scholarly journals Comparison of microbiome and culture techniques for determination of gastrointestinal microbial communities in ceca of chickens

2018 ◽  
Author(s):  
Kimberly Wilson ◽  
Whitney Briggs ◽  
Audrey Duff ◽  
Chasser Kaylin ◽  
Xialoun Sun ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Illumina Miseq ◽  
Intestinal Microbiome ◽  
P Value ◽  
Culture Techniques ◽  
Operational Taxonomic Units ◽  
Colony Forming Units ◽  
Miseq Platform ◽  
Next Generation Sequencing Ngs

The use of 16S next generation sequencing (NGS) technology to identify the relative abundance of microbial communities have become the standard when studying the intestinal microbiome. The increased use is due to the ability to identify a proportion of bacteria that cannot be observed with culture-based methods. However, culture-based techniques are acceptable to identify key bacterial groups, yet may grossly underestimate the microbial community in question. Since there is limited research comparing NGS results to colony forming units (CFU), the objective of this study was to compare total Enterobacteriaceae and lactic acid bacteria (LAB) recovery with culture techniques (CFU/g ceca) to total number of reads from operational taxonomic units (OTU) categorized as Enterobacteriaceae or LAB from Illumina MiSeq platform from matched chick cecal samples at three and 10 days of age. Both CFU recovery (1.09x109 ± 2.42x108; 1.37x108 ± 5.57x107) and reads (5460 ± 1164 ; 282 ± 163) belonging to Enterobacteriaceae decreased by 10 days of age (p < 0.001). Similarly, LAB reads decreased over time (21,128 ± 2262; 6220 ± 817, respectively p < 0.0001). However, LAB CFU recovery increased by 10 days (1.18x108 ± 1.91x107; 1.62x109 ± 5.00x108, respectively p < 0.01). At three days the Pearson’s correlation was -0.082 between CFU of culturable Enterobacteriaceae to reads and culturable LAB CFU to reads at 0.097, showing no correlation (p = 0.606, 0.551; respectively). By 10 days, no correlation of reads and CFU occurred with Enterobacteriaceae (r=-0.049; p-value = 0.769) while with LAB the correlation was 0.290 (p = 0.066) at 10 days. The CFU may be appropriate to identify a few families that change due to treatment or product. Without identifying viable cells to DNA recovered from NGS, there will always be the question whether the reads within the binned OTU in the intestinal tract is accurate.

scholarly journals Phylogenetic Core Groups: a promising concept in search of a consistent methodological framework

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Alberto Pascual-García
Keyword(s):  
Microbial Communities ◽  
Microbial Ecology ◽  
Environmental Conditions ◽  
The Novel ◽  
Methodological Framework ◽  
Operational Taxonomic Units ◽  
Traditional Classification ◽  
Taxonomical Classification ◽  
Novel Concept

AbstractIn this comment, we analyse the conceptual framework proposed by Aguirre de Cárcer (Microbiome 7:142, 2019), introducing the novel concept of Phylogenetic Core Groups (PCGs). This notion aims to complement the traditional classification in operational taxonomic units (OTUs), widely used in microbial ecology, to provide a more intrinsic taxonomical classification which avoids the use of pre-determined thresholds. However, to introduce this concept, the author frames his proposal in a wider theoretical framework based on a conceptualization of selection that we argue is a tautology. This blurs the subsequent formulation of an assembly principle for microbial communities, favouring that some contradictory examples introduced to support the framework appear aligned in their conclusions. And more importantly, under this framework and its derived methodology, it is not possible to infer PCGs from data in a consistent way. We reanalyse the proposal to identify its logical and methodological flaws and, through the analysis of synthetic scenarios, we propose a number of methodological refinements to contribute towards the determination of PCGs in a consistent way. We hope our analysis will promote the exploration of PCGs as a potentially valuable tool, helping to bridge the gap between environmental conditions and community composition in microbial ecology.

scholarly journals Metabarcoding Analyses of Gut Microbiota of Nile Tilapia (Oreochromis niloticus) from Lake Awassa and Lake Chamo, Ethiopia

2020 ◽  
Vol 8 (7) ◽  
pp. 1040
Author(s):  
Negash Kabtimer Bereded ◽  
Manuel Curto ◽  
Konrad J. Domig ◽  
Getachew Beneberu Abebe ◽  
Solomon Workneh Fanta ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Oreochromis Niloticus ◽  
Nile Tilapia ◽  
Large Scale ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Operational Taxonomic Units ◽  
Nile Tilapia Oreochromis Niloticus ◽  
Miseq Platform ◽  
Ethiopian Lakes

