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16s rRNA Sequencing

16S ribosomal  (rRNA) sequencing using next generation sequencing is a method used to identify and compare bacteria and archaea present within almost any type of sample. 16S rRNA gene sequencing is a well-established method for studying phylogeny and taxonomy of samples from complex microbiomes or environments that are difficult or impossible to study.

 

 

 

 

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Sci Rep. 2016 Sep 27;6:33965. doi: 10.1038/srep33965.

Probiotic legacy effects on gut microbial assembly in tilapia larvae.

Giatsis C1, Sipkema D2, Ramiro-Garcia J2,3,4, Bacanu GM2, Abernathy J5, Verreth J1, Smidt H2, Verdegem M1.

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Abstract

The exposure of fish to environmental free-living microbes and its effect on early colonization in the gut have been studied in recent years. However, little is known regarding how the host and environment interact to shape gut communities during early life. Here, we tested whether the early microbial exposure of tilapia larvae affects the gut microbiota at later life stages. The experimental period was divided into three stages: axenic, probiotic and active suspension. Axenic tilapia larvae were reared either under conventional conditions (active suspension systems) or exposed to a single strain probiotic (Bacillus subtilis) added to the water. Microbial characterization by Illumina HiSeq sequencing of 16S rRNA gene amplicons showed the presence of B. subtilis in the gut during the seven days of probiotic application. Although B. subtilis was no longer detected in the guts of fish exposed to the probiotic after day 7, gut microbiota of the exposed tilapia larvae remained significantly different from that of the control treatment. Compared with the control, fish gut microbiota under probiotic treatment was less affected by spatial differences resulting from tank replication, suggesting that the early probiotic contact contributed to the subsequent observation of low inter-individual variation.

PMID: 27670882 DOI: 10.1038/srep33965

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Select item 27670106

 

2.

Environ Sci Technol. 2016 Sep 27. [Epub ahead of print]

Structure, Variation, and Co-occurrence of Soil Microbial Communities in Abandoned Sites of a Rare Earth Elements Mine.

Chao Y, Liu W, Chen Y, Chen W, Zhao L, Ding Q, Wang S, Tang YT, Zhang T, Qiu RL.

Abstract

Mining activity for rare earth elements (REEs) has caused serious environmental pollution, particularly for soil ecosystems. However, the effects of REEs on soil microbiota are still poorly understood. In this study, soils were collected from abandoned sites of a REEs mine, and the structure, diversity, and co-occurrence patterns of soil microbiota were evaluated by Illumina high-throughput sequencing targeting 16S rRNA genes. Although microbiota developed significantly along with the natural restoration, the microbial structure on the site abandoned for 10 years still significantly differed from that on the unmined site. Potential plant growth promoting bacteria (PGPB) were identified by comparing 16S sequences against a self-constructed PGPB database via BLAST, and it was found that siderophore-producing and phosphorus-solubilizing bacteria were more abundant in the studied soils than in reference soils. Canonical correspondence analysis indicated that species richness of plant community was the prime factor affecting microbial structure, followed by limiting nutrients (total carbon and total nitrogen) and REEs content. Further co-occurring network analysis revealed nonrandom assembly patterns of microbiota in the studied soils. These results increase our understanding of microbial variation and assembly pattern during natural restoration in REE contaminated soils.

PMID: 27670106 DOI: 10.1021/acs.est.6b02284

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Select item 27666090

MR DNA 16s sequencing service

3.

Sci Rep. 2016 Sep 26;6:34090. doi: 10.1038/srep34090.

Evaluating digestion efficiency in full-scale anaerobic digesters by identifying active microbial populations through the lens of microbial activity.

Mei R1, Narihiro T1,2, Nobu MK1, Kuroda K1,3, Liu WT1.

