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      A Refined View of Airway Microbiome in Chronic Obstructive Pulmonary Disease at Species and Strain-Levels

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          Abstract

          Little is known about the underlying airway microbiome diversity in chronic obstructive pulmonary disease (COPD) at in-depth taxonomic levels. Here we present the first insights on the COPD airway microbiome at species and strain-levels. The full-length 16S rRNA gene was characterized from sputum in 98 COPD patients and 27 age-matched healthy controls, using the Pacific Biosciences sequencing platform. Individual species within the same genus exhibited reciprocal relationships with COPD and disease severity. Species dominant in health can be taken over by another species within the same genus but with potentially increasing pathogenicity in severe COPD patients. Ralstonia mannitolilytica, an opportunistic pathogen, was significantly increased in frequent exacerbators (fold-change = 4.94, FDR P = 0.005). There were distinct patterns of interaction between bacterial species and host inflammatory mediators according to neutrophilic or eosinophilic inflammations, two major airway inflammatory phenotypes in COPD. Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, and Neisseria meningitidis were associated with enhanced Th1, Th17 and pro-inflammatory mediators, while a group of seven species including Tropheryma whipplei were specifically associated with Th2 mediators related to eosinophilia. We developed an automated pipeline to assign strain-level taxonomy leveraging bacterial intra-genomic 16S allele frequency. Using this pipeline we further resolved three non-typeable H. influenzae strains PittEE, PittGG and 86-028NP with reasonable precision and uncovered strain-level variation related to airway inflammation. In particular, 86-028NP and PittGG strains exhibited inverse associations with Th2 chemokines CCL17 and CCL13, suggesting their abundances may inversely predict eosinophilic inflammation. A systematic comparison of 16S hypervariable regions indicated V1V3 instead of the commonly used V4 region was the best surrogate for airway microbiome. The full-length 16S data augmented the power of functional inference, which slightly better recapitulated the actual metagenomes. This led to the unique identification of butyrate-producing and nitrate reduction pathways as depleted in COPD. Our analysis uncovered finer-scale airway microbial diversity that was previously underappreciated, thus enabled a refined view of the airway microbiome in COPD.

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          Most cited references38

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          The lung tissue microbiome in chronic obstructive pulmonary disease.

          Based on surface brushings and bronchoalveolar lavage fluid, Hilty and coworkers demonstrated microbiomes in the human lung characteristic of asthma and chronic obstructive pulmonary disease (COPD), which have now been confirmed by others. To extend these findings to human lung tissue samples. DNA from lung tissue samples was obtained from nonsmokers (n = 8); smokers without COPD (n = 8); patients with very severe COPD (Global Initiative for COPD [GOLD] 4) (n = 8); and patients with cystic fibrosis (CF) (n = 8). The latter served as a positive control, with sterile water as a negative control. All bacterial community analyses were based on polymerase chain reaction amplifying 16S rRNA gene fragments. Total bacterial populations were measured by quantitative polymerase chain reaction and bacterial community composition was assessed by terminal restriction fragment length polymorphism analysis and pyrotag sequencing. Total bacterial populations within lung tissue were small (20-1,252 bacterial cells per 1,000 human cells) but greater in all four sample groups versus the negative control group (P < 0.001). Terminal restriction fragment length polymorphism analysis and sequencing distinguished three distinct bacterial community compositions: one common to the nonsmoker and smoker groups, a second to the GOLD 4 group, and the third to the CF-positive control group. Pyrotag sequencing identified greater than 1,400 unique bacterial sequences and showed an increase in the Firmicutes phylum in GOLD 4 patients versus all other groups (P < 0.003) attributable to an increase in the Lactobacillus genus (P < 0.0007). There is a detectable bacterial community within human lung tissue that changes in patients with very severe COPD.
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            Advancements in Next-Generation Sequencing

            The term next-generation sequencing is almost a decade old, but it remains the colloquial way to describe highly parallel or high-output sequencing methods that produce data at or beyond the genome scale. Since the introduction of these technologies, the number of applications and methods that leverage the power of genome-scale sequencing has increased at an exponential pace. This review highlights recent concepts, technologies, and methods from next-generation sequencing to illustrate the breadth and depth of the applications and research areas that are driving progress in genomics.
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              High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

              Abstract Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.
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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
                30 July 2020
                2020
                : 11
                : 1758
                Affiliations
                [1] 1Institute of Ecological Science, School of Life Sciences, South China Normal University , Guangzhou, China
                [2] 2State Key Laboratory of Organ Failure Research, Microbiome Medicine Center, Division of Laboratory Medicine, Zhujiang Hospital, Southern Medical University , Guangzhou, China
                [3] 3State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University , Guangzhou, China
                [4] 4Department of Medicine, Firestone Institute for Respiratory Health at St. Joseph’s Healthcare, McMaster University , Hamilton, ON, Canada
                [5] 5Institute for Lung Health, NIHR Leicester Biomedical Research Centre, Department of Respiratory Sciences, University of Leicester , Leicester, United Kingdom
                [6] 6Pulmonary and Critical Care Department, Shenzhen Institute of Respiratory Diseases, Shenzhen People’s Hospital , Shenzhen, China
                Author notes

                Edited by: Spyridon Ntougias, Democritus University of Thrace, Greece

                Reviewed by: Mohib Uddin, AstraZeneca, Sweden; Eduard Monso, Corporació Sanitària Parc Taulí, Spain

                *Correspondence: Zhenyu Liang, 490458234@ 123456qq.com

                These authors have contributed equally to this work

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

                Article
                10.3389/fmicb.2020.01758
                7406711
                32849386
                67ac8443-ebd4-4821-859c-ddd2e1d3a963
                Copyright © 2020 Wang, Liu, Wang, Yang, Wang, Chen, Stampfli, Zhou, Shu, Brightling, Liang and Chen.

                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
                : 11 March 2020
                : 06 July 2020
                Page count
                Figures: 5, Tables: 2, Equations: 0, References: 67, Pages: 16, Words: 0
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                airway microbiome,copd,full-length 16s sequencing,pacbio,airway inflammation

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