The Nile tilapia (Oreochromis niloticus) gut harbors a diverse microbial community; however, their variation across gut regions, lumen and mucosa is not fully elucidated. In this study, gut microbiota of all samples across gut regions and sample types (luminal content and mucosa) were analyzed and compared from two Ethiopian lakes. Microbiota were characterized using 16S rRNA Illumina MiSeq platform sequencing. A total of 2061 operational taxonomic units (OTUs) were obtained and the results indicated that Nile tilapia from Lake Chamo harbored a much more diversified gut microbiota than Lake Awassa. In addition, the gut microbiota diversity varied significantly across the gut region based on the Chao1, Shannon and Simpson index. The microbiome analyses of all samples in the midgut region showed significantly higher values for alpha diversity (Chao 1, Shannon and Simpson). Beta diversity analysis revealed a clear separation of samples according to sampling areas and gut regions. The most abundant genera were Clostridium_sensu_stricto and Clostridium_XI genera across all samples. Between the two sampling lakes, two phyla, Phylum Fusobacteria and Cyanobacteria, were found to be significantly different. On the other hand, six phyla (Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria and Cyanobacteria) were significantly different across gut regions. In this study, we found that all samples shared a large core microbiota, comprising a relatively large number of OTUs, which was dominated by Proteobacteria, Firmicutes, Cyanobacteria, Fusobacteria and Actinobacteria. This study has established the bases for future large-scale investigations of gut microbiota of fishes in Ethiopian lakes.

scholarly journals Intestinal Microbiota of Fattening Pigs Offered Non-Fermented and Fermented Liquid Feed with and without the Supplementation of Non-Fermented Coarse Cereals

2020 ◽  
Vol 8 (5) ◽  
pp. 638 ◽  
Author(s):  
Sebastian Bunte ◽  
Richard Grone ◽  
Birgit Keller ◽  
Christoph Keller ◽  
Eric Galvez ◽  
...  
Keyword(s):  
Hypervariable Region ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Bacterial Composition ◽  
Operational Taxonomic Units ◽  
Liquid Feed ◽  
Faecal Samples ◽  
Genus Lactobacillus ◽  
Miseq Platform ◽  
Small And Large Intestine

Introducing high numbers of lactic acid bacteria into the gastrointestinal tract of pigs via fermented liquid feed (FLF) could have an impact on intestinal bacterial ecosystems. Twenty piglets were allocated into four groups and fed a botanically identical liquid diet that was offered either non-fermented (twice), fully fermented or partially fermented but supplemented with 40% of non-fermented coarse cereals. Microbiota studies were performed on the small and large intestine digesta and faecal samples. A 16S rRNA gene amplification was performed within the hypervariable region V4 and sequenced with the Illumina MiSeq platform. R (version 3.5.2) was used for the statistical analyses. The digesta of the small intestines of pigs fed FLF were dominated by Lactobacillaceae (relative abundance up to 95%). In the colonic contents, the abundance of Lactobacillaceae was significantly higher only in the pigs fed the FLF supplemented with non-fermented coarse cereals. Additionally, the digesta of the small and large intestines as well as in the faeces of the pigs fed the FLF supplemented with non-fermented coarse cereals were significantly enriched for two operational taxonomic units (OTUs) belonging to the genus Lactobacillus and Bifidobacterium. The FLF supplemented with non-fermented coarse cereals had probiotic and prebiotic-like impacts on the intestinal and faecal bacterial composition of pigs.

scholarly journals Influences of pH and Iron Concentration on the Salivary Microbiome in Individual Humans with and without Caries

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Jianye Zhou ◽  
Nan Jiang ◽  
Zhenzhen Wang ◽  
Longqing Li ◽  
Jumei Zhang ◽  
...  
Keyword(s):  
Synergistic Effects ◽  
Iron Concentration ◽  
Illumina Miseq ◽  
Distribution Patterns ◽  
Disease Diagnosis ◽  
Sequencing Data ◽  
Research Directions ◽  
Operational Taxonomic Units ◽  
Salivary Ph ◽  
Miseq Platform

