DOCSLIB.ORG

Improved High-Throughput Sequencing of the Human Oral Microbiome: from Illumina to Pacbio

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Improved High-Throughput Sequencing of the Human Oral Microbiome: from Illumina to Pacbio Hindawi Canadian Journal of Infectious Diseases and Medical Microbiology Volume 2020, Article ID 6678872, 13 pages https://doi.org/10.1155/2020/6678872 Research Article Improved High-Throughput Sequencing of the Human Oral Microbiome: From Illumina to PacBio Jie Zhang , Lingkai Su , Yuan Wang , and Shuli Deng e Affiliated Hospital of Stomatology, School of Stomatology, Zhejiang University School of Medicine and Key Laboratory of Oral Biomedical Research of Zhejiang Province, Hangzhou, Zhejiang 310006, China Correspondence should be addressed to Shuli Deng; [email protected] Received 21 October 2020; Revised 6 November 2020; Accepted 18 November 2020; Published 11 December 2020 Academic Editor: Tingtao Chen Copyright © 2020 Jie Zhang et al. +is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. A comprehensive understanding of the commensal microflora and its relation to health is essential for preventing and combating diseases. +e aim of this study was to examine the structure of the oral microbiome by using different sequencing technologies. Material and Methods. Five preschool children with no symptoms of oral and systemic diseases were recruited. Samples of saliva were collected. A 468 bp insert size library was constructed on the MiSeq platform and then subjected to 300 bp paired-end sequencing. Libraries with longer insert sizes, including a full-length 16S rDNA gene, were sequenced on the PacBio RS II platform. Results. A total of 122.6 Mb of raw data, including 244,967 high-quality sequences, were generated by the MiSeq platform, while 134.6 Mb of raw data, including 70,030 high-quality reads, were generated by the PacBio RS II platform. Clustering of the unique sequences into OTUs at 3% dissimilarity resulted in an average of 225 OTUs on the MiSeq platform; however, the number of OTUs generated on the PacBio RS II platform was 449, far greater than the number of OTUs generated on the MiSeq platform. A total of 437 species belonging to 10 phyla and 60 genera were detected by the PacBio RS II platform, while 163 species belonging to 12 phyla and 72 genera were detected by the MiSeq platform. Conclusions. +e oral microflora of healthy Chinese children were analyzed. Compared with traditional 16S rRNA sequencing technology, the PacBio system, despite providing a lower amount of clean data, surpassed the resolution of the MiSeq platform by improving the read length and annotating the nucleotide sequences at the species or strain level. +is trial is registered with NCT02341352. 1. Introduction for understanding the biology and functional character- ization of oral microorganisms. +e human oral microbiome comprises over 700 prevalent +e emergence of the next-generation sequencers (NGS) taxa at the species level, including a large number of op- and their sequencing by synthesis have drastically trans- portunistic pathogens involved in periodontal, respiratory, formed the way scientists delve into the relationship between cardiovascular, and systemic diseases [1–5]. Identification microbiome and related diseases [8]. Since then, many of oral microorganisms at the species level is the basis and studies have used the NGS technologies, such as Roche/454 prerequisite for analyzing microbial communities of the [9], ABI/Solid, Illumina [10], and its upgrade platforms oral cavity. +e 16S rRNA gene is considered the gold including Illumina/HiSeq and MiSeq for microbial ecosys- standard for phylogenetic studies of microbial communi- tem analysis [9–15]. When it comes to the resolution and ties and high-throughput sequencing of the 16S rRNA gene accuracy of the sequencing results, lengths and quantity of could provide snapshots of microbial communities, re- reads are very important factors [16–18]. Unfortunately, the vealing phylogeny and the abundances of microbial pop- NGS came with this drawback. Compared with the previous ulations across diverse ecosystems [6, 7]. For this reason, methods (e.g., Sanger sequencing), the reads generated are the sequencing techniques had become an important tool short. +is became a major challenge for the assembly, 2 Canadian Journal of Infectious Diseases and Medical Microbiology especially in the case of large repetitive genomes [19]. +us, 2.2. Saliva Sampling and Isolation of Bacterial DNAs. +e in spite of the low cost and extremely high-throughput, the subjects were instructed neither to eat and drink nor to NGS platform is sometimes less accurate as a result of short perform any oral hygiene procedure two hours before read lengths and long repeats present in multiple copies [17]. sampling. Saliva samples were collected from all subjects in Besides, although the explosion of sequence data brought the morning between 9 : 00 am and 11 : 00 am. about by high-throughput sequencing technologies is Unstimulated saliva samples were collected according to highlighting a richness of microbes not previously antici- a protocol, modified from a previous study. +e children pated, not all of the novel organisms discovered by the NGS were initially asked to rinse their mouth thoroughly with can be named by taxonomists because the existing tools are deionized water prior to sampling, followed by collection of not sufficient to provide species names or phylogenetic at least 5 mL unstimulated saliva in a plastic cup. Finally, the information for the millions of short reads [20]. Operational samples were transferred into sterile cryogenic vials. +en, taxonomic units (OTUs) at the 97% similarity is recognized the samples were placed into liquid nitrogen and stored at as providing differentiation of bacterial organisms below the −80°C until use. genus level [12]; however, it was still inaccurate for the Bacterial DNAs were extracted using the E.Z.N.A.