scholarly journals EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach

2021 ◽  
Vol 9 (8) ◽  
pp. 1649
Author(s):  
Véronica L. Roman ◽  
Christophe Merlin ◽  
Marko P. J. Virta ◽  
Xavier Bellanger
Keyword(s):  
Microbial Communities ◽  
Single Cell ◽  
Operational Taxonomic Unit ◽  
Rrna Gene ◽  
Environmental Matrices ◽  
Taxonomic Assignment ◽  
Integrative And Conjugative Elements ◽  
Functional Sequence ◽  
Methodological Improvement ◽  
New Gene

EpicPCR (Emulsion, Paired Isolation and Concatenation PCR) is a recent single-cell genomic method based on a fusion-PCR allowing us to link a functional sequence of interest to a 16S rRNA gene fragment and use the mass sequencing of the resulting amplicons for taxonomic assignment of the functional sequence-carrying bacteria. Although it is interesting because it presents the highest efficiency for assigning a bacterial host to a marker, epicPCR remains a complex multistage procedure with technical difficulties that may easily impair the approach depth and quality. Here, we described how to adapt epicPCR to new gene targets and environmental matrices while identifying the natural host range of SXT/R391 integrative and conjugative elements in water microbial communities from the Meurthe River (France). We notably show that adding a supplementary PCR step allowed us to increase the amplicon yield and thus the number of reads obtained after sequencing. A comparison of operational taxonomic unit (OTU) identification approaches when using biological and technical replicates demonstrated that, although OTUs can be validated when obtained from three out of three technical replicates, up to now, results obtained from two or three biological replicates give a similar and even a better confidence level in OTU identification, while allowing us to detect poorly represented SXT/R391 hosts in microbial communities.

scholarly journals MicroPheno: Predicting environments and host phenotypes from 16S rRNA gene sequencing using a k-mer based representation of shallow sub-samples

2018 ◽  
Author(s):  
Ehsaneddin Asgari ◽  
Kiavash Garakani ◽  
Alice Carolyn McHardy ◽  
Mohammad R.K. Mofrad
Keyword(s):  
Deep Learning ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Gene Sequencing ◽  
Operational Taxonomic Unit ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Sequence Alignments ◽  
Rrna Gene Sequencing

Motivation: Microbial communities play important roles in the function and maintenance of various biosystems, ranging from the human body to the environment. A major challenge in microbiome research is the classification of microbial communities of different environments or host phenotypes. The most common and cost-effective approach for such studies to date is 16S rRNA gene sequencing. Recent falls in sequencing costs have increased the demand for simple, efficient, and accurate methods for rapid detection or diagnosis with proved applications in medicine, agriculture, and forensic science. We describe a reference- and alignment-free approach for predicting environments and host phenotypes from 16S rRNA gene sequencing based on k-mer representations that benefits from a bootstrapping framework for investigating the sufficiency of shallow sub-samples. Deep learning methods as well as classical approaches were explored for predicting environments and host phenotypes. Results: k-mer distribution of shallow sub-samples outperformed the computationally costly Operational Taxonomic Unit (OTU) features in the tasks of body-site identification and Crohn's disease prediction. Aside from being more accurate, using k-mer features in shallow sub-samples allows (i) skipping computationally costly sequence alignments required in OTU-picking, and (ii) provided a proof of concept for the sufficiency of shallow and short-length 16S rRNA sequencing for phenotype prediction. In addition, k-mer features predicted representative 16S rRNA gene sequences of 18 ecological environments, and 5 organismal environments with high macro-F1 scores of 0.88 and 0.87. For large datasets, deep learning outperformed classical methods such as Random Forest and SVM. Availability: The software and datasets are available at https://llp.berkeley.edu/micropheno.

