A Comparison of rpoB and 16S rRNA as Markers in Pyrosequencing Studies of Bacterial Diversity
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{"title"=>"A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity", "type"=>"journal", "authors"=>[{"first_name"=>"Michiel", "last_name"=>"Vos", "scopus_author_id"=>"56228683600"}, {"first_name"=>"Christopher", "last_name"=>"Quince", "scopus_author_id"=>"8897703900"}, {"first_name"=>"Agata S.", "last_name"=>"Pijl", "scopus_author_id"=>"6603145573"}, {"first_name"=>"Mattias", "last_name"=>"de Hollander", "scopus_author_id"=>"24166120100"}, {"first_name"=>"George A.", "last_name"=>"Kowalchuk", "scopus_author_id"=>"7003778501"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"364268081", "sgr"=>"84857067661", "issn"=>"19326203", "pmid"=>"22355318", "scopus"=>"2-s2.0-84857067661", "doi"=>"10.1371/journal.pone.0030600", "isbn"=>"1932-6203"}, "id"=>"5182b652-f193-3ea9-a835-725bafcd1861", "abstract"=>"<sec> <title>Background</title> <p>The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding <italic>rpoB</italic> marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the <italic>rpoB</italic> gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria.</p> </sec> <sec> <title>Methodology/Principal Findings</title> <p>Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the <italic>rpoB</italic> marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster.</p> </sec> <sec> <title>Conclusions/Significance</title> <p>The <italic>rpoB</italic> marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed.</p> </sec>", "link"=>"http://www.mendeley.com/research/comparison-rpob-16s-rrna-markers-pyrosequencing-studies-bacterial-diversity", "reader_count"=>244, "reader_count_by_academic_status"=>{"Unspecified"=>8, "Professor > Associate Professor"=>9, "Librarian"=>1, "Student > Doctoral Student"=>13, "Researcher"=>66, "Student > Ph. D. Student"=>66, "Student > Postgraduate"=>14, "Other"=>8, "Student > Master"=>33, "Student > Bachelor"=>15, "Lecturer"=>3, "Lecturer > Senior Lecturer"=>1, "Professor"=>7}, "reader_count_by_user_role"=>{"Unspecified"=>8, "Professor > Associate Professor"=>9, "Librarian"=>1, "Student > Doctoral Student"=>13, "Researcher"=>66, "Student > Ph. D. Student"=>66, "Student > Postgraduate"=>14, "Other"=>8, "Student > Master"=>33, "Student > Bachelor"=>15, "Lecturer"=>3, "Lecturer > Senior Lecturer"=>1, "Professor"=>7}, "reader_count_by_subject_area"=>{"Unspecified"=>15, "Environmental Science"=>20, "Biochemistry, Genetics and Molecular Biology"=>26, "Mathematics"=>1, "Agricultural and Biological Sciences"=>163, "Medicine and Dentistry"=>3, "Arts and Humanities"=>1, "Chemistry"=>2, "Social Sciences"=>1, "Computer Science"=>1, "Immunology and Microbiology"=>8, "Earth and Planetary Sciences"=>3}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Chemistry"=>{"Chemistry"=>2}, "Social Sciences"=>{"Social Sciences"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>8}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>163}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>26}, "Mathematics"=>{"Mathematics"=>1}, "Unspecified"=>{"Unspecified"=>15}, "Environmental Science"=>{"Environmental Science"=>20}, "Arts and Humanities"=>{"Arts and Humanities"=>1}}, "reader_count_by_country"=>{"Argentina"=>1, "United States"=>9, "United Kingdom"=>3, "Spain"=>1, "India"=>1, "Greece"=>1, "Canada"=>3, "Czech Republic"=>1, "Netherlands"=>1, "Sweden"=>1, "Belgium"=>1, "Brazil"=>5, "Denmark"=>1, "Mexico"=>3, "France"=>3, "Nigeria"=>1, "Chile"=>2, "Germany"=>4, "Estonia"=>1}, "group_count"=>7}

