify the constituent numerically dominant populations in each community 16S rRNA gene libraries were prepared following PCR amplification using the 8f and 926r primers. From each library, the DNA sequences of approximately 200 16S rRNA clones were determined and subjected to phylogenetic analyses. The diversity and kinds of organisms that comprise the vaginal microbial community varied among women. Species of Lactobacillus appeared to dominate the communities in four of the five women. However, the community of one woman was dominated by Atopobium sp., whereas a second woman had appreciable numbers of Megasphaera sp., Atopobium sp. and Leptotrichia sp., none of which have previously been shown to be common members of the vaginal ecosystem. Of the women whose communities were dominated by lactobacilli, there were two distinct clusters, each of which consisted of a single species. One class consisted of two women with genetically divergent clones that were related to Lactobacillus crispatus, whereas the second group of two women had clones of Lactobacillus iners that were highly related to a single phylotype. These surprising results suggest that culture-independent methods can provide new insights into the diversity of bacterial species found in the human vagina, and this information could prove to be pivotal in understanding risk factors for various infectious diseases.

scholarly journals PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip

2011 ◽  
Vol 77 (18) ◽  
pp. 6313-6322 ◽  
Author(s):  
Kristen M. DeAngelis ◽  
Cindy H. Wu ◽  
Harry R. Beller ◽  
Eoin L. Brodie ◽  
Romy Chakraborty ◽  
...  
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Pcr Amplification ◽  
Community Analysis ◽  
High Density ◽  
Microbial Community Analysis ◽  
Taxon Richness ◽  
Tropical Forest Soil ◽  
Global Biogeochemical Cycles ◽  
Direct Hybridization

ABSTRACTEnvironmental microbial community analysis typically involves amplification by PCR, despite well-documented biases. We have developed two methods of PCR-independent microbial community analysis using the high-density microarray PhyloChip: direct hybridization of 16S rRNA (dirRNA) or rRNA converted to double-stranded cDNA (dscDNA). We compared dirRNA and dscDNA communities to PCR-amplified DNA communities using a mock community of eight taxa, as well as experiments derived from three environmental sample types: chromium-contaminated aquifer groundwater, tropical forest soil, and secondary sewage in seawater. Community profiles by both direct hybridization methods showed differences that were expected based on accompanying data but that were missing in PCR-amplified communities. Taxon richness decreased in RNA compared to that in DNA communities, suggesting a subset of 20% in soil and 60% in groundwater that is active; secondary sewage showed no difference between active and inactive populations. Direct hybridization of dscDNA and RNA is thus a viable alternative to PCR-amplified microbial community analysis, providing identification of the active populations within microbial communities that attenuate pollutants, drive global biogeochemical cycles, or proliferate disease states.

scholarly journals A coexistence theory in microbial communities

2018 ◽  
Vol 5 (9) ◽  
pp. 180476 ◽  
Author(s):  
Marina Dohi ◽  
Akihiko Mougi
Keyword(s):  
Microbial Community ◽  
Equilibrium State ◽  
Microbial Communities ◽  
Critical Role ◽  
Abundance Ratio ◽  
The Other ◽  
Natural Ecosystems ◽  
Trade Off ◽  
Coexistence Theory ◽  
Different Types

Microbes are widespread in natural ecosystems where they create complex communities. Understanding the functions and dynamics of such microbial communities is a very important theme not only for ecology but also for humankind because microbes can play major roles in our health. Yet, it remains unclear how such complex ecosystems are maintained. Here, we present a simple theory on the dynamics of a microbial community. Bacteria preferring a particular pH in their environment indirectly inhibit the growth of the other types of bacteria by changing the pH to their optimum value. This pH-driven interaction always causes a state of bistability involving different types of bacteria that can be more or less abundant. Furthermore, a moderate abundance ratio of different types of bacteria can confer enhanced resilience to a specific equilibrium state, particularly when a trade-off relationship exists between growth and the ability of bacteria to change the pH of their environment. These results suggest that the balance of the composition of microbiota plays a critical role in maintaining microbial communities.

scholarly journals Microbial community-level features linked to divergent carbon flows during early litter decomposition in a constant environment

2019 ◽  
Author(s):  
Renee Johansen ◽  
Michaeline Albright ◽  
Deanna Lopez ◽  
La Verne Gallegos-Graves ◽  
Andreas Runde ◽  
...  
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Litter Decomposition ◽  
Plant Litter ◽  
Carbon Flow ◽  
Community Level ◽  
Natural Ecosystems ◽  
Long Term Storage ◽  
Plant Litter Decomposition ◽  
Underlying Mechanisms