™ Soil reason that this level of clustering defines either microbial DNA Kit (Qiagen, Omega, USA), according to the in- species or strains. structions of the manufacturer. +e enriched microbial +ird-generation sequencing (TGS), PacBio single DNAs were purified by ethanol precipitation. DNA con- molecule, real-time (SMRT) sequencing technology cir- centration was measured using NanoDrop, and its molecular cumvented this problem by greatly increasing read lengths size was estimated by agarose gel electrophoresis. DNAs that have the ability to sequence the full length of the 16S were stored at −20°C until use. rRNA gene [16, 18]. It involves a DNA fragment sequenced by a single DNA polymerase molecule connected to the bottom of a zero-mode waveguide [18]. During DNA 2.3. PCR Amplification of the 16S rRNA Gene. PCR ampli- synthesis, each of the nucleotides is illuminated upon in- fication of the 16S rRNA gene hypervariable V3-V4 regions corporation, which can enable for identification. +e PacBio was performed with universal bacterial primers 338F (5′- RS II can yield average sequence reads of greater than ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGAC- 2500 bp; however, some research data show that circular TACHVGGGTWTCTAAT-3′). +e V1-V9 hypervariable consensus sequencing (CCS) of shorter fragments region was performed with primers 27F (5′-AGAGTTT- (<1500 bp) can decrease the sequencing errors [21]. Some GATCCTGGCTCAG-3′) and 1492R (5′- studies have shown that the longer reads generated from GGTTACCTTGTTACGACTT-3′). +e products were sequencing the entire 16S rRNA gene provide a higher extracted with the AxyPrep DNA Gel Extraction kit (Qiagen, resolution of organisms and higher estimates of richness USA) and were then examined by agarose gel electropho- [17]. A previous study has shown that PacBio outperformed resis. According to the electrophoretic results, the PCR the other sequencers such as Roche 454 and MiSeq in terms products were quantified by Quantifluo™-ST (Promega, of the length of contigs and reconstructed the greatest USA). +en, the products from different samples were then portion of the genome when sequencing the genome of mixed at equal ratios for pyrosequencing on the two dif- Vibrio parahaemolyticus [22]. However, there have been few ferent platforms. studies that aim at comparing the next-generation se- quencing technology with PacBio RS II in oral microbiome. 2.4. DNA Library Construction and Sequencing. In this study, we explore the microbiota of oral cavity using Construction of DNA library was carried out by following sequences amplified V3-V4 and the V1-V9 small subunit the manufacturer’s instructions (Illumina and PacBio). A ribosomal RNA (16S) hypervariable regions by two different 468 bp insert size library was constructed on the MiSeq platforms. +e aim of this study was to evaluate the per- platform and then applied to 300 bp paired-end sequencing. formance of TGS technology PacBio RS II in comparison Libraries with longer insert size (1540 bp) were performed with NGS technology Illumina/MiSeq for the structure of on the PacBio RS II platform, including full length of 16S oral microbiome in 5 healthy preschool children in China. rDNA gene. Barcoded 16S rRNA amplicons (V3-V4 and V1- V9 hypervariable regions) of the five Chinese children were 2. Materials and Methods sequenced on MiSeq and PacBio RS II platforms, respec- tively. Raw data were generated, and low-quality reads were 2.1. Patient Information. Five preschool children aged 63–74 then removed by quality control (Figure 1). months, lacking evidence of oral and systematic diseases were recruited based on a list of exclusion criteria on Nov 26,
Load more

Recommended publications
  • Phylogenomic Analysis Supports the Ancestral Presence of LPS-Outer Membranes in the Firmicutes
    Phylogenomic analysis supports the ancestral presence of LPS-outer membranes in the Firmicutes. Luisa Cs Antunes, Daniel Poppleton, Andreas Klingl, Alexis Criscuolo, Bruno Dupuy, Céline Brochier-Armanet, Christophe Beloin, Simonetta Gribaldo To cite this version: Luisa Cs Antunes, Daniel Poppleton, Andreas Klingl, Alexis Criscuolo, Bruno Dupuy, et al.. Phy- logenomic analysis supports the ancestral presence of LPS-outer membranes in the Firmicutes.. eLife, eLife Sciences Publication, 2016, 5, pp.e14589. 10.7554/eLife.14589.020. pasteur-01362343 HAL Id: pasteur-01362343 https://hal-pasteur.archives-ouvertes.fr/pasteur-01362343 Submitted on 8 Sep 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License RESEARCH ARTICLE Phylogenomic analysis supports the ancestral presence of LPS-outer membranes in the Firmicutes Luisa CS Antunes1†, Daniel Poppleton1†, Andreas Klingl2, Alexis Criscuolo3, Bruno Dupuy4, Ce´ line Brochier-Armanet5, Christophe Beloin6, Simonetta Gribaldo1* 1Unite´ de