ABSTRACT This study aimed to identify the differences in the oral microbial communities in saliva from patients with and without caries by performing sequencing with the Illumina MiSeq platform, as well as to further assess their relationships with environmental factors (salivary pH and iron concentration). Forty-three volunteers were selected, including 21 subjects with and 22 without caries, from one village in Gansu, China. Based on 966,255 trimmed sequences and clustering at the 97% similarity level, 1,303 species-level operational taxonomic units were generated. The sequencing data for the two groups revealed that (i) particular distribution patterns (synergistic effects or competition) existed in the subjects with and without caries at both the genus and species levels and (ii) both the salivary pH and iron concentration had significant influences on the microbial community structure. IMPORTANCE The significant influences of the oral environment observed in this study increase the current understanding of the salivary microbiome in caries. These results will be useful for expanding research directions and for improving disease diagnosis, prognosis, and therapy.

scholarly journals PSII-16 Evidence for stratification of rumen wall microbial communities revealed by 16S rRNA based amplicon sequencing

2019 ◽  
Vol 97 (Supplement_2) ◽  
pp. 226-227
Author(s):  
Lucas Koester ◽  
Mark Lyte ◽  
Stephan Schmitz-Esser ◽  
Heather Allen
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Temporal Stability ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rumen Content ◽  
Rrna Gene ◽  
Operational Taxonomic Units ◽  
Abundant Otus ◽  
Dorsal Portion

Abstract Rumen content (RC) stratifies based on particle size and density consisting of the less dense forage within the dorsal and the denser particles in the ventral portions of the rumen and is in constant contact with the microbial communities present on the rumen wall (RW) epithelium. Little is known about the nutrient requirements and functional processes of RW microbial communities. Our hypothesis is that the RW microbial communities stratify mirroring the stratification of RC due to different available nutrients. Five fistulated, milking Holstein cows of the same management conditions were sampled at four rumen layers corresponding to the RC stratification. Epithelial biopsies were taken through the fistula; the uppermost aligned with the dorsal portion of the RC (A), and three other sites, each 10 cm ventral to the previous (B, C and D). Each cow and stratification layer was sampled five times over four months to analyze temporal stability of the RW microbial communities. DNA was extracted using the Qiagen Powerlyzer Powersoil kit and used for 16S rRNA gene Illumina MiSeq sequencing. Sequences were clustered into operational taxonomic units (OTU) based on a 99% similarity cutoff using MOTHUR. After quality control, 2.0 million reads remained for 90 samples which were clustered into 5,016 OTUs with 10 or more reads. 99.2% of the reads were bacterial, whereas 0.8% affiliated to Archaea. Statistical analysis revealed that among the 20 most abundant OTUs, phylotypes classified as Desulfobulbus, unclassified_Cardiobacteraceae, Mogibacterium, Lachnospiraceae-UCG008 and Methanobrevibacter were significantly different in abundance between sites A compared to D. On a whole community level, analysis of molecular variance (AMOVA) revealed significant differences between groups A, C and D. Our data reveal first evidence that a stratification of RW microbiota is present in dairy cattle and also reveal high temporal stability of RW microbiota.

scholarly journals Dietary Bovine Milk Exosomes Elicit Changes in Microbial Communities in C57BL/6 Mice

2018 ◽  
Author(s):  
Fang Zhou ◽  
Henry A. Paz ◽  
Jiang Shu ◽  
Mahrou Sadri ◽  
Juan Cui ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Gut Microbiome ◽  
Bovine Milk ◽  
Cell Communication ◽  
Intestinal Microbiome ◽  
Operational Taxonomic Units ◽  
Health And Disease ◽  
Cecum Content ◽  
First Time ◽  
Bovine Species

ABSTRACTExosomes and exosome-like vesicles participate in cell-to-cell communication in animals, plant and bacteria. Dietary exosomes in bovine milk are bioavailable in non-bovine species, but a fraction of milk exosomes reaches the large intestine. We hypothesized that milk exosomes alter the composition of the gut microbiome in mice. C57BL/6 mice were fed AIN-93G diets, defined by their content of bovine milk exosomes and RNA cargos: exosome/RNA depleted (ERD) versus exosome/RNA-sufficient (ERS) diets. Feeding was initiated at age three weeks and cecum content was collected at ages 7, 15 and 47 weeks. Microbial communities were identified by 16S rRNA gene sequencing. The dietary intake of exosomes and age had significant effects on the microbial communities in the cecum. At the phylum level, the abundance of Verrucomicrobia was greater in mice fed ERD compared to ERS, and the abundance of both Firmicutes and Tenericutes was smaller in mice fed ERD compared to ERS at age 47 weeks. At the family level, the abundance of Anaeroplasmataceae was greater in mice fed ERD compared to ERS, and the abundance of Bifidobacteriaceae, Lachnospiraceae, and Dehalobacteriaceae was significantly greater in mice fed ERS than mice fed ERD at age 15 weeks. Exosome feeding significantly altered the abundance of 52 operational taxonomic units; diet effects were particularly strong in the Lachnospiraceae, Ruminococcaceae and the Verrucomicrobiaceae families. We conclude that exosomes in bovine milk alter microbial communities in non-bovine species, suggesting that exosomes and their cargos participate in the crosstalk between bacterial and animal kingdoms.IMPORTANCEVirtually all living cells, including bacteria communicate through exosomes, which can be found in all body fluids. Exosomes and the RNA cargos have been implicated in all aspects of health and disease, e.g., metastasis of cancer, neuronal signaling and embryonic development. Previously, we reported that exosomes and their microRNA cargos are not solely derived from endogenous synthesis, but may also be obtained from dietary sources such as bovine milk in non-bovine mammals. Here, we report for the first time that bovine milk exosomes communicate with the intestinal microbiome and alters microbial communities in mice. This is the first report suggesting that the gut microbiome facilitates the signaling by dietary exosomes across kingdoms: animal (cow) → bacteria → animal (mouse).

