scholarly journals Diet Breadth Affects Bacterial Identity but Not Diversity in the Pollen Provisions of Closely Related Polylectic and Oligolectic Bees

Insects ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 645
Author(s):  
Jason A. Rothman ◽  
Diana L. Cox-Foster ◽  
Corey Andrikopoulos ◽  
Quinn S. McFrederick
Keyword(s):  
Host Species ◽  
High Throughput Sequencing ◽  
Microbial Community Composition ◽  
Diet Breadth ◽  
Solitary Bees ◽  
Rrna Gene ◽  
Osmia Lignaria ◽  
Future Studies ◽  
Functional Consequences ◽  
Oligolectic Bees

Mounting evidence suggests that microbes found in the pollen provisions of wild and solitary bees are important drivers of larval development. As these microbes are also known to be transmitted via the environment, most likely from flowers, the diet breadth of a bee may affect the diversity and identity of the microbes that occur in its pollen provisions. Here, we tested the hypothesis that, due to the importance of floral transmission of microbes, diet breadth affects pollen provision microbial community composition. We collected pollen provisions at four sites from the polylectic bee Osmia lignaria and the oligolectic bee Osmia ribifloris. We used high-throughput sequencing of the bacterial 16S rRNA gene to characterize the bacteria found in these provisions. We found minimal overlap in the specific bacterial variants in pollen provisions across the host species, even when the bees were constrained to foraging from the same flowers in cages at one site. Similarly, there was minimal overlap in the specific bacterial variants across sites, even within the same host species. Together, these findings highlight the importance of environmental transmission and host specific sorting influenced by diet breadth for microbes found in pollen provisions. Future studies addressing the functional consequences of this filtering, along with tests for differences between more species of oligoletic and polylectic bees will provide rich insights into the microbial ecology of solitary bees.

scholarly journals Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform

mSystems ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Johanna B. Holm ◽  
Michael S. Humphrys ◽  
Courtney K. Robinson ◽  
Matthew L. Settles ◽  
Sandra Ott ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sequence Data ◽  
Microbial Community Composition ◽  
Pcr Amplification ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Illumina Hiseq ◽  
Illumina Miseq Platform ◽  
Miseq Platform

ABSTRACT Amplification, sequencing, and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300-bp paired-end reads of higher quality than those produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-step PCR amplification protocol is also described that allows for targeting of different amplicon regions, and enhances amplification success from samples with low bacterial bioburden. IMPORTANCE Amplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high-throughput sequencing have made it a widely adopted approach, especially for projects that necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per-sample cost relative to those of the Illumina MiSeq platform without sacrificing amplicon length. To make this method more flexible for various amplicon-targeted regions as well as improve amplification from low-biomass samples, we also present and validate a 2-step PCR library preparation method.

scholarly journals Ultra-high throughput multiplexing and sequencing of >500 bp amplicon regions on the Illumina HiSeq 2500 platform

2018 ◽  
Author(s):  
Johanna B. Holm ◽  
Michael S. Humphrys ◽  
Courtney K. Robinson ◽  
Matthew L. Settles ◽  
Sandra Ott ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Microbial Community Composition ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Illumina Hiseq ◽  
Illumina Miseq Platform ◽  
Miseq Platform

AbstractAmplification, sequencing and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300 bp paired-end reads of higher quality than produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-Step PCR amplification protocol is also described that allows for targeting of different amplicon regions, thus improving amplification success from low bacterial bioburden samples.ImportanceAmplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high throughput sequencing have made it a widely-adopted approach, especially for projects which necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per sample cost relative to the Illumina MiSeq platform, without sacrificing amplicon length. To make this method more flexible to various amplicon targeted regions as well as improve amplification from low biomass samples, we also present and validate a 2-Step PCR library preparation method.

scholarly journals CoMiniGut—a small volumein vitrocolon model for the screening of gut microbial fermentation processes

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4268 ◽  
Author(s):  
Maria Wiese ◽  
Bekzod Khakimov ◽  
Sebastian Nielsen ◽  
Helena Sørensen ◽  
Frans van den Berg ◽  
...  
Keyword(s):  
Relative Abundance ◽  
High Throughput Sequencing ◽  
Microbial Community Composition ◽  
Short Chain Fatty Acids ◽  
Rrna Gene ◽  
Microbial Fermentation ◽  
Data Sets ◽  
Microbial Composition ◽  
Working Volume