A peek in the micro-sized world: a review of design principles, engineering tools, and applications of engineered microbial community

2020 ◽  
Vol 48 (2) ◽  
pp. 399-409
Author(s):  
Baizhen Gao ◽  
Rushant Sabnis ◽  
Tommaso Costantini ◽  
Robert Jinkerson ◽  
Qing Sun
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Environmental Remediation ◽  
Real Life ◽  
Design Principles ◽  
Microbial Interactions ◽  
Potential Applications ◽  
New Gene ◽  
Global Biogeochemical Cycles ◽  
Synthetic Microbial Communities

Microbial communities drive diverse processes that impact nearly everything on this planet, from global biogeochemical cycles to human health. Harnessing the power of these microorganisms could provide solutions to many of the challenges that face society. However, naturally occurring microbial communities are not optimized for anthropogenic use. An emerging area of research is focusing on engineering synthetic microbial communities to carry out predefined functions. Microbial community engineers are applying design principles like top-down and bottom-up approaches to create synthetic microbial communities having a myriad of real-life applications in health care, disease prevention, and environmental remediation. Multiple genetic engineering tools and delivery approaches can be used to ‘knock-in' new gene functions into microbial communities. A systematic study of the microbial interactions, community assembling principles, and engineering tools are necessary for us to understand the microbial community and to better utilize them. Continued analysis and effort are required to further the current and potential applications of synthetic microbial communities.

scholarly journals A Comparative Study on the Microbial Communities of Rhynchophorus ferrugineus (Red Palm Weevil)-Infected and Healthy Palm Trees

2021 ◽  
Author(s):  
N. Alshammari ◽  
Meshari Alazmi ◽  
Naimah A. Alanazi ◽  
Abdel Moneim E. Sulieman ◽  
Vajid N. Veettil ◽  
...  
Keyword(s):  
Microbial Communities ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rhynchophorus Ferrugineus ◽  
Tree Trunk ◽  
Red Palm Weevil ◽  
Healthy Tree ◽  
Palm Trees ◽  
Infected Tree ◽  
Palm Weevil

AbstractSeveral studies have investigated palm trees’ microbiota infected with red palm weevil (RPW) (Rhynchophorus ferrugineus), the major pest of palm trees. This study compared the microbial communities of infected and uninfected palm trees in the Hail region, Northern Saudi Arabia, determined by high-throughput 16S rRNA gene sequencing by Illumina MiSeq. The results indicated that taxonomic diversity variation was higher for infected tree trunk than the healthy tree trunk. Soil samples from the vicinity of healthy and infected trees did not have a significant variation in bacterial diversity. Myxococcota, Acidobacteriota, and Firmicutes were the dominant phyla in RPW-infected tree trunk, and Pseudomonadaceae was the most prominent family. This study is the first report on the characterization of RPW-infected and healthy palm trees’ microbiome.

scholarly journals Structure of Microbial Communities When Complementary Effluents Are Anaerobically Digested

2021 ◽  
Vol 11 (3) ◽  
pp. 1293
Author(s):  
Ana Eusébio ◽  
André Neves ◽  
Isabel Paula Marques
Keyword(s):  
Anaerobic Digestion ◽  
Microbial Communities ◽  
Volatile Fatty Acids ◽  
Olive Mill Wastewater ◽  
Organic Load ◽  
Rrna Gene ◽  
Bacterial Populations ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Olive Mill

Olive oil and pig productions are important industries in Portugal that generate large volumes of wastewater with high organic load and toxicity, raising environmental concerns. The principal objective of this study is to energetically valorize these organic effluents—piggery effluent and olive mill wastewater—through the anaerobic digestion to the biogas/methane production, by means of the effluent complementarity concept. Several mixtures of piggery effluent were tested, with an increasing percentage of olive mill wastewater. The best performance was obtained for samples of piggery effluent alone and in admixture with 30% of OMW, which provided the same volume of biogas (0.8 L, 70% CH4), 63/75% COD removal, and 434/489 L CH4/kg SVin, respectively. The validation of the process was assessed by molecular evaluation through Next Generation Sequencing (NGS) of the 16S rRNA gene. The structure of the microbial communities for both samples, throughout the anaerobic process, was characterized by the predominance of bacterial populations belonging to the phylum Firmicutes, mainly Clostridiales, with Bacteroidetes being the subdominant populations. Archaea populations belonging to the genus Methanosarcina became predominant throughout anaerobic digestion, confirming the formation of methane mainly from acetate, in line with the greatest removal of volatile fatty acids (VFAs) in these samples.

scholarly journals Dissimilarity of microbial diversity of pond water, shrimp intestine and sediment in Aquamimicry system

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Shenzheng Zeng ◽  
Sukontorn Khoruamkid ◽  
Warinphorn Kongpakdee ◽  
Dongdong Wei ◽  
Lingfei Yu ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Well Being ◽  
Pacific White Shrimp ◽  
Shrimp Farming ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Shrimp Culture ◽  
Water And Sediment ◽  
Surrounding Environment ◽  
Shrimp Production