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Figshare

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  • {"files"=>["https://ndownloader.figshare.com/files/682216"], "description"=>"<p>The 1% and 2.3% cut-offs for 16S rRNA and <i>rpoB</i> are chosen to reflect species definitions (see text).</p>", "links"=>[], "tags"=>["curves", "otu", "proteobacteria"], "article_id"=>352705, "categories"=>["Biological Sciences", "Ecology", "Genetics", "Evolutionary Biology"], "users"=>["Michiel Vos", "Christopher Quince", "Agata S. Pijl", "Mattias de Hollander", "George A. Kowalchuk"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0030600.g002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Rarefaction_curves_showing_mean_expected_OTU_number_for_Proteobacteria_as_a_function_of_sample_size_/352705", "title"=>"Rarefaction curves showing mean expected OTU number for Proteobacteria as a function of sample size.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-15 00:45:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/682378"], "description"=>"<p>* = The parametric total diversity estimates are given as lower 95% confidence interval : median : upper 95% confidence interval.</p>", "links"=>[], "tags"=>["sampling", "defined", "reads", "proteobacterial"], "article_id"=>352866, "categories"=>["Biological Sciences", "Ecology", "Genetics", "Evolutionary Biology"], "users"=>["Michiel Vos", "Christopher Quince", "Agata S. Pijl", "Mattias de Hollander", "George A. Kowalchuk"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0030600.t002", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_90_sampling_effort_defined_as_number_of_reads_required_to_observe_90_of_the_true_diversity_for_proteobacterial_species_/352866", "title"=>"90% sampling effort, defined as number of reads required to observe 90% of the true diversity, for proteobacterial species.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-02-15 00:47:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/682302"], "description"=>"<p>A: a Minimum Spanning Tree for all sequences more than 94% similar to abundant Anaeromyxobacter sequence R100. Circle size equates with the number of sequences, bar length equates with the number of nucleotide substitutions between sequences. B: a NeighbourNet phylogenetic network based on the same sequences as in A.</p>", "links"=>[], "tags"=>["genetics and genomics", "Computational biology", "ecology", "Evolutionary biology"], "article_id"=>352789, "categories"=>["Biological Sciences", "Ecology", "Genetics", "Evolutionary Biology"], "users"=>["Michiel Vos", "Christopher Quince", "Agata S. Pijl", "Mattias de Hollander", "George A. Kowalchuk"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0030600.g003", "stats"=>{"downloads"=>2, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Population_level_analyses_/352789", "title"=>"Population-level analyses.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-02-15 00:46:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/682417"], "description"=>"<p>* = The parametric total diversity estimates are given as lower 95% confidence interval : median : upper 95% confidence interval.</p>", "links"=>[], "tags"=>["richness", "sequences", "classified"], "article_id"=>352905, "categories"=>["Biological Sciences", "Ecology", "Genetics", "Evolutionary Biology"], "users"=>["Michiel Vos", "Christopher Quince", "Agata S. Pijl", "Mattias de Hollander", "George A. Kowalchuk"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0030600.t001", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Estimation_of_species_richness_for_sequences_classified_as_Proteobacteria_/352905", "title"=>"Estimation of species richness for sequences classified as Proteobacteria.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-02-15 00:48:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/347670", "https://ndownloader.figshare.com/files/347729", "https://ndownloader.figshare.com/files/347779"], "description"=>"<div><h3>Background</h3><p>The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding <em>rpoB</em> marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the <em>rpoB</em> gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria.</p> <h3>Methodology/Principal Findings</h3><p>Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the <em>rpoB</em> marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster.</p> <h3>Conclusions/Significance</h3><p>The <em>rpoB</em> marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed.</p> </div>", "links"=>[], "tags"=>["16s", "rrna", "markers", "pyrosequencing", "studies", "bacterial"], "article_id"=>128750, "categories"=>["Biological Sciences", "Ecology", "Genetics", "Evolutionary Biology"], "users"=>["Michiel Vos", "Christopher Quince", "Agata S. Pijl", "Mattias de Hollander", "George A. Kowalchuk"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0030600.s001", "https://dx.doi.org/10.1371/journal.pone.0030600.s002", "https://dx.doi.org/10.1371/journal.pone.0030600.s003"], "stats"=>{"downloads"=>4, "page_views"=>40, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_Comparison_of_rpoB_and_16S_rRNA_as_Markers_in_Pyrosequencing_Studies_of_Bacterial_Diversity/128750", "title"=>"A Comparison of <em>rpoB</em> and 16S rRNA as Markers in Pyrosequencing Studies of Bacterial Diversity", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-02-15 02:25:50"}

PMC Usage Stats | Further Information

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Relative Metric

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