Driven by the growing recognition of the influence of the gut microbiota (GM) on human health and disease, there is a rapidly increasing interest in understanding how dietary components, pharmaceuticals and pre- and probiotics influence GM.In vitrocolon models represent an attractive tool for this purpose. With the dual objective of facilitating the investigation of rare and expensive compounds, as well as an increased throughput, we have developed a prototypein vitroparallel gut microbial fermentation screening tool with a working volume of only 5 ml consisting of five parallel reactor units that can be expanded with multiples of five to increase throughput. This allows e.g., the investigation of interpersonal variations in gut microbial dynamics and the acquisition of larger data sets with enhanced statistical inference. The functionality of thein vitrocolon model, Copenhagen MiniGut (CoMiniGut) was first demonstrated in experiments with two common prebiotics using the oligosaccharide inulin and the disaccharide lactulose at 1% (w/v). We then investigated fermentation of the scarce and expensive human milk oligosaccharides (HMOs) 3-Fucosyllactose, 3-Sialyllactose, 6-Sialyllactose and the more common Fructooligosaccharide in fermentations with infant gut microbial communities. Investigations of microbial community composition dynamics in the CoMiniGut reactors by MiSeq-based 16S rRNA gene amplicon high throughput sequencing showed excellent experimental reproducibility and allowed us to extract significant differences in gut microbial composition after 24 h of fermentation for all investigated substrates and fecal donors. Furthermore, short chain fatty acids (SCFAs) were quantified for all treatments and donors. Fermentations with inulin and lactulose showed that inulin leads to a microbiota dominated by obligate anaerobes, with high relative abundance of Bacteroidetes, while the more easily fermented lactulose leads to higher relative abundance of Proteobacteria. The subsequent study on the influence of HMOs on two infant GM communities, revealed the strongest bifidogenic effect for 3′SL for both infants. Inter-individual differences of infant GM, especially with regards to the occurrence of Bacteroidetes and differences in bifidobacterial species composition, correlated with varying degrees of HMO utilization foremost of 6′SL and 3′FL, indicating species and strain related differences in HMO utilization which was also reflected in SCFAs concentrations, with 3′SL and 6′SL resulting in significantly higher butyrate production compared to 3′FL. In conclusion, the increased throughput of CoMiniGut strengthens experimental conclusions through elimination of statistical interferences originating from low number of repetitions. Its small working volume moreover allows the investigation of rare and expensive bioactives.

scholarly journals The Madness of Microbiome: Attempting To Find Consensus “Best Practice” for 16S Microbiome Studies

2018 ◽  
Vol 84 (7) ◽  
Author(s):  
Jolinda Pollock ◽  
Laura Glendinning ◽  
Trong Wisedchanwet ◽  
Mick Watson
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Best Practice ◽  
High Throughput Sequencing ◽  
Microbial Community Composition ◽  
Short Review ◽  
Rrna Gene ◽  
Processing Step ◽  
Sequencing Platforms ◽  
Processing Steps

ABSTRACTThe development and continuous improvement of high-throughput sequencing platforms have stimulated interest in the study of complex microbial communities. Currently, the most popular sequencing approach to study microbial community composition and dynamics is targeted 16S rRNA gene metabarcoding. To prepare samples for sequencing, there are a variety of processing steps, each with the potential to introduce bias at the data analysis stage. In this short review, key information from the literature pertaining to each processing step is described, and consequently, general recommendations for future 16S rRNA gene metabarcoding experiments are made.

scholarly journals A conceptual framework modeling of functional microbial communities in wastewater treatment electro-bioreactors

2020 ◽  
Vol 82 (12) ◽  
pp. 3047-3061
Author(s):  
Nancy A. ElNaker ◽  
Abdelsattar M. Sallam ◽  
El-Sayed M. El-Sayed ◽  
H. El Ghandoor ◽  
M. S. Talaat ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
Conceptual Framework ◽  
Microbial Communities ◽  
Conceptual Model ◽  
High Throughput Sequencing ◽  
Microbial Community Composition ◽  
Operating Conditions ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Operational Parameters

Abstract Understanding the microbial ecology of a system allows linking members of the community and their metabolic functions to the performance of the wastewater bioreactor. This study provided a comprehensive conceptual framework for microbial communities in wastewater treatment electro-bioreactors (EBRs). The model was based on data acquired from monitoring the effect of altering different bioreactor operational parameters, such as current density and hydraulic retention time, on the microbial communities of an EBR and its nutrient removal efficiency. The model was also based on the 16S rRNA gene high-throughput sequencing data analysis and bioreactor efficiency data. The collective data clearly demonstrated that applying various electric currents affected the microbial community composition and stability and the reactor efficiency in terms of chemical oxygen demand, N and P removals. Moreover, a schematic that recommends operating conditions that are tailored to the type of wastewater that needs to be treated based on the functional microbial communities enriched at specific operating conditions was suggested. In this study, a conceptual model as a simplified representation of the behavior of microbial communities in EBRs was developed. The proposed conceptual model can be used to predict how biological treatment of wastewater in EBRs can be improved by varying several operating conditions.

scholarly journals Phosphorus forms in the sediment of seagrass meadows affected mainly by fungi rather than bacteria: a preliminary study based on 31P-NMR and high-throughput sequencing