scholarly journals Efficient and stable metabarcoding sequencing from DNBSEQ-G400 sequencer examined by large fungal community analysis

2020 ◽  
Author(s):  
Xiaohuan Sun ◽  
Jingjing Wang ◽  
Chao Fang ◽  
Jiguang Li ◽  
Mo Han ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Large Scale ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Soil Samples ◽  
Its2 Region ◽  
Sequencing Platform ◽  
Sequencing Bias ◽  
Miseq Platform ◽  
Sequencing Platforms

ABSTRACTMetabarcoding has become the de facto method for characterizing the structure of microbial communities in complex environmental samples. To determine how sequencing platform may influence microbial community characterization, we present a large-scale comparison of two sequencing platforms; Illumina MiSeq and a new platform DNBSEQ-G400 developed by MGI Tech. The accuracy of DNBSEQ-G400 on bacterial and fungal mock samples and compared sequencing consistency and precision between DNBSEQ-G400 and MiSeq platforms by sequencing the fungal ITS2 region from 1144 soil samples with 3 technical replicates. The DNBSEQ-G400 showed a high accuracy in reproducing mock communities containing different proportions of bacteria and fungi, respectively. The taxonomic profiles of the 1144 soil samples generated by the two DNBSEQ-G400 modes closely resembled each other and were highly correlated with those generated by the MiSeq platform. Analyses of technical replicates demonstrated a run bias against certain taxa on the MiSeq but not DNBSEQ-G400 platform. Based on lower cost, greater capacity, and less bias, we conclude that DNBSEQ-G400 is an optimal platform for short-term metabarcoding of microbial communities.IMPORTANCEExperimental steps that generate sequencing bias during amplicon sequencing have been intensively evaluated, including the choice of primer pair, polymerase, PCR cycle and technical replication. However, few studies have assessed the accuracy and precision of different sequencing platforms. Here, we compared the performance of newly released DNBSEQ-G400 sequencer with that of the commonly used Illumina MiSeq platform by leveraging amplicon sequencing of a large number of soil samples. Significant sequencing bias among major fungal genera was found in parallel MiSeq runs, which can be easily neglected without the use of sequencing controls. We emphasize the importance of technical controls in large-scale sequencing efforts and provide DNBSEQ-G400 as an alternative with increased sequencing capacity and more stable reproducibility for amplicon sequencing.

scholarly journals Characterization of New ATM Deletion Associated with Hereditary Breast Cancer

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 136
Author(s):  
Sandra Parenti ◽  
Claudio Rabacchi ◽  
Marco Marino ◽  
Elena Tenedini ◽  
Lucia Artuso ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Germ Line ◽  
Illumina Miseq ◽  
Dna And Rna ◽  
Atm Gene ◽  
Miseq Platform ◽  
Ngs Data Analysis ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data

Next-generation sequencing (NGS)-based cancer risk screening with multigene panels has become the most successful method for programming cancer prevention strategies. ATM germ-line heterozygosity has been described to increase tumor susceptibility. In particular, families carrying heterozygous germ-line variants of ATM gene have a 5- to 9-fold risk of developing breast cancer. Recent studies identified ATM as the second most mutated gene after CHEK2 in BRCA-negative patients. Nowadays, more than 170 missense variants and several truncating mutations have been identified in ATM gene. Here, we present the molecular characterization of a new ATM deletion, identified thanks to the CNV algorithm implemented in the NGS analysis pipeline. An automated workflow implementing the SOPHiA Genetics’ Hereditary Cancer Solution (HCS) protocol was used to generate NGS libraries that were sequenced on Illumina MiSeq Platform. NGS data analysis allowed us to identify a new inactivating deletion of exons 19–27 of ATM gene. The deletion was characterized both at the DNA and RNA level.

