Amplification by PCR Artificially Reduces the Proportion of the Rare Biosphere in Microbial Communities
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{"title"=>"Amplification by PCR artificially reduces the proportion of the rare biosphere in microbial communities", "type"=>"journal", "authors"=>[{"first_name"=>"Juan M.", "last_name"=>"Gonzalez", "scopus_author_id"=>"56808320000"}, {"first_name"=>"Maria C.", "last_name"=>"Portillo", "scopus_author_id"=>"26646979800"}, {"first_name"=>"Pedro", "last_name"=>"Belda-Ferre", "scopus_author_id"=>"41761038000"}, {"first_name"=>"Alex", "last_name"=>"Mira", "scopus_author_id"=>"7004449568"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-84855692788", "doi"=>"10.1371/journal.pone.0029973", "sgr"=>"84855692788", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"22253843", "issn"=>"19326203", "pui"=>"364073936"}, "id"=>"ed0f5080-0f59-34c5-bae2-afd9269b50fb", "abstract"=>"The microbial world has been shown to hold an unimaginable diversity. The use of rRNA genes and PCR amplification to assess microbial community structure and diversity present biases that need to be analyzed in order to understand the risks involved in those estimates. Herein, we show that PCR amplification of specific sequence targets within a community depends on the fractions that those sequences represent to the total DNA template. Using quantitative, real-time, multiplex PCR and specific Taqman probes, the amplification of 16S rRNA genes from four bacterial species within a laboratory community were monitored. Results indicate that the relative amplification efficiency for each bacterial species is a nonlinear function of the fraction that each of those taxa represent within a community or multispecies DNA template. Consequently, the low-proportion taxa in a community are under-represented during PCR-based surveys and a large number of sequences might need to be processed to detect some of the bacterial taxa within the 'rare biosphere'. The structure of microbial communities from PCR-based surveys is clearly biased against low abundant taxa which are required to decipher the complete extent of microbial diversity in nature.", "link"=>"http://www.mendeley.com/research/amplification-pcr-artificially-reduces-proportion-rare-biosphere-microbial-communities", "reader_count"=>165, "reader_count_by_academic_status"=>{"Unspecified"=>3, "Professor > Associate Professor"=>5, "Researcher"=>43, "Student > Doctoral Student"=>8, "Student > Ph. D. Student"=>57, "Student > Postgraduate"=>6, "Student > Master"=>18, "Other"=>9, "Student > Bachelor"=>4, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>10}, "reader_count_by_user_role"=>{"Unspecified"=>3, "Professor > Associate Professor"=>5, "Researcher"=>43, "Student > Doctoral Student"=>8, "Student > Ph. D. Student"=>57, "Student > Postgraduate"=>6, "Student > Master"=>18, "Other"=>9, "Student > Bachelor"=>4, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>10}, "reader_count_by_subject_area"=>{"Unspecified"=>9, "Engineering"=>6, "Environmental Science"=>14, "Biochemistry, Genetics and Molecular Biology"=>6, "Mathematics"=>1, "Agricultural and Biological Sciences"=>115, "Medicine and Dentistry"=>6, "Business, Management and Accounting"=>1, "Physics and Astronomy"=>1, "Computer Science"=>1, "Immunology and Microbiology"=>4, "Earth and Planetary Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>6}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>6}, "Physics and Astronomy"=>{"Physics and Astronomy"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>4}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>115}, "Computer Science"=>{"Computer Science"=>1}, "Business, Management and Accounting"=>{"Business, Management and Accounting"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>6}, "Mathematics"=>{"Mathematics"=>1}, "Unspecified"=>{"Unspecified"=>9}, "Environmental Science"=>{"Environmental Science"=>14}}, "reader_count_by_country"=>{"United States"=>13, "Japan"=>1, "United Kingdom"=>2, "Spain"=>7, "Canada"=>2, "Netherlands"=>1, "Sweden"=>1, "Belgium"=>1, "Norway"=>1, "Brazil"=>2, "Denmark"=>2, "Mexico"=>3, "Slovenia"=>1, "Chile"=>1, "France"=>2, "Germany"=>1, "Estonia"=>1}, "group_count"=>10}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/693765"], "description"=>"<p>The plots correspond to amplification from different multispecies DNA mixtures representing different fraction of total DNA template, from 0.01% to 100%. The bacterial species are: <i>Bacillus subtilis</i>, <i>Deinococcus radiodurans</i>, <i>Escherichia coli</i>, and <i>Pseudomonas aeruginosa</i>. The percentage of the examined bacterial species for each reaction set is indicated on the right.</p>", "links"=>[], "tags"=>["pcr", "amplification", "dna", "bacterial"], "article_id"=>364205, "categories"=>["Molecular Biology", "Ecology"], "users"=>["Juan M. Gonzalez", "Maria C. Portillo", "Pedro Belda-Ferre", "Alex Mira"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029973.g001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Real_time_PCR_amplification_of_DNA_for_four_bacterial_species_/364205", "title"=>"Real-time PCR amplification of DNA for four bacterial species.