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Abstract

Anaerobic digestion is a common technology to biologically stabilize wasted solids produced in municipal wastewater treatment. Its efficiency is usually evaluated by calculating the reduction in volatile solids, which assumes no biomass growth associated with digestion. To determine whether this assumption is valid and further evaluate digestion efficiency, this study sampled 35 digester sludge from different reactors at multiple time points together with the feed biomass in a full-scale water reclamation plant at Chicago, Illinois. The microbial communities were characterized using Illumina sequencing technology based on 16S rRNA and 16S rRNA gene (rDNA). 74 core microbial populations were identified and represented 58.7% of the entire digester community. Among them, active populations were first identified using the ratio of 16S rRNA and 16S rDNA (rRNA/rDNA) for individual populations, but this approach failed to generate consistent results. Subsequently, a recently proposed mass balance model was applied to calculate the specific growth rate (μ), and this approach successfully identified active microbial populations in digester (positive μ) that could play important roles than those with negative μ. It was further estimated that 82% of microbial populations in the feed sludge were digested in comparison with less than 50% calculated using current equations.

PMID: 27666090 DOI: 10.1038/srep34090

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Select item 27650378

 

4.

Mar Genomics. 2016 Sep 16. pii: S1874-7787(16)30091-5. doi: 10.1016/j.margen.2016.09.001. [Epub ahead of print]

Choice of molecular barcode will affect species prevalence but not bacterial community composition.

Lebret K1, Schroeder J2, Balestreri C2, Highfield A2, Cummings D3, Smyth T3, Schroeder D4.

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Abstract

The rapid advancement of next generation sequencing protocols in recent years has led to the diversification in the methods used to study microbial communities; however, how comparable the data generated from these different methods are, remains unclear. In this study we compared the taxonomic composition and seasonal dynamics of the bacterial community determined by two distinct 16s amplicon sequencing protocols: sequencing of the V6 region of the 16s rRNA gene using 454 pyrosequencing vs the V4 region of the 16s rRNA gene using the Illumina Hiseq 2500 platform. Significant differences between relative abundances at all taxonomic levels were observed; however, their seasonal dynamics between phyla were largely consistent between methods. This study highlights that care must be taken when comparing datasets generated from different methods.

Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

KEYWORDS:

16S; Illumina; Marine bacterial community; Pyrosequencing

PMID: 27650378 DOI: 10.1016/j.margen.2016.09.001

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Select item 27646687

 

5.

Sci Rep. 2016 Sep 20;6:33600. doi: 10.1038/srep33600.

A Hardy Plant Facilitates Nitrogen Removal via Microbial Communities in Subsurface Flow Constructed Wetlands in Winter.

Wang P1,2, Zhang H1, Zuo J1, Zhao D1, Zou X1, Zhu Z1,2, Jeelani N1, Leng X1,2, An S1,2.

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Abstract

The plants effect in subsurface flow constructed wetlands (SSF-CWs) is controversial, especially at low temperatures. Consequently, several SSF-CWs planted with Iris pseudacorus (CWI) or Typha orientalis Presl. (CWT) and several unplanted ones (CWC) were set up and fed with secondary effluent of sewage treatment plant during the winter in Eastern China. The 16S rDNA Illumina Miseq sequencing analysis indicated the positive effects of I. pseudacorus on the bacterial community richness and diversity in the substrate. Moreover, the community compositions of the bacteria involved with denitrification presented a significant difference in the three systems. Additionally, higher relative abundances of nitrifying bacteria (0.4140%, 0.2402% and 0.4318% for Nitrosomonas, Nitrosospira and Nitrospira, respectively) were recorded in CWI compared with CWT (0.2074%, 0.0648% and 0.0181%, respectively) and CWC (0.3013%, 0.1107% and 0.1185%, respectively). Meanwhile, the average removal rates of NH4(+)-N and TN in CWI showed a prominent advantage compared to CWC, but no distinct advantage was found in CWT. The hardy plant I. pseudacorus, which still had active root oxygen release in cold temperatures, positively affected the abundance of nitrifying bacteria in the substrate, and accordingly was supposed to contribute to a comparatively high nitrogen removal efficiency of the system during the winter.

PMID: 27646687 PMCID: PMC5028706 DOI: 10.1038/srep33600

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Select item 27645138

 

6.

Microb Ecol. 2016 Sep 19. [Epub ahead of print]

Microbial Community Dynamics and Response to Plant Growth-Promoting Microorganisms in the Rhizosphere of Four Common Food Crops Cultivated in Hydroponics.