AbstractDuring plant litter decomposition in soils, carbon has two general fates: return to the atmosphere via microbial respiration or transport into soil where long-term storage may occur. Discovering microbial community features that drive carbon fate from litter decomposition may improve modeling and management of soil carbon. This concept assumes there are features (or underlying processes) that are widespread among disparate communities, and therefore amenable to modeling. We tested this assumption using an epidemiological approach in which two contrasting patterns of carbon flow in laboratory microcosms were delineated as functional states and diverse microbial communities representing each state were compared to discover shared features linked to carbon fate. Microbial communities from 206 soil samples from the southwestern United States were inoculated on plant litter in microcosms, and carbon flow was measured as cumulative carbon dioxide (CO2) and dissolved organic carbon (DOC) after 44 days. Carbon flow varied widely among the microcosms, with a 2-fold range in cumulative CO2efflux and a 5-fold range in DOC quantity. Bacteria, not fungi, were the strongest drivers of DOC variation. The most significant community-level feature linked to DOC abundance was bacterial richness—the same feature linked to carbon fate in human-gut microbiome studies. This proof-of-principle study under controlled conditions suggests common features driving carbon flow in disparate microbial communities can be identified, motivating further exploration of underlying mechanisms that may influence carbon fate in natural ecosystems.

scholarly journals Variation in Melitaea cinxia gut microbiota is phylogenetically highly structured but only mildly driven by host plant microbiota, sex or parasitism

2019 ◽  
Author(s):  
G. Minard ◽  
G Tikhonov ◽  
O. Ovaskainen ◽  
M. Saastamoinen
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Host Plant ◽  
Natural Populations ◽  
Melitaea Cinxia ◽  
Microbiota Composition ◽  
Natural Ecosystems ◽  
Phylogenetic Structure ◽  
Plant Microbiota ◽  
The Impact

Originality-Significance StatementThe factors contributing to the assembly of microbiota in animals are extremely complex, and thus a comprehensive understanding of the mechanisms shaping host-associated microbial communities in natural ecosystems requires extensive ecological studies and appropriate statistical methods. In this study, we investigated the bacterial microbiota associated with the caterpillars of the Glanville fritillary (Melitaea cinxia), which is a long-term-studied ecological model system. We assessed the structure of variation in both occurrence and abundance of gut microbial communities of individuals collected in the wild with joint-species modelling, with the aim to relate the microbial community structure with multiple potentially impacting covariates: host plant microbiota and metabolites, hosts’ sex, potential parasitoid infection, and family structure. These covariates exhibited substantial correlation with multiple microbial taxa’s occurrences, which correlations were consistent for phylogenetically related groups of taxa, but varied across the whole microbial community; on the contrary, only few correlations were found with taxa’s abundances. The dominating co-occurrence pattern of microbiota assembly, which effectively split caterpillar individuals into two distinct groups, was, however, unrelated to any of the considered covariates.SummaryUnderstanding of what ecological factors shape intraspecific variation of insect microbiota is still relatively poor. In Lepidopteran caterpillars, microbiota is assumed to be mainly composed of transient bacterial symbionts acquired from the host plant. We sampled Glanville fritillary (Melitaea cinxia) caterpillars from natural populations to describe the microbiome and to identify potential factors that determine the structure of the microbial community, including the sex of the host, the impact of parasitoid infection, and the possible link between host plant and caterpillar microbiota. Our results demonstrate high variability of microbiota composition even among caterpillars that shared the same host plant individual. The observed variation in microbiota composition is partially attributed to the measured properties of the host or its plant microbial and chemical composition, and is aligned with microbial phylogenetic structure, with related taxa exhibiting similar patterns. However, the prevailing part of the observed variation was not associated with any of the assessed characteristics, although it followed a pronounced segregation structure: in some caterpillars the microbial communities were dominated by several related Enterobacteriaceae taxa, while in others these taxa were absent. Our results challenge previous findings that the host plant properties are the major drivers of microbiota communities of insect herbivores.

scholarly journals Microbial community profiling applications in plant protection

2010 ◽  
Vol 63 ◽  
pp. 271-271
Author(s):  
E.M. Gerard ◽  
J. Monk ◽  
S.A. Wakelin ◽  
M. O?Callaghan
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Ammonia Oxidation ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Plant Protection ◽  
Antibiotic Production ◽  
Pcr Amplification ◽  
Disease Suppression ◽  
Rrna Genes ◽  
Cell Functions

The function and diversity of microbial communities associated with plants insects and soils directly impacts on plant health and production Although community level investigations appear daunting significant gains are being made through the application of molecular biology An important technique involves polymerase chain reaction (PCR) amplification of genes from DNA/RNA isolated from the environment removing limitations caused by microbial cultivation PCR amplicons are then separated using denaturing gradient gel electrophoresis (DGGE) providing a fingerprint of the diversity of that gene within the microbial community This PCRDGGE based method originally targeted the ribosomal RNA (rRNA) genes present in all microorganisms Other genetic markers are now used including general markers coding for conserved proteins involved in core cell functions or genes essential to activities defining specific functional groups such as ammonia oxidation nitrogen fixation or antibiotic production Reversetranscription PCRDGGE on community RNA can be used to profile metabolically active populations PCRDGGE allows for the rapid comparison of multiple samples and when used in combination with other approaches provides robust information of environmental microbial communities Recent uses in plant protection research include examination of effects of pesticides and biocontrol agents on microbial populations soil disease suppression plant rhizosphere communities microbemineral interactions microbeinsect interactions insect microbiota predator prey studies and community responses to changing farming practices

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