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      Characterization of oral and cloacal microbial communities in cold-stunned Kemp’s ridley sea turtles ( Lepidochelys kempii) during the time course of rehabilitation

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          Abstract

          Microbial communities of animals play a role in health and disease, including immunocompromised conditions. In the northeastern United States, cold-stunning events often cause endangered Kemp’s ridley turtles ( Lepidochelys kempii) to become stranded on beaches in autumn. These sea turtles are admitted to rehabilitation facilities when rescued alive and are presumed immunocompromised secondary to hypothermia. To better understand the role that microbes play in the health of cold-stunned sea turtles, we characterized the oral and cloacal microbiome from Kemp’s ridley turtles at multiple timepoints during rehabilitation, from admission to pre-release, by using Illumina sequencing to analyze the 16S rRNA gene. Microbial communities were distinct between body sites and among turtles that survived and those that died. We found that clinical parameters such as presence of pneumonia or values for various blood analytes did not correlate with oral or cloacal microbial community composition. We also investigated the effect of antibiotics on the microbiome during rehabilitation and prior to release and found that the type of antibiotic altered the microbial community composition, yet overall taxonomic diversity remained the same. The microbiome of cold-stunned Kemp’s ridley turtles gradually changed through the course of rehabilitation with environment, antibiotics, and disease status all playing a role in those changes and ultimately the release status of the turtles.

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          DADA2: High resolution sample inference from Illumina amplicon data

          We present DADA2, a software package that models and corrects Illumina-sequenced amplicon errors. DADA2 infers sample sequences exactly, without coarse-graining into OTUs, and resolves differences of as little as one nucleotide. In several mock communities DADA2 identified more real variants and output fewer spurious sequences than other methods. We applied DADA2 to vaginal samples from a cohort of pregnant women, revealing a diversity of previously undetected Lactobacillus crispatus variants.
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            phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data

            Background The analysis of microbial communities through DNA sequencing brings many challenges: the integration of different types of data with methods from ecology, genetics, phylogenetics, multivariate statistics, visualization and testing. With the increased breadth of experimental designs now being pursued, project-specific statistical analyses are often needed, and these analyses are often difficult (or impossible) for peer researchers to independently reproduce. The vast majority of the requisite tools for performing these analyses reproducibly are already implemented in R and its extensions (packages), but with limited support for high throughput microbiome census data. Results Here we describe a software project, phyloseq, dedicated to the object-oriented representation and analysis of microbiome census data in R. It supports importing data from a variety of common formats, as well as many analysis techniques. These include calibration, filtering, subsetting, agglomeration, multi-table comparisons, diversity analysis, parallelized Fast UniFrac, ordination methods, and production of publication-quality graphics; all in a manner that is easy to document, share, and modify. We show how to apply functions from other R packages to phyloseq-represented data, illustrating the availability of a large number of open source analysis techniques. We discuss the use of phyloseq with tools for reproducible research, a practice common in other fields but still rare in the analysis of highly parallel microbiome census data. We have made available all of the materials necessary to completely reproduce the analysis and figures included in this article, an example of best practices for reproducible research. Conclusions The phyloseq project for R is a new open-source software package, freely available on the web from both GitHub and Bioconductor.
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              Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms

              DNA sequencing continues to decrease in cost with the Illumina HiSeq2000 generating up to 600 Gb of paired-end 100 base reads in a ten-day run. Here we present a protocol for community amplicon sequencing on the HiSeq2000 and MiSeq Illumina platforms, and apply that protocol to sequence 24 microbial communities from host-associated and free-living environments. A critical question as more sequencing platforms become available is whether biological conclusions derived on one platform are consistent with what would be derived on a different platform. We show that the protocol developed for these instruments successfully recaptures known biological results, and additionally that biological conclusions are consistent across sequencing platforms (the HiSeq2000 versus the MiSeq) and across the sequenced regions of amplicons.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SoftwareRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: Project administrationRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                27 May 2021
                2021
                : 16
                : 5
                : e0252086
                Affiliations
                [1 ] Animal Health Department, New England Aquarium, Boston, Massachusetts, United States of America
                [2 ] University of Massachusetts, Boston, Massachusetts, United States of America
                [3 ] Rescue & Rehabilitation Department, New England Aquarium, Boston, Massachusetts, United States of America
                [4 ] Department of Marine and Environmental Sciences, Marine Science Center, Northeastern University, Nahant, Massachusetts, United States of America
                University of Illinois College of Medicine, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0002-3966-9487
                Article
                PONE-D-21-02853
                10.1371/journal.pone.0252086
                8159006
                34043685
                ce0361a9-7707-4051-897a-b06679fbab59
                © 2021 McNally 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
                : 26 January 2021
                : 7 May 2021
                Page count
                Figures: 9, Tables: 1, Pages: 22
                Funding
                Funded by: Dr. Robert W. Spayne Research Grant 2015
                Award Recipient :
                Funded by: Nancy Goranson Endowment Fund 2015
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100004184, Northeastern University;
                Award Recipient :
                Sequencing costs were supported with Dr. Robert W. Spayne Research Grant 2015 and the Nancy Goranson Endowment Fund 2015 from University of Massachusetts Boston to KLM and start-up funds to JLB from Northeastern University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Amniotes
                Reptiles
                Testudines
                Turtles
                Biology and Life Sciences
                Zoology
                Animals
                Vertebrates
                Amniotes
                Reptiles
                Testudines
                Turtles
                Medicine and Health Sciences
                Pharmacology
                Drugs
                Antimicrobials
                Antibiotics
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobials
                Antibiotics
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbiome
                Biology and Life Sciences
                Genetics
                Genomics
                Microbial Genomics
                Microbiome
                Biology and Life Sciences
                Microbiology
                Microbial Genomics
                Microbiome
                Medicine and Health Sciences
                Health Care
                Convalescence
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobial Resistance
                Antibiotic Resistance
                Medicine and Health Sciences
                Pharmacology
                Antimicrobial Resistance
                Antibiotic Resistance
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Blood
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Blood
                Biology and Life Sciences
                Physiology
                Body Fluids
                Blood
                Biology and Life Sciences
                Ecology
                Ecological Metrics
                Species Diversity
                Shannon Index
                Ecology and Environmental Sciences
                Ecology
                Ecological Metrics
                Species Diversity
                Shannon Index
                Medicine and Health Sciences
                Pulmonology
                Pneumonia
                Custom metadata
                All sequencing data and metadata are available at NCBI’s Sequence Read Archive, under BioProject accession number: PRJNA690527. Additional relevant data is within the paper and its Supporting Information files.

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