Sheridan C1, Depuydt P1, De Ro M1, Petit C1, Van Gysegem E1, Delaere P1, Dixon M2, Stasiak M2, Aciksöz SB3, Frossard E3, Paradiso R4, De Pascale S4, Ventorino V4, De Meyer T5, Sas B6, Geelen D7.

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Abstract

Plant growth promoting microorganisms (PGPMs) of the plant root zone microbiome have received limited attention in hydroponic cultivation systems. In the framework of a project aimed at the development of a biological life support system for manned missions in space, we investigated the effects of PGPMs on four common food crops (durum and bread wheat, potato and soybean) cultivated in recirculating hydroponic systems for a whole life cycle. Each crop was inoculated with a commercial PGPM mixture and the composition of the microbial communities associated with their root rhizosphere, rhizoplane/endosphere and with the recirculating nutrient solution was characterised through 16S- and ITS-targeted Illumina MiSeq sequencing. PGPM addition was shown to induce changes in the composition of these communities, though these changes varied both between crops and over time. Microbial communities of PGPM-treated plants were shown to be more stable over time. Though additional development is required, this study highlights the potential benefits that PGPMs may confer to plants grown in hydroponic systems, particularly when cultivated in extreme environments such as space.

KEYWORDS:

Dynamics; Hydroponics; Plant; Plant growth-promoting organisms; Rhizosphere; Space

PMID: 27645138 DOI: 10.1007/s00248-016-0855-0

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Select item 27641252

 

7.

Bioresour Technol. 2016 Sep 14. pii: S0960-8524(16)31308-6. doi: 10.1016/j.biortech.2016.09.051. [Epub ahead of print]

Microbial community analysis using MiSeq sequencing in a novel configuration fluidized bed reactor for effective denitrification.

Rungkitwatananukul P1, Nomai S1, Hirakata Y2, Pungrasmi W3, Puprasert C1, Hatamoto M2, Yamaguchi T2.

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Abstract

A novel configured fluidized bed reactor (FBR) with granular rubber as the fluidized media was operated without internal recirculation to achieve denitrification. This FBR could operate under a low hydraulic retention time (HRT) of 50min due to the low rubber media density and absence of recirculation. Synthetic nitrate-rich wastewater with a fixed nitrate (NO3--N) concentration and varying COD concentrations was fed into the FBR. The nitrate removal profile showed a rapid nitrate reduction at the bottom of the reactor with a high performance under the low HRT. Different microbial communities were identified using Illumina Miseq sequencing. The dominant microorganisms belonged to the Beta- and Gamma-proteobacteria classes and played important roles in nitrate reduction. Acidovorax was abundant at low COD: NO3--N ratios, while Rhizobium and Zoogloea were dominant at high COD: NO3--N ratios. The COD: NO3--N ratio strongly influenced the composition of the microbial community including the dominant species.

Copyright © 2016 Elsevier Ltd. All rights reserved.

KEYWORDS:

16S rRNA gene sequencing; Denitrification; Fluidized bed reactor; Low COD to nitrate ratio; Microbial community analysis

PMID: 27641252 DOI: 10.1016/j.biortech.2016.09.051

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Select item 27635335

MR DNA 16s sequencing service

8.

PeerJ. 2016 Aug 24;4:e2367. doi: 10.7717/peerj.2367. eCollection 2016.

Tree phyllosphere bacterial communities: exploring the magnitude of intra- and inter-individual variation among host species.

Laforest-Lapointe I1, Messier C2, Kembel SW1.

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Abstract

BACKGROUND:

The diversity and composition of the microbial community of tree leaves (the phyllosphere) varies among trees and host species and along spatial, temporal, and environmental gradients. Phyllosphere community variation within the canopy of an individual tree exists but the importance of this variation relative to among-tree and among-species variation is poorly understood. Sampling techniques employed for phyllosphere studies include picking leaves from one canopy location to mixing randomly selected leaves from throughout the canopy. In this context, our goal was to characterize the relative importance of intra-individual variation in phyllosphere communities across multiple species, and compare this variation to inter-individual and interspecific variation of phyllosphere epiphytic bacterial communities in a natural temperate forest in Quebec, Canada.

