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      The Bacterial and Fungal Microbiota of Nelore Steers Is Dynamic Across the Gastrointestinal Tract and Its Fecal-Associated Microbiota Is Correlated to Feed Efficiency

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          Abstract

          The ruminant gastrointestinal tract (GIT) microbiome plays a major role in the health, physiology and production traits of the host. In this work, we characterized the bacterial and fungal microbiota of the rumen, small intestine (SI), cecum and feces of 27 Nelore steers using next-generation sequencing and evaluated biochemical parameters within the GIT segments. We found that only the bacterial microbiota clustered according to each GIT segment. Bacterial diversity and richness as well as volatile fatty acid concentration was lowest in the SI. Taxonomic grouping of bacterial operational taxonomic units (OTUs) revealed that Lachnospiraceae (24.61 ± SD 6.58%) and Ruminococcaceae (20.87 ± SD 4.22%) were the two most abundant taxa across the GIT. For the fungi, the family Neocallismastigaceae dominated in all GIT segments, with the genus Orpinomyces being the most abundant. Twenty-eight bacterial and six fungal OTUs were shared across all GIT segments in at least 50% of the steers. We also evaluated if the fecal-associated microbiota of steers showing negative and positive residual feed intake (n-RFI and p-RFI, respectively) was associated with their feed efficiency phenotype. Diversity indices for both bacterial and fungal fecal microbiota did not vary between the two feed efficiency groups. Differences in the fecal bacterial composition between high and low feed efficiency steers were primarily assigned to OTUs belonging to the families Lachnospiraceae and Ruminococcaceae and to the genus Prevotella. The fungal OTUs shared across the GIT did not vary between feed efficiency groups, but 7 and 3 OTUs were found only in steers with positive and negative RFI, respectively. These results provide further insights into the composition of the Nelore GIT microbiota, which could have implications for improving animal health and productivity. Our findings also reveal differences in fecal-associated bacterial OTUs between steers from different feed efficiency groups, suggesting that fecal sampling may represent a non-invasive strategy to link the bovine microbiota with productivity phenotypes.

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          Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.

          Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.
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            The core gut microbiome, energy balance and obesity.

            Metagenomics is an emerging field focused on characterizing the structures, functions and dynamic operations of microbial communities sampled in their native habitats without the need for culture. Here, we present findings from a 16S rRNA gene sequence- and whole community DNA shotgun sequencing-based analysis of the adult human gut microbiomes of lean and obese mono- and dizygotic twins. Our findings indicate that a core microbiome can be found at the gene level, despite large variation in community membership, and that variations from the core are associated with obesity. These findings have implications for ongoing Human Microbiome Project(s), and highlight important challenges to the field of metagenomics.
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              The gut mycobiome of the Human Microbiome Project healthy cohort

              Background Most studies describing the human gut microbiome in healthy and diseased states have emphasized the bacterial component, but the fungal microbiome (i.e., the mycobiome) is beginning to gain recognition as a fundamental part of our microbiome. To date, human gut mycobiome studies have primarily been disease centric or in small cohorts of healthy individuals. To contribute to existing knowledge of the human mycobiome, we investigated the gut mycobiome of the Human Microbiome Project (HMP) cohort by sequencing the Internal Transcribed Spacer 2 (ITS2) region as well as the 18S rRNA gene. Results Three hundred seventeen HMP stool samples were analyzed by ITS2 sequencing. Fecal fungal diversity was significantly lower in comparison to bacterial diversity. Yeast dominated the samples, comprising eight of the top 15 most abundant genera. Specifically, fungal communities were characterized by a high prevalence of Saccharomyces, Malassezia, and Candida, with S. cerevisiae, M. restricta, and C. albicans operational taxonomic units (OTUs) present in 96.8, 88.3, and 80.8% of samples, respectively. There was a high degree of inter- and intra-volunteer variability in fungal communities. However, S. cerevisiae, M. restricta, and C. albicans OTUs were found in 92.2, 78.3, and 63.6% of volunteers, respectively, in all samples donated over an approximately 1-year period. Metagenomic and 18S rRNA gene sequencing data agreed with ITS2 results; however, ITS2 sequencing provided greater resolution of the relatively low abundance mycobiome constituents. Conclusions Compared to bacterial communities, the human gut mycobiome is low in diversity and dominated by yeast including Saccharomyces, Malassezia, and Candida. Both inter- and intra-volunteer variability in the HMP cohort were high, revealing that unlike bacterial communities, an individual’s mycobiome is no more similar to itself over time than to another person’s. Nonetheless, several fungal species persisted across a majority of samples, evidence that a core gut mycobiome may exist. ITS2 sequencing data provided greater resolution of the mycobiome membership compared to metagenomic and 18S rRNA gene sequencing data, suggesting that it is a more sensitive method for studying the mycobiome of stool samples. Electronic supplementary material The online version of this article (10.1186/s40168-017-0373-4) contains supplementary material, which is available to authorized users.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                25 June 2019
                2019
                : 10
                : 1263
                Affiliations
                [1] 1Departamento de Microbiologia, Universidade Federal de Viçosa , Viçosa, Brazil
                [2] 2Department of Bacteriology, University of Wisconsin–Madison , Madison, WI, United States
                [3] 3Departamento de Zootecnia, Universidade Federal de Viçosa , Viçosa, Brazil
                [4] 4Instituto de Zootecnia, Centro APTA Bovinos de Corte , São Paulo, Brazil
                Author notes

                Edited by: Guillermina Hernandez-Raquet, Institut National de la Recherche Agronomique (INRA), France

                Reviewed by: Stephan Schmitz-Esser, Iowa State University, United States; Fuyong Li, University of Alberta, Canada; Hui Yang, Jiangxi Agricultural University, China

                *Correspondence: Hilario Cuquetto Mantovani, hcm6@ 123456ufv.br

                This article was submitted to Systems Microbiology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2019.01263
                6603086
                31293524
                845a9104-80a6-4efb-9c7a-74d44a1eac63
                Copyright © 2019 Lopes, La Reau, Duarte, Detmann, Bento, Mercadante, Bonilha, Suen and Mantovani.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 08 January 2019
                : 21 May 2019
                Page count
                Figures: 7, Tables: 2, Equations: 0, References: 63, Pages: 14, Words: 0
                Funding
                Funded by: Fundação de Amparo à Pesquisa do Estado de Minas Gerais 10.13039/501100004901
                Funded by: Conselho Nacional de Desenvolvimento Científico e Tecnológico 10.13039/501100003593
                Funded by: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior 10.13039/501100002322
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                git microbiota,beef cattle,16s rrna gene,its1,fecal samples,residual feed intake,next-generation sequencing

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