2020 ◽  
Vol 49 (4) ◽  
pp. 408-420
Author(s):  
Muqiu Zhao ◽  
Hui Wang ◽  
Shuai Wang ◽  
Qiuying Han ◽  
Yunfeng Shi
Keyword(s):  
High Throughput ◽  
Relative Abundance ◽  
Tidal Flat ◽  
High Throughput Sequencing ◽  
Coastal Wetland ◽  
Microbial Community Composition ◽  
Rrna Gene ◽  
Wetland Ecosystems ◽  
Seagrass Meadows ◽  
P Cycling

AbstractMicroorganisms play an important role in the circulation of phosphorus (P) in the sediment of coastal wetland ecosystems. In this study, solution 31P nuclear magnetic resonance (NMR) was used to determine different forms of P in the sediments of four different seagrass meadows and a bare tidal flat, while high-throughput 16S and ITS rRNA gene sequencing was used to determine the microbial community composition. The solution 31P-NMR spectra revealed six forms of the P compounds detected by the NaOH-EDTA extraction of sediments, where Ortho-P was the most dominant P compound, followed by Mono-P. The Po compounds were more varied in the seagrass meadow sediments and more abundant compared to the bare tidal flat. Bacterial communities in the sediments collected from E. acoroides and fungal communities in the bare tidal flat were relatively different from those at the other sites. The relative abundance of P-cycling-related fungi belonging to the phylum Ascomycota was 26.20% and was much higher than that of bacteria (only 0.29%) belonging to the class Bacilli. Mono-P was the major factor determining the distribution of P-cycling-related fungi and negatively correlated with the relative abundance of Aspergillus and Trichoderma. We believe that fungi can affect P forms in the sediment of seagrass meadows more than bacteria.

scholarly journals Benchmarking laboratory processes to characterise low-biomass respiratory microbiota

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raiza Hasrat ◽  
Jolanda Kool ◽  
Wouter A. A. de Steenhuijsen Piters ◽  
Mei Ling J. N. Chu ◽  
Sjoerd Kuiling ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Composition ◽  
Microbial Community Composition ◽  
Positive Control ◽  
Rrna Gene ◽  
Elution Buffer ◽  
Community Profile ◽  
Microbial Community Profile ◽  
Microbial Dna ◽  
Pcr Conditions

AbstractThe low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available laboratory protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community profile, we amplified the 16S rRNA gene of respiratory samples using various bacterial loads and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 or V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community profile of low biomass samples. Purification methods and MiSeq reagent kits provided nearly similar microbiota profiles (paired Bray–Curtis dissimilarity median: 0.03 and 0.05, respectively). While profiles of positive controls were significantly influenced by the type of dilution solvent, the theoretical profile of the Zymo mock was most accurately analysed when the Zymo mock was diluted in elution buffer (difference compared to the theoretical Zymo mock: 21.6% for elution buffer, 29.2% for Milli-Q, and 79.6% for DNA/RNA shield). Microbiota profiles of DNA blanks formed a distinct cluster compared to low biomass samples, demonstrating that low biomass samples can accurately be distinguished from DNA blanks. In summary, to accurately characterise the microbial community composition we recommend 1. amplification of the obtained microbial DNA with 30 PCR cycles, 2. purifying amplicon pools by two consecutive AMPure XP steps and 3. sequence the pooled amplicons by V3 MiSeq reagent kit. The benchmarked standardized laboratory workflow presented here ensures comparability of results within and between low biomass microbiome studies.

scholarly journals 2,4-Dichlorophenoxyacetic acid degradation in methanogenic mixed cultures obtained from Brazilian Amazonian soil samples

2021 ◽  
Author(s):  
Gunther Brucha ◽  
Andrea Aldas-Vargas ◽  
Zacchariah Ross ◽  
Peng Peng ◽  
Siavash Atashgahi ◽  
...  
Keyword(s):  
Microbial Community Composition ◽  
High Mobility ◽  
16S Rrna Gene Sequencing ◽  
Dichlorophenoxyacetic Acid ◽  
Rrna Gene ◽  
Deep Soil ◽  
Anoxic Environments ◽  
Top Soil ◽  
Significant Enrichment ◽  
2,4 Dichlorophenoxyacetic Acid