Abstract The Pacific white shrimp, with the largest production in shrimp industry, has suffered from multiple severe viral and bacterial diseases, which calls for a more reliable and environmentally friendly system to promote shrimp culture. The “Aquamimicry system”, mimicking the nature of aquatic ecosystems for the well-being of aquatic animals, has effectively increased shrimp production and been adapted in many countries. However, the microbial communities in the shrimp intestine and surrounding environment that act as an essential component in Aquamimicry remain largely unknown. In this study, the microbial composition and diversity alteration in shrimp intestine, surrounding water and sediment at different culture stages were investigated by high throughput sequencing of 16S rRNA gene, obtaining 13,562 operational taxonomic units (OTUs). Results showed that the microbial communities in shrimp intestine and surrounding environment were significantly distinct from each other, and 23 distinguished taxa for each habitat were further characterized. The microbial communities differed significantly at different culture stages, confirmed by a great number of OTUs dramatically altered during the culture period. A small part of these altered OTUs were shared between shrimp intestine and surrounding environment, suggesting that the microbial alteration of intestine was not consistent with that of water and sediment. Regarding the high production of Aquamimicry farm used as a case in this study, the dissimilarity between intestinal and surrounding microbiota might be considered as a potential indicator for healthy status of shrimp farming, which provided hints on the appropriate culture practices to improve shrimp production.

scholarly journals Microbial drivers of methane emissions from unrestored industrial salt ponds

2021 ◽  
Author(s):  
Jinglie Zhou ◽  
Susanna M. Theroux ◽  
Clifton P. Bueno de Mesquita ◽  
Wyatt H. Hartman ◽  
Ye Tian ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Methane Flux ◽  
Methane Emissions ◽  
Metagenomic Data ◽  
Rrna Gene ◽  
Salt Ponds ◽  
Salt Pond ◽  
Reference Wetlands ◽  
Reference Wetland ◽  
The Impact

AbstractWetlands are important carbon (C) sinks, yet many have been destroyed and converted to other uses over the past few centuries, including industrial salt making. A renewed focus on wetland ecosystem services (e.g., flood control, and habitat) has resulted in numerous restoration efforts whose effect on microbial communities is largely unexplored. We investigated the impact of restoration on microbial community composition, metabolic functional potential, and methane flux by analyzing sediment cores from two unrestored former industrial salt ponds, a restored former industrial salt pond, and a reference wetland. We observed elevated methane emissions from unrestored salt ponds compared to the restored and reference wetlands, which was positively correlated with salinity and sulfate across all samples. 16S rRNA gene amplicon and shotgun metagenomic data revealed that the restored salt pond harbored communities more phylogenetically and functionally similar to the reference wetland than to unrestored ponds. Archaeal methanogenesis genes were positively correlated with methane flux, as were genes encoding enzymes for bacterial methylphosphonate degradation, suggesting methane is generated both from bacterial methylphosphonate degradation and archaeal methanogenesis in these sites. These observations demonstrate that restoration effectively converted industrial salt pond microbial communities back to compositions more similar to reference wetlands and lowered salinities, sulfate concentrations, and methane emissions.

scholarly journals Collection of a Bacterial Community Reconstructed from Marine Metagenomes Derived from Jinhae Bay, South Korea

Data ◽  
2021 ◽  
Vol 6 (5) ◽  
pp. 44
Author(s):  
Jae-Hyun Lim ◽  
Il-Nam Kim
Keyword(s):  
Microbial Community ◽  
South Korea ◽  
Microbial Communities ◽  
Marine Bacteria ◽  
Sampling Site ◽  
Community Change ◽  
Rrna Gene ◽  
Water Column Stratification ◽  
Metagenomics Data ◽  
Jinhae Bay