METHODS:

We targeted five dominant temperate forest tree species including angiosperms and gymnosperms: Acer saccharum, Acer rubrum, Betula papyrifera, Abies balsamea and Picea glauca. For one randomly selected tree of each species, we sampled microbial communities at six distinct canopy locations: bottom-canopy (1-2 m height), the four cardinal points of mid-canopy (2-4 m height), and the top-canopy (4-6 m height). We also collected bottom-canopy leaves from five additional trees from each species.

RESULTS:

Based on an analysis of bacterial community structure measured via Illumina sequencing of the bacterial 16S gene, we demonstrate that 65% of the intra-individual variation in leaf bacterial community structure could be attributed to the effect of inter-individual and inter-specific differences while the effect of canopy location was not significant. In comparison, host species identity explains 47% of inter-individual and inter-specific variation in leaf bacterial community structure followed by individual identity (32%) and canopy location (6%).

DISCUSSION:

Our results suggest that individual samples from consistent positions within the tree canopy from multiple individuals per species can be used to accurately quantify variation in phyllosphere bacterial community structure. However, the considerable amount of intra-individual variation within a tree canopy ask for a better understanding of how changes in leaf characteristics and local abiotic conditions drive spatial variation in the phyllosphere microbiome.

KEYWORDS:

Bioindicator; Inter-individual variation; Interspecific variation; Intra-individual variation; Microbiome; Phyllosphere; Plant-bacteria interaction; Temperate forest

PMID: 27635335 PMCID: PMC5012278 DOI: 10.7717/peerj.2367

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Select item 27633927

 

9.

FEMS Microbiol Ecol. 2016 Sep 14. pii: fiw196. [Epub ahead of print]

First deep screening of bacterial assemblages associated with corals of the Tropical Eastern Pacific.

Hernández-Zulueta J1, Araya R2, Vargas-Ponce O3, Díaz-Pérez L4, Rodríguez-Troncoso AP5, Ceh J2, Ríos-Jara E4, Rodríguez-Zaragoza FA6.

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Abstract

Bacterial assemblages associated with the hermatypic coralsPocillopora damicornis and Pocillopora verrucosa, the surrounding seawater and the sediment at six coral reef sites in the north section of the Tropical Eastern Pacificwere assessed using MiSeq Illumina sequencing of the V4 region of the 16S rDNA. The bacterial microbiota in both coralsspecies, seawater and sediment were stableto seasonal variations. Bacterial assemblages between the same substrates were not significantly different from each otherin the six sites sampled. Interestingly, the bacterial composition between substrates within the same site was significantly different, or not, depending on the conservation status of the site. Moreover, we found species-specific bacterial OTUs in both coral species. Analyzing the relationship between bacterial composition and environmental variables revealed a positive correlationbetween bacterial assemblagesanddissolved oxygen, ammonium and silicate.

© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

KEYWORDS:

16S rDNA; Tropical Eastern Pacific; bacterial assemblages; corals; high-throughput sequencing; microbial ecology

PMID: 27633927 DOI: 10.1093/femsec/fiw196

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Select item 27625705

 

10.

Gut Pathog. 2016 Sep 13;8(1):42. doi: 10.1186/s13099-016-0124-2. eCollection 2016.

Gut microbiome alterations in patients with stage 4 hepatitis C.

Aly AM1, Adel A1, El-Gendy AO2, Essam TM3, Aziz RK3.

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Abstract

BACKGROUND:

Hepatitis C virus (HCV) causes debilitating liver diseases, which may progress to cirrhosis and cancer, and claims 500,000 annual lives worldwide. While HCV epidemiology, pathophysiology, and therapy are being deeply studied, rare attention is given to reciprocal interactions between HCV infection , HCV-induced chronic liver diseases, and the human gut microbiome. As Egypt has the world's highest prevalence of HCV infections, we launched this study to monitor differences in the gut microbial community composition of Egyptian HCV patients that may affect, or result from, the patients' liver state.