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 10:01:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/694120"], "description"=>"<p>Two artificial communities (I and II) are shown. They were estimated by following a random amplification (black squares) and the proposed model (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029973#pone-0029973-g002\" target=\"_blank\">Figure 2</a>) (white squares) of the relative amplification efficiency as a function of the fraction that each OTU represents within the total community. A, Community I; B, Community II.</p>", "links"=>[], "tags"=>["ratios", "otu", "ranking"], "article_id"=>364556, "categories"=>["Molecular Biology", "Ecology"], "users"=>["Juan M. Gonzalez", "Maria C. Portillo", "Pedro Belda-Ferre", "Alex Mira"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029973.g004", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Product_to_template_ratios_versus_OTU_ranking_for_two_artificial_communities_/364556", "title"=>"Product-to-template ratios <i>versus</i> OTU ranking for two artificial communities.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 10:03:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/694305"], "description"=>"<p>Two communities (1 and 2) from the human oral cavity were analyzed. The product was represented by the amplicon analysis data and the template was considered the results obtained from a metagenome analysis lacking PCR amplification. A, Experimental community 1; B, Experimental community 2.</p>", "links"=>[], "tags"=>["ratios", "ranked", "genera"], "article_id"=>364739, "categories"=>["Molecular Biology", "Ecology"], "users"=>["Juan M. Gonzalez", "Maria C. Portillo", "Pedro Belda-Ferre", "Alex Mira"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029973.g006", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Product_to_template_ratios_versus_ranked_genera_for_two_experimental_communities_/364739", "title"=>"Product-to-template ratios <i>versus</i> ranked genera for two experimental communities.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 10:04:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/694436"], "description"=>"1<p>BHQ, Blackhole Quencher.</p>", "links"=>[], "tags"=>["primers", "labeled", "probes"], "article_id"=>364866, "categories"=>["Molecular Biology", "Ecology"], "users"=>["Juan M. Gonzalez", "Maria C. Portillo", "Pedro Belda-Ferre", "Alex Mira"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029973.t002", "stats"=>{"downloads"=>2, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PCR_primers_and_labeled_probes_utilized_in_the_present_study_/364866", "title"=>"PCR primers and labeled probes utilized in the present study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-20 10:05:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/694190"], "description"=>"<p>The curves shown correspond to the results of 16S rRNA gene analyses from a metagenome study through direct sequencing (no PCR amplification) (black dashed lines) and from a microbial survey based on the 16S rRNA amplicon analysis after PCR amplification (solid lines). A, Experimental community 1; B, Experimental community 2.</p>", "links"=>[], "tags"=>["curves", "genus", "microbial", "communities"], "article_id"=>364623, "categories"=>["Molecular Biology", "Ecology"], "users"=>["Juan M. Gonzalez", "Maria C. Portillo", "Pedro Belda-Ferre", "Alex Mira"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029973.g005", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Rarefaction_curves_at_the_genus_level_obtained_for_two_experimental_microbial_communities_from_the_human_oral_cavity_/364623", "title"=>"Rarefaction curves at the genus level obtained for two experimental microbial communities from the human oral cavity.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 10:03:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/694469"], "description"=>"<p>Maximum sequencing effort considered was the processing of 10<sup>8</sup> sequences.</p>1<p>Approximated sequencing effort required to detect an OTU present at the indicated frequency through PCR-based surveys.</p>2<p>Taxa requiring a sequencing effort higher than 10<sup>8</sup> sequences were considered to remain undetected (ND).</p>", "links"=>[], "tags"=>["communities", "ii", "estimates", "sequencing", "taxa", "pcr-based", "surveys"], "article_id"=>364892, "categories"=>["Molecular Biology", "Ecology"], "users"=>["Juan M. Gonzalez", "Maria C. Portillo", "Pedro Belda-Ferre", "Alex Mira"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029973.t003", "stats"=>{"downloads"=>4, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Composition_of_artificial_communities_I_and_II_and_estimates_of_the_sequencing_effort_expected_to_detect_these_taxa_through_PCR_based_surveys_according_to_the_proposed_model_/364892", "title"=>"Composition of artificial communities I and II and estimates of the sequencing effort expected to detect these taxa through PCR-based surveys according to the proposed model.