Marine bacteria are known to play significant roles in marine biogeochemical cycles regarding the decomposition of organic matter. Despite the increasing attention paid to the study of marine bacteria, research has been too limited to fully elucidate the complex interaction between marine bacterial communities and environmental variables. Jinhae Bay, the study area in this work, is the most anthropogenically eutrophied coastal bay in South Korea, and while its physical and biogeochemical characteristics are well described, less is known about the associated changes in microbial communities. In the present study, we reconstructed a metagenomics data based on the 16S rRNA gene to investigate temporal and vertical changes in microbial communities at three depths (surface, middle, and bottom) during a seven-month period from June to December 2016 at one sampling site (J1) in Jinhae Bay. Of all the bacterial data, Proteobacteria, Bacteroidetes, and Cyanobacteria were predominant from June to November, whereas Firmicutes were predominant in December, especially at the middle and bottom depths. These results show that the composition of the microbial community is strongly associated with temporal changes. Furthermore, the community compositions were markedly different between the surface, middle, and bottom depths in summer, when water column stratification and bottom water hypoxia (low dissolved oxygen level) were strongly developed. Metagenomics data contribute to improving our understanding of important relationships between environmental characteristics and microbial community change in eutrophication-induced and deoxygenated coastal areas.

scholarly journals Impact of Substratum Surface on Microbial Community Structure and Treatment Performance in Biological Aerated Filters

2013 ◽  
Vol 80 (1) ◽  
pp. 177-183 ◽  
Author(s):  
Lavane Kim ◽  
Eulyn Pagaling ◽  
Yi Y. Zuo ◽  
Tao Yan
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Bacterial Species ◽  
Community Analysis ◽  
Microbial Community Analysis ◽  
Rrna Gene ◽  
Content Type ◽  
Property Change ◽  
And Control ◽  
Start Ups

ABSTRACTThe impact of substratum surface property change on biofilm community structure was investigated using laboratory biological aerated filter (BAF) reactors and molecular microbial community analysis. Two substratum surfaces that differed in surface properties were created via surface coating and used to develop biofilms in test (modified surface) and control (original surface) BAF reactors. Microbial community analysis by 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis (DGGE) showed that the surface property change consistently resulted in distinct profiles of microbial populations during replicate reactor start-ups. Pyrosequencing of the bar-coded 16S rRNA gene amplicons surveyed more than 90% of the microbial diversity in the microbial communities and identified 72 unique bacterial species within 19 bacterial orders. Among the 19 orders of bacteria detected,BurkholderialesandRhodocyclalesof theBetaproteobacteriaclass were numerically dominant and accounted for 90.5 to 97.4% of the sequence reads, and their relative abundances in the test and control BAF reactors were different in consistent patterns during the two reactor start-ups. Three of the five dominant bacterial species also showed consistent relative abundance changes between the test and control BAF reactors. The different biofilm microbial communities led to different treatment efficiencies, with consistently higher total organic carbon (TOC) removal in the test reactor than in the control reactor. Further understanding of how surface properties affect biofilm microbial communities and functional performance would enable the rational design of new generations of substrata for the improvement of biofilm-based biological treatment processes.

scholarly journals Assessing high-throughput environmental DNA extraction methods for meta-barcode characterization of aquatic microbial communities

2018 ◽  
Vol 17 (1) ◽  
pp. 37-49 ◽  
Author(s):  
Abdolrazagh Hashemi Shahraki ◽  
Subba Rao Chaganti ◽  
Daniel Heath
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
High Throughput ◽  
Environmentally Friendly ◽  
Environmental Dna ◽  
Magnetic Bead ◽  
Extraction Methods ◽  
Environmental Research ◽  
Rrna Gene

Abstract The characterization of microbial community dynamics using genomic methods is rapidly expanding, impacting many fields including medical, ecological, and environmental research and applications. One of the biggest challenges for such studies is the isolation of environmental DNA (eDNA) from a variety of samples, diverse microbes, and widely variable community compositions. The current study developed environmentally friendly, user safe, economical, and high throughput eDNA extraction methods for mixed aquatic microbial communities and tested them using 16 s rRNA gene meta-barcoding. Five different lysis buffers including (1) cetyltrimethylammonium bromide (CTAB), (2) digestion buffer (DB), (3) guanidinium isothiocyanate (GITC), (4) sucrose lysis (SL), and (5) SL-CTAB, coupled with four different purification methods: (1) phenol-chloroform-isoamyl alcohol (PCI), (2) magnetic Bead-Robotic, (3) magnetic Bead-Manual, and (4) membrane-filtration were tested for their efficacy in extracting eDNA from recreational freshwater samples. Results indicated that the CTAB-PCI and SL-Bead-Robotic methods yielded the highest genomic eDNA concentrations and succeeded in detecting the core microbial community including the rare microbes. However, our study recommends the SL-Bead-Robotic eDNA extraction protocol because this method is safe, environmentally friendly, rapid, high-throughput and inexpensive.

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