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      Complexity and Dynamics of the Winemaking Bacterial Communities in Berries, Musts, and Wines from Apulian Grape Cultivars through Time and Space

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          Abstract

          Currently, there is very little information available regarding the microbiome associated with the wine production chain. Here, we used an amplicon sequencing approach based on high-throughput sequencing (HTS) to obtain a comprehensive assessment of the bacterial community associated with the production of three Apulian red wines, from grape to final product. The relationships among grape variety, the microbial community, and fermentation was investigated. Moreover, the winery microbiota was evaluated compared to the autochthonous species in vineyards that persist until the end of the winemaking process. The analysis highlighted the remarkable dynamics within the microbial communities during fermentation. A common microbial core shared among the examined wine varieties was observed, and the unique taxonomic signature of each wine appellation was revealed. New species belonging to the genus Halomonas were also reported. This study demonstrates the potential of this metagenomic approach, supported by optimized protocols, for identifying the biodiversity of the wine supply chain. The developed experimental pipeline offers new prospects for other research fields in which a comprehensive view of microbial community complexity and dynamics is desirable.

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          Most cited references30

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          FLASH: fast length adjustment of short reads to improve genome assemblies.

          Next-generation sequencing technologies generate very large numbers of short reads. Even with very deep genome coverage, short read lengths cause problems in de novo assemblies. The use of paired-end libraries with a fragment size shorter than twice the read length provides an opportunity to generate much longer reads by overlapping and merging read pairs before assembling a genome. We present FLASH, a fast computational tool to extend the length of short reads by overlapping paired-end reads from fragment libraries that are sufficiently short. We tested the correctness of the tool on one million simulated read pairs, and we then applied it as a pre-processor for genome assemblies of Illumina reads from the bacterium Staphylococcus aureus and human chromosome 14. FLASH correctly extended and merged reads >99% of the time on simulated reads with an error rate of <1%. With adequately set parameters, FLASH correctly merged reads over 90% of the time even when the reads contained up to 5% errors. When FLASH was used to extend reads prior to assembly, the resulting assemblies had substantially greater N50 lengths for both contigs and scaffolds. The FLASH system is implemented in C and is freely available as open-source code at http://www.cbcb.umd.edu/software/flash. t.magoc@gmail.com.
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            Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate.

            Wine grapes present a unique biogeography model, wherein microbial biodiversity patterns across viticultural zones not only answer questions of dispersal and community maintenance, they are also an inherent component of the quality, consumer acceptance, and economic appreciation of a culturally important food product. On their journey from the vineyard to the wine bottle, grapes are transformed to wine through microbial activity, with indisputable consequences for wine quality parameters. Wine grapes harbor a wide range of microbes originating from the surrounding environment, many of which are recognized for their role in grapevine health and wine quality. However, determinants of regional wine characteristics have not been identified, but are frequently assumed to stem from viticultural or geological factors alone. This study used a high-throughput, short-amplicon sequencing approach to demonstrate that regional, site-specific, and grape-variety factors shape the fungal and bacterial consortia inhabiting wine-grape surfaces. Furthermore, these microbial assemblages are correlated to specific climatic features, suggesting a link between vineyard environmental conditions and microbial inhabitation patterns. Taken together, these factors shape the unique microbial inputs to regional wine fermentations, posing the existence of nonrandom "microbial terroir" as a determining factor in regional variation among wine grapes.
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              Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome

              Background DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled. Methodology/Principal Findings In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used. Conclusions/Significance Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                14 June 2016
                2016
                : 11
                : 6
                : e0157383
                Affiliations
                [1 ]Institute of Biomembranes and Bioenergetics, CNR, Bari, Italy
                [2 ]Institute of Sciences of Food Production, CNR, Operative Unit of Lecce, Lecce (Le), Italy
                [3 ]Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari “A. Moro” Bari, Italy
                [4 ]Institute of Sciences of Food Production, CNR, Bari, Italy
                [5 ]GBS BAO Advanced Analytics Services and MBLab, IBM, Bari, Italy
                [6 ]Algorithms, Bioinformatics, Complexity and Formal Methods Research Group, Polytechnic University of Catalonia, Barcelona, Spain
                [7 ]Institute for Biomedical Technologies, CNR, Bari, Italy
                Wilfrid Laurier University, CANADA
                Author notes

                Competing Interests: The authors have the following interests: Gaetano Scioscia [GS] is employed by IBM Italy SpA. He serves as investigators for the research and educational project DM19410 ‘Laboratorio di Bioinformatica per la Biodiversita’ Molecolare’ MBLab. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

                Conceived and designed the experiments: MM FG MS GP. Performed the experiments: MM MI CM GC GS. Analyzed the data: BF GV MS GP. Contributed reagents/materials/analysis tools: GM AT ES. Wrote the paper: MM BF FG MS GP.

                Article
                PONE-D-16-04179
                10.1371/journal.pone.0157383
                4907434
                27299312
                55fb1dc7-6de4-435d-84e4-e34743b09380
                © 2016 Marzano et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 1 February 2016
                : 27 May 2016
                Page count
                Figures: 6, Tables: 1, Pages: 19
                Funding
                This study was partially supported by the following Projects: ‘‘Laboratorio di Bioinformatica per la Biodiversità Molecolare’’ (MBLab) (Project code DM19410), “Biotecnologie degli alimenti per l’innovazione e la competitività delle principali filiere regionali: estensione della conservabilità e aspetti funzionali -BiotecA” (Project code QCBRAJ6), “Lifewatch” (CNR, Italy) and “Micromap” (PON01_02589). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. For what concerns the IBM employee involved in this paper, his activities in this study have been conducted on top of their regular work for the Company, since this research branch is outside his working subjects and goals. So this study was conducted by the IBM author in total autonomy. IBM provided support in the form of salaries for author [GS], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of this author are articulated in the ‘author contributions’ section.
                Categories
                Research Article
                Biology and Life Sciences
                Nutrition
                Diet
                Beverages
                Alcoholic Beverages
                Wine
                Medicine and Health Sciences
                Nutrition
                Diet
                Beverages
                Alcoholic Beverages
                Wine
                Biology and Life Sciences
                Biochemistry
                Metabolism
                Metabolic Processes
                Fermentation
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                Agriculture
                Crop Science
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                Grapes
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                Custom metadata
                All relevant data are within the paper and its Supporting Information files. Moreover, all the sequencing raw data are available at NCBI SRA repository using the SRA accession number SRP072913.

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