Abstract2,4-Dichlorophenoxyacetic acid (2,4-D) is the third most applied pesticide in Brazil to control broadleaf weeds in crop cultivation and pastures. Due to 2,4-D’s high mobility and long half-life under anoxic conditions, this herbicide has high probability for groundwater contamination. Bioremediation is an attractive solution for 2,4-D contaminated anoxic environments, but there is limited understanding of anaerobic 2,4-D biodegradation. In this study, methanogenic enrichment cultures were obtained from Amazonian top soil (0—40 cm) and deep soil (50 -80 cm below ground) that biotransform 2,4-D (5 µM) to 4-chlorophenol and phenol. When these cultures were transferred (10% v/v) to fresh medium containing 40 µM or 160 µM 2,4-D, the rate of 2,4-D degradation decreased, and biotransformation did not proceed beyond 4-chlorophenol and 2,4-dichlorophenol in the top and deep soil cultures, respectively. 16S rRNA gene sequencing and qPCR of a selection of microbes revealed no significant enrichment of known organohalide-respiring bacteria. Furthermore, a member of the genus Cryptanaerobacter was identified as possibly responsible for phenol conversion to benzoate in the top soil inoculated culture. Overall, these results demonstrate the effect of 2,4-D concentration on biodegradation and microbial community composition, which are both important factors when developing pesticide bioremediation technologies.

scholarly journals Gut Microbiome Changes in Captive Plateau Zokors (Eospalax baileyi)

2021 ◽  
Vol 17 ◽  
pp. 117693432199635
Author(s):  
Daoxin Liu ◽  
Pengfei Song ◽  
Jingyan Yan ◽  
Haijing Wang ◽  
Zhenyuan Cai ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Gut Microbiome ◽  
High Throughput Sequencing ◽  
Tibet Plateau ◽  
Rrna Gene ◽  
Diversity Analysis ◽  
Bacterial Phyla ◽  
In Captivity ◽  
Relative Abundances ◽  
Plateau Zokor

Wild-caught animals must cope with drastic lifestyle and dietary changes after being induced to captivity. How the gut microbiome structure of these animals will change in response receives increasing attention. The plateau zokor ( Eospalax baileyi), a typic subterranean rodent endemic to the Qinghai-Tibet plateau, spends almost the whole life underground and is well adapted to the environmental pressures of both plateau and underground. However, how the gut microbiome of the plateau zokor will change in response to captivity has not been reported to date. This study compared the microbial community structure and functions of 22 plateau zokors before (the WS group) and after being kept in captivity for 15 days (the LS group, fed on carrots) using the 16S rRNA gene via high-throughput sequencing technology. The results showed that the LS group retained 973 of the 977 operational taxonomic units (OTUs) in the WS group, and no new OTUs were found in the LS group. The dominant bacterial phyla were Bacteroides and Firmicutes in both groups. In alpha diversity analysis, the Shannon, Sobs, and ACE indexes of the LS group were significantly lower than those of the WS group. A remarkable difference ( P < 0.01) between groups was also detected in beta diversity analysis. The UPGMA clustering, NMDS, PCoA, and Anosim results all showed that the intergroup difference was significantly greater than the intragroup difference. And compared with the WS group, the intragroup difference of the gut microbiota in the LS group was much larger, which failed to support the assumption that similar diets should drive convergence of gut microbial communities. PICRUSt revealed that although some functional categories displayed significant differences between groups, the relative abundances of these categories were very close in both groups. Based on all the results, we conclude that as plateau zokors enter captivity for a short time, although the relative abundances of different gut microbiota categories shifted significantly, they can maintain almost all the OTUs and the functions of the gut microbiota in the wild. So, the use of wild-caught plateau zokors in gut microbial studies is acceptable if the time in captivity is short.

scholarly journals The Fecal Bacterial Microbiota in Horses with Equine Recurrent Uveitis

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 745
Author(s):  
Michelle Martin de Bustamante ◽  
Diego Gomez ◽  
Jennifer MacNicol ◽  
Ralph Hamor ◽  
Caryn Plummer
Keyword(s):  
Beta Diversity ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Diversity Indices ◽  
Rrna Gene ◽  
Linear Discriminant ◽  
Alpha And Beta Diversity ◽  
Bacterial Microbiota ◽  
Healthy Control ◽  
Equine Recurrent Uveitis

The objective of this study was to describe and compare the fecal bacterial microbiota of horses with equine recurrent uveitis (ERU) and healthy horses using next-generation sequencing techniques. Fecal samples were collected from 15 client-owned horses previously diagnosed with ERU on complete ophthalmic examination. For each fecal sample obtained from a horse with ERU, a sample was collected from an environmentally matched healthy control with no evidence of ocular disease. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene. The relative abundance of predominant taxa, and alpha and beta diversity indices were calculated and compared between groups. The phyla Firmicutes, Bacteroidetes, Verrucomicrobia, and Proteobacteria predominated in both ERU and control horses, accounting for greater than 60% of sequences. Based on linear discriminant analysis effect size (LEfSe), no taxa were found to be enriched in either group. No significant differences were observed in alpha and beta diversity indices between groups (p > 0.05 for all tests). Equine recurrent uveitis is not associated with alteration of the gastrointestinal bacterial microbiota when compared with healthy controls.

Sign in / Sign up

Export Citation Format

Share Document