scholarly journals The determination of the stability of nitrogen-fixing microorganisms of the soil of Ecuador to toxic metals CrO42–, Ni2+, Cu2+

2018 ◽  
Vol 23 ◽  
pp. 267-272
Author(s):  
O. Yu. Bielikova ◽  
N. A. Matvieieva ◽  
L. S. Yastremskaya ◽  
A. B. Tashyrev
Keyword(s):  
Microbial Communities ◽  
Toxic Metals ◽  
Maximum Permissible Concentration ◽  
Lag Phase ◽  
Nitrogen Fixing ◽  
Natural Ecosystems ◽  
Colony Forming Units ◽  
High Concentrations ◽  
The Stability

Aim. The aim of the work was to determine the stability of nitrogen-fixing microorganisms isolated from the rhizosphere of bromelia (Ecuador), to the effect of toxic metals. Methods. Microorganisms were isolated on the Ashby nutrient medium. The selected strains were cultured on a medium with Cu2+ (Cu (ІІ) citrate) from 50 to 500 mg/l by cation in steps of 50; Ni2+ (NiCl2) from 20 to 200 mg/l by cation in steps of 20; Cr (VI) (K2CrO4) from 20 to 100 mg/l with Cr (VI) in increments of 20. The growth of microorganisms in the presence of metals was characterized by the maximum permissible concentration (MPC) of metals, duration of lag phase and the number of colony-forming units (CFU) of nitrogen-fixing microorganisms with increasing concentration of toxic metals. Results. The selected dominant nitrogen-fixing microorganisms from the soil of Ecuador were resistant to toxic metals (Cu2+, Ni2+, Cr (VI)) in high concentrations. It was found that MPC for microbial communities of nitrogen-fixing microorganisms were: 40 mg/l Cr (VI), 300 mg/l Cu2+ and 100 mg/l Ni2+. Conclusions. It was shown that the selected dominant nitrogen-fixing microorganisms from the Ecuadorian soil were resistant to toxic metals (Cu2+, Ni2+, Cr (VI)) in high concentrations, which in 4–30 times exceed the damage or bactericidal concentrations for the majority of known organophosphate microorganisms of natural ecosystems. Keywords: metalresistance, nitrogen-fixing microorganisms, Cu2+, Ni2+, Cr (VI).

scholarly journals Sequencing SARS-CoV-2 in a Malaria Research Laboratory in Mali, West Africa: The Road to Sequencing the First SARS-CoV-2 Genome in Mali

Processes ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 2169
Author(s):  
Antoine Dara ◽  
Bourema Kouriba ◽  
Amadou Daou ◽  
Abdoul Karim Sangare ◽  
Djibril Kassogue ◽  
...  
Keyword(s):  
Illumina Miseq ◽  
The Road ◽  
Genomic Epidemiology ◽  
Laboratory Setup ◽  
The United Kingdom ◽  
Illumina Miseq Platform ◽  
Miseq Platform ◽  
Next Generation Sequencing Ngs ◽  
High Quality Genome ◽  
Generation Sequencing

Next-generation sequencing (NGS) has become a necessary tool for genomic epidemiology. Even though the utility of genomics in human health has been proved, genomic surveillance has never been as important as during the COVID-19 pandemic. This has been demonstrated by the recent use of genomic surveillance to detect new variants of SARS-CoV-2 in the United Kingdom, South Africa, and Brazil. Until recently, Malian scientists did not have access to any local NGS platform, and samples had to be shipped abroad for sequencing. Here, we report on how we adapted a laboratory setup for Plasmodium research to generate the first complete SARS-CoV-2 genome locally. Total RNA underwent a library preparation using an Illumina TruSeq stranded RNA kit. A metagenomics sequencing was performed on an Illumina MiSeq platform, which was followed by bioinformatic analyses on a local server in Mali. We recovered a full genome of SARS-CoV-2 of 29 kb with an average depth coverage of 200×. We have demonstrated our capacity to generate a high-quality genome with limited resources and highlight the need to develop genomics capacity locally to solve health problems. We discuss challenges related to access to reagents during a pandemic period and propose some home-made solutions.

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