RESULTS:

To this end, we analyzed stool samples from six stage 4-HCV patients and eight healthy individuals by high-throughput 16S rRNA gene sequencing using Illumina MiSeq. Overall, the alpha-diversity of the healthy persons' gut microbiomes was higher than those of the HCV patients. Whereas members of phylum Bacteroidetes were more abundant in HCV patients, healthy individuals had higher abundance of Firmicutes, Proteobacteria, and Actinobacteria. Genus-level analysis showed differential abundance of Prevotella and Faecalibacterium (higher in HCV patients) vs. Ruminococcus and Clostridium (healthy group), indicating that the higher abundance of Bacteroidetes in HCV patients is most likely due to Prevotella overabundance. The probiotic genus, Bifidobacterium, was only observed in the microbiotas of healthy individuals.

CONCLUSIONS:

To the best of our knowledge, this study provides a first overview of major phyla and genera differentiating stage 4-HCV patients from healthy individuals and suggests possible microbiome remodeling in chronic hepatitis C, possibly shaped by bacterial translocation as well as the liver's impaired role in digestion and protein synthesis. Future studies will investigate the microbiome composition and functional capabilities in more patients while tracing some potential biomarker taxa (e.g., Prevotella, Faecalibacterium vs. Bifidobacterium).

KEYWORDS:

Gastro-intestinal tract; High-throughput sequencing; Infectious diseases; Liver disease; Microbiome; Next-generation sequencing; Virology

PMID: 27625705 PMCID: PMC5020480 DOI: 10.1186/s13099-016-0124-2

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Select item 27620079

 

11.

Water Environ Res. 2016 Oct;88(10):930-53. doi: 10.2175/106143016X14696400494371.

Molecular Biological Methods in Environmental Engineering.

Zhang G1, Wei L, Chang CC, Zhang Y, Wei D.

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Abstract

Bacteria, acting as catalysts, perform the function of degrading pollutants. Molecular biological techniques play an important role in research on the community analysis, the composition and the functions of complex microbial communities. The development of secondary high-throughput pyrosequencing techiniques enhances the understanding of the composition of the microbial community. The literatures of 2015 indicated that 16S rDNA gene as genetic tag is still the important method for bacteria identification and classification. 454 high throughput sequencing and Illumina MiSeq sequencing have been the primary and widely recognized methods to analyze the microbial. This review will provide environmental engineers and microbiologists an overview of important advancements in molecular techniques and highlight the application of these methods in diverse environments.

PMID: 27620079 DOI: 10.2175/106143016X14696400494371

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Select item 27611781

MR DNA 16s sequencing service

12.

PLoS One. 2016 Sep 9;11(9):e0162302. doi: 10.1371/journal.pone.0162302. eCollection 2016.

Urinary Microbiota Associated with Preterm Birth: Results from the Conditions Affecting Neurocognitive Development and Learning in Early Childhood (CANDLE) Study.

Ollberding NJ1, Völgyi E2,3, Macaluso M1, Kumar R4, Morrow C5, Tylavsky FA3, Piyathilake CJ6.

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Abstract

Preterm birth (PTB) is the leading cause of infant morbidity and mortality. Genitourinary infection is implicated in the initiation of spontaneous PTB; however, examination of the urinary microbiota in relation to preterm delivery using next-generation sequencing technologies is lacking. In a case-control study nested within the Conditions Affecting Neurocognitive Development and Learning in Early Childhood (CANDLE) study, we examined associations between the urinary microbiota and PTB. A total of 49 cases (delivery < 37 weeks gestation) and 48 controls (delivery ≥ 37 weeks gestation) balanced on health insurance type were included in the present analysis. Illumina sequencing of the 16S rRNA gene V4 region was performed on urine samples collected during the second trimester. We observed no difference in taxa richness, evenness, or community composition between cases and controls or for gestational age modeled as a continuous variable. Operational taxonomic units (OTUs) classified to Prevotella, Sutterella, L. iners, Blautia, Kocuria, Lachnospiraceae, and S.marcescens were enriched among cases (FDR corrected p≤ 0.05). A urinary microbiota clustering partition dominated by S. marcescens was also associated with PTB (OR = 3.97, 95% CI: 1.19-13.24). These data suggest a limited role for the urinary microbiota in PTB when measured during the second trimester by 16S rRNA gene sequencing. The enrichment among cases in several organisms previously reported to be associated with genitourinary pathology requires confirmation in future studies to rule out the potential for false positive findings.

PMID: 27611781 DOI: 10.1371/journal.pone.0162302

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