Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies
Supplemental File 1
AbstractBacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969- 983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies. © 2009 Wang, Qian.
CitationWang Y, Qian P-Y (2009) Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies. PLoS ONE 4: e7401. doi:10.1371/journal.pone.0007401.
SponsorsKAUST Global Partnership. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PublisherPublic Library of Science (PLoS)
PubMed Central IDPMC2754607
CollectionsPublications Acknowledging KAUST Support
- Design and experimental application of a novel non-degenerate universal primer set that amplifies prokaryotic 16S rRNA genes with a low possibility to amplify eukaryotic rRNA genes.
- Authors: Mori H, Maruyama F, Kato H, Toyoda A, Dozono A, Ohtsubo Y, Nagata Y, Fujiyama A, Tsuda M, Kurokawa K
- Issue date: 2014
- Metagenomic 16S rDNA Illumina tags are a powerful alternative to amplicon sequencing to explore diversity and structure of microbial communities.
- Authors: Logares R, Sunagawa S, Salazar G, Cornejo-Castillo FM, Ferrera I, Sarmento H, Hingamp P, Ogata H, de Vargas C, Lima-Mendez G, Raes J, Poulain J, Jaillon O, Wincker P, Kandels-Lewis S, Karsenti E, Bork P, Acinas SG
- Issue date: 2014 Sep
- Comparing bacterial communities inferred from 16S rRNA gene sequencing and shotgun metagenomics.
- Authors: Shah N, Tang H, Doak TG, Ye Y
- Issue date: 2011
- Usefulness of the MicroSeq 500 16S ribosomal DNA-based bacterial identification system for identification of clinically significant bacterial isolates with ambiguous biochemical profiles.
- Authors: Woo PC, Ng KH, Lau SK, Yip KT, Fung AM, Leung KW, Tam DM, Que TL, Yuen KY
- Issue date: 2003 May
- Comparison of 16S rRNA and protein-coding genes as molecular markers for assessing microbial diversity (Bacteria and Archaea) in ecosystems.
- Authors: Roux S, Enault F, Bronner G, Debroas D
- Issue date: 2011 Dec
Showing items related by title, author, creator and subject.
Bacterial niche-specific genome expansion is coupled with highly frequent gene disruptions in deep-sea sedimentsWang, Yong; Yang, Jiang Ke; Lee, On On; Li, Tie Gang; Al-Suwailem, Abdulaziz M.; Danchin, Antoine; Qian, Pei-Yuan (Public Library of Science (PLoS), 2011-12-21)The complexity and dynamics of microbial metagenomes may be evaluated by genome size, gene duplication and the disruption rate between lineages. In this study, we pyrosequenced the metagenomes of microbes obtained from the brine and sediment of a deep-sea brine pool in the Red Sea to explore the possible genomic adaptations of the microbes in response to environmental changes. The microbes from the brine and sediments (both surface and deep layers) of the Atlantis II Deep brine pool had similar communities whereas the effective genome size varied from 7.4 Mb in the brine to more than 9 Mb in the sediment. This genome expansion in the sediment samples was due to gene duplication as evidenced by enrichment of the homologs. The duplicated genes were highly disrupted, on average by 47.6% and 70% for the surface and deep layers of the Atlantis II Deep sediment samples, respectively. The disruptive effects appeared to be mainly due to point mutations and frameshifts. In contrast, the homologs from the Atlantis II Deep brine sample were highly conserved and they maintained relatively small copy numbers. Likely, the adaptation of the microbes in the sediments was coupled with pseudogenizations and possibly functional diversifications of the paralogs in the expanded genomes. The maintenance of the pseudogenes in the large genomes is discussed. © 2011 Wang et al.
Bacterial profiling of White Plague Disease across corals and oceans indicates a conserved and distinct disease microbiomeRoder, C.; Arif, C.; Daniels, C.; Weil, E.; Voolstra, Christian R. (Wiley-Blackwell, 2014-01-29)Coral diseases are characterized by microbial community shifts in coral mucus and tissue, but causes and consequences of these changes are vaguely understood due to the complexity and dynamics of coral-associated bacteria. We used 16S rRNA gene microarrays to assay differences in bacterial assemblages of healthy and diseased colonies displaying White Plague Disease (WPD) signs from two closely related Caribbean coral species, Orbicella faveolata and Orbicella franksi. Analysis of differentially abundant operational taxonomic units (OTUs) revealed strong differences between healthy and diseased specimens, but not between coral species. A subsequent comparison to data from two Indo-Pacific coral species (Pavona duerdeni and Porites lutea) revealed distinct microbial community patterns associated with ocean basin, coral species and health state. Coral species were clearly separated by site, but also, the relatedness of the underlying bacterial community structures resembled the phylogenetic relationship of the coral hosts. In diseased samples, bacterial richness increased and putatively opportunistic bacteria were consistently more abundant highlighting the role of opportunistic conditions in structuring microbial community patterns during disease. Our comparative analysis shows that it is possible to derive conserved bacterial footprints of diseased coral holobionts that might help in identifying key bacterial species related to the underlying etiopathology. Furthermore, our data demonstrate that similar-appearing disease phenotypes produce microbial community patterns that are consistent over coral species and oceans, irrespective of the putative underlying pathogen. Consequently, profiling coral diseases by microbial community structure over multiple coral species might allow the development of a comparative disease framework that can inform on cause and relatedness of coral diseases. 2013 The Authors Molecular Ecology John Wiley & Sons Ltd.
Unraveling plant responses to bacterial pathogens through proteomicsZimaro, Tamara; Gottig, Natalia; Garavaglia, Betiana S.; Gehring, Christoph A; Ottado, Jorgelina (Hindawi Publishing Corporation, 2011-11-03)Plant pathogenic bacteria cause diseases in important crops and seriously and negatively impact agricultural production. Therefore, an understanding of the mechanisms by which plants resist bacterial infection at the stage of the basal immune response or mount a successful specific R-dependent defense response is crucial since a better understanding of the biochemical and cellular mechanisms underlying these interactions will enable molecular and transgenic approaches to crops with increased biotic resistance. In recent years, proteomics has been used to gain in-depth understanding of many aspects of the host defense against pathogens and has allowed monitoring differences in abundance of proteins as well as posttranscriptional and posttranslational processes, protein activation/inactivation, and turnover. Proteomics also offers a window to study protein trafficking and routes of communication between organelles. Here, we summarize and discuss current progress in proteomics of the basal and specific host defense responses elicited by bacterial pathogens. Copyright 2011 Tamara Zimaro et al.