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      Oligotyping: differentiating between closely related microbial taxa using 16S rRNA gene data

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          Abstract

          1. Bacteria comprise the most diverse domain of life on Earth, where they occupy nearly every possible ecological niche and play key roles in biological and chemical processes. Studying the composition and ecology of bacterial ecosystems and understanding their function are of prime importance. High-throughput sequencing technologies enable nearly comprehensive descriptions of bacterial diversity through 16S ribosomal RNA gene amplicons. Analyses of these communities generally rely upon taxonomic assignments through reference data bases or clustering approaches using de facto sequence similarity thresholds to identify operational taxonomic units. However, these methods often fail to resolve ecologically meaningful differences between closely related organisms in complex microbial data sets.

          2. In this paper, we describe oligotyping, a novel supervised computational method that allows researchers to investigate the diversity of closely related but distinct bacterial organisms in final operational taxonomic units identified in environmental data sets through 16S ribosomal RNA gene data by the canonical approaches.

          3. Our analysis of two data sets from two different environments demonstrates the capacity of oligotyping at discriminating distinct microbial populations of ecological importance.

          4. Oligotyping can resolve the distribution of closely related organisms across environments and unveil previously overlooked ecological patterns for microbial communities. The URL http://oligotyping.org offers an open-source software pipeline for oligotyping.

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          Most cited references37

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          Life in extreme environments.

          Each recent report of liquid water existing elsewhere in the Solar System has reverberated through the international press and excited the imagination of humankind. Why? Because in the past few decades we have come to realize that where there is liquid water on Earth, virtually no matter what the physical conditions, there is life. What we previously thought of as insurmountable physical and chemical barriers to life, we now see as yet another niche harbouring 'extremophiles'. This realization, coupled with new data on the survival of microbes in the space environment and modelling of the potential for transfer of life between celestial bodies, suggests that life could be more common than previously thought. Here we examine critically what it means to be an extremophile, and the implications of this for evolution, biotechnology and especially the search for life in the Universe.
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            Performance comparison of benchtop high-throughput sequencing platforms.

            Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).
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              A molecular view of microbial diversity and the biosphere.

              N Pace (1997)
              Over three decades of molecular-phylogenetic studies, researchers have compiled an increasingly robust map of evolutionary diversification showing that the main diversity of life is microbial, distributed among three primary relatedness groups or domains: Archaea, Bacteria, and Eucarya. The general properties of representatives of the three domains indicate that the earliest life was based on inorganic nutrition and that photosynthesis and use of organic compounds for carbon and energy metabolism came comparatively later. The application of molecular-phylogenetic methods to study natural microbial ecosystems without the traditional requirement for cultivation has resulted in the discovery of many unexpected evolutionary lineages; members of some of these lineages are only distantly related to known organisms but are sufficiently abundant that they are likely to have impact on the chemistry of the biosphere.
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                Author and article information

                Journal
                Methods Ecol Evol
                Methods Ecol Evol
                mee3
                Methods in Ecology and Evolution
                BlackWell Publishing Ltd (Oxford, UK )
                2041-210X
                2041-210X
                December 2013
                23 October 2013
                : 4
                : 12
                : 1111-1119
                Affiliations
                [1 ]Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory Woods Hole, MA, 02543, USA
                Author notes
                *Correspondence author. E-mail: meren@ 123456mbl.edu
                Article
                10.1111/2041-210X.12114
                3864673
                24358444
                fefab029-2c12-4cfc-9bac-8040561234fe
                © 2013 The Authors. Methods in Ecology and Evolution © 2013 British Ecological Society.

                This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

                History
                : 08 March 2013
                : 20 August 2013
                Categories
                Taxa

                16s,bacterial taxonomy,microbial diversity,otu clustering,shannon entropy

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