    [Show full text]
  • Veillonella, Firmicutes: Microbes Disguised As Gram Negatives
    Standards in Genomic Sciences (2013) 9:431-448 DOI:10.4056/sig s.2981345 Veillonella, Firmicutes: Microbes disguised as Gram negatives Tammi Vesth 1, Aslı Ozen 1,5, Sandra C. Andersen1, Rolf Sommer Kaas1, Oksana Lukjancenko1, Jon Bohlin2, Intawat Nookaew3, Trudy M. Wassenaar4 and David W. Ussery6* 1Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark 2Norwegian School of Veterinary Science, Department of Food Safety and Infection Biolog y, Oslo, Norway 3Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden 4Molecular Microbiology and Genomics Consultants, Zotzenheim, Germany 5The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark 6Comparative Genomics Group, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA *Correspondence: Dave Ussery ([email protected];) The Firmicutes represent a major component of the intestinal microflora. The intestinal Firmicutes a re a large, diverse group of organisms, many of which are poorly characterized due to their anaerobic growth requirements. Although most Firmicutes are Gram positive, members of the class Negativicutes, including the genus Veillonella, stain Gram negative. Veillonella are among the most abundant organisms of the oral and intestinal microflora of animals and humans, in spite of being strict anaerobes. In this work, the genomes of 24 Negativicutes, including eight Veillonella spp ., a re c ompa re d to 2 0 othe r Firmicutes genomes; a further 101 prokaryotic genomes were included, cover- ing 26 phyla. Thus a total of 145 prokaryotic genomes were analyzed by various methods to investi- gate the apparent conflict of the Veillonella Gram stain and their taxonomic position within the Firmicutes.
    [Show full text]
  • Phylogenetic Characterization of Subgingival Plaque Associated with Aggressive Or Chronic Periodontitis
    Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2011 Phylogenetic characterization of subgingival plaque associated with aggressive or chronic periodontitis Lüdin, N D Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-53536 Dissertation Originally published at: Lüdin, N D. Phylogenetic characterization of subgingival plaque associated with aggressive or chronic periodontitis. 2011, University of Zurich, Faculty of Medicine. Universitt Zürich Zentrum für Zahnmedizin Institut für Orale Biologie Abteilung für Orale Mikrobiologie und Immunologie Direktor a.i.: Prof. Dr. D. Wyler Arbeit unter der Leitung von Prof. Dr. sc. nat. R. Gmür Phylogenetic characterization of subgingival plaque associated with aggressive or chronic periodontitis INAUGURAL- DISSERTATION zur Erlangung der Doktorwürde der Zahnmedizin der Medizinischen Fakultt der Universitt Zürich vorgelegt von Nicole Dominique Lüdin von Neerach ZH Genehmigt auf Antrag von Prof. Dr. med. dent. T. Imfeld Zürich 2011 Contents Contents Abstract......................................................................................................................................................... 3 Introduction .................................................................................................................................................. 5 Materials and Methods ..............................................................................................................................
    [Show full text]
  • Oral Bacteriome of HIV‐1‐Infected Children from Rio De
    PROF. LUCIO SOUZA GONÇALVES (Orcid ID : 0000-0002-4388-6310) Article type : Original Article Clinical Periodontology Oral bacteriome of HIV-1-infected children from Rio de Janeiro – Brazil: Next- generation DNA sequencing analysis Running title: Oral bacteriome of HIV-infected children 1 Lucio Souza Gonçalves, DDS, MSc, PhD 1,2 Dennis de Carvalho Ferreira, DDS, MSc, PhD 7 Nicholas C. K. Heng, BSc, PhD 1 Fabio Vidal, DDS, MSc, PhD 3 Henrique Fragoso dos Santos, DDS, MSc, PhD 7 Diogo Godoy Zanicott, BDS, MClinDent, PhD 4 Mariana Vasconcellos, DDS, MSc 2 Mayra Stambovsky, DDS, MSc 8 Blair Lawley, PhD 6 Norma de Paula Motta Rubini, PhD 5 Katia Regina Netto dos Santos, MSc, PhD 9 Gregory J Seymour, BDS, MDSc, PhD 1Programa de Pós-Graduação em Odontologia, Universidade Estácio de Sá, Rio de Janeiro, Brazil. Author Manuscript This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/JCPE.13176 This article is protected by copyright. All rights reserved 2 Universidade Veiga de Almeida, Rio de Janeiro, Brazil. 3Universidade Federal Fluminense, RJ, Brazil. 4 Universidade Federal do Rio de Janeiro, Rio de janeiro, Brazil. 5Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. 6Departmento de Imunologia, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, Brazil. 7Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand 8Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand 9School of Dentistry, The University of Queensland, Brisbane, Australia Conflict of interest: The authors declare that they have no competing interests.
    [Show full text]