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-20 10:05:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/693962"], "description"=>"<p>A, Community I with a total of 11 OTUs and C, Community II with a total of 248 OTUs. The curves for the initial community (black dashed lines) and post-PCR communities (solid lines) according to the proposed model (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029973#pone-0029973-g002\" target=\"_blank\">Figure 2</a>) are shown. The portion of the curves included in the dotted squares are amplified on the right. B and D correspond to the lowest sampling sizes for A and C, respectively.</p>", "links"=>[], "tags"=>["curves"], "article_id"=>364407, "categories"=>["Molecular Biology", "Ecology"], "users"=>["Juan M. Gonzalez", "Maria C. Portillo", "Pedro Belda-Ferre", "Alex Mira"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029973.g003", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Rarefaction_curves_obtained_for_two_different_artificial_communities_/364407", "title"=>"Rarefaction curves obtained for two different artificial communities.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 10:02:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/694383"], "description"=>"<p>For the artificial communities, the analysis includes the original communities, and the communities obtained after a theoretically random amplification and after simulating the proposed amplification model. For the experimental communities, results of the 16S rRNA analysis from metagenome analysis through direct sequencing (lacking amplification by PCR) and 16S rRNA amplicon analysis after PCR amplification are presented.</p>1<p>Number of taxa detected during the analyses. For the artificial communities 10<sup>8</sup> sequences were screened. For the experimental communities around 10<sup>3</sup> sequences were processed.</p>", "links"=>[], "tags"=>["indices"], "article_id"=>364822, "categories"=>["Molecular Biology", "Ecology"], "users"=>["Juan M. Gonzalez", "Maria C. Portillo", "Pedro Belda-Ferre", "Alex Mira"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029973.t004", "stats"=>{"downloads"=>3, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparison_of_diversity_indices_estimated_from_two_artificial_and_two_experimental_communities_/364822", "title"=>"Comparison of diversity indices estimated from two artificial and two experimental communities.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-20 10:04:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/694495"], "description"=>"<p>Bacterial species used in this study including the number of 16S rRNA genes per genome and their genome length.</p>", "links"=>[], "tags"=>["16s", "rrna", "genes", "genome"], "article_id"=>364927, "categories"=>["Molecular Biology", "Ecology"], "users"=>["Juan M. Gonzalez", "Maria C. Portillo", "Pedro Belda-Ferre", "Alex Mira"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029973.t001", "stats"=>{"downloads"=>2, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Bacterial_species_used_in_this_study_including_the_number_of_16S_rRNA_genes_per_genome_and_their_genome_length_/364927", "title"=>"Bacterial species used in this study including the number of 16S rRNA genes per genome and their genome length.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-20 10:05:30"}
  • {"files"=>["https://ndownloader.figshare.com/files/693846"], "description"=>"<p><i>Bacillus subtilis</i> (black squares), <i>Deinococcus radiodurans</i> (white triangles), <i>Escherichia coli</i> (white squares), and <i>Pseudomonas aeruginosa</i> (black triangles), at different proportions within the community. Linear (A) and logarithmic (B) scales are shown to see the curve fitting for large and low initial proportions of the target sequences within the community.</p>", "links"=>[], "tags"=>["amplification", "16s", "rrna", "genes", "bacterial"], "article_id"=>364287, "categories"=>["Molecular Biology", "Ecology"], "users"=>["Juan M. Gonzalez", "Maria C. Portillo", "Pedro Belda-Ferre", "Alex Mira"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029973.g002", "stats"=>{"downloads"=>2, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Relationship_between_the_relative_amplification_efficiency_of_16S_rRNA_genes_from_four_bacterial_species_/364287", "title"=>"Relationship between the relative amplification efficiency of 16S rRNA genes from four bacterial species.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 10:01:56"}

PMC Usage Stats | Further Information

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  • {"unique-ip"=>"8", "full-text"=>"18", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"9"}
  • {"unique-ip"=>"13", "full-text"=>"13", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2015", "month"=>"10"}
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  • {"unique-ip"=>"10", "full-text"=>"6", "pdf"=>"3", "abstract"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"9"}
  • {"unique-ip"=>"13", "full-text"=>"8", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"1", "year"=>"2014", "month"=>"10"}
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