A Direct PCR Approach to Accelerate Analyses of Human-Associated Microbial Communities
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{"title"=>"A Direct PCR Approach to Accelerate Analyses of Human-Associated Microbial Communities", "type"=>"journal", "authors"=>[{"first_name"=>"Gilberto E.", "last_name"=>"Flores", "scopus_author_id"=>"36857955400"}, {"first_name"=>"Jessica B.", "last_name"=>"Henley", "scopus_author_id"=>"55355101700"}, {"first_name"=>"Noah", "last_name"=>"Fierer", "scopus_author_id"=>"8638392900"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"365608654", "sgr"=>"84866040222", "issn"=>"19326203", "pmid"=>"22962617", "scopus"=>"2-s2.0-84866040222", "doi"=>"10.1371/journal.pone.0044563", "isbn"=>"1932-6203 (Electronic)\\n1932-6203 (Linking)"}, "id"=>"901150bc-9a64-3b96-9f78-1162d2b105ac", "abstract"=>"Since the composition of the human microbiome is highly variable both within and between individuals, researchers are increasingly reliant on high-throughput molecular approaches to identify linkages between the composition of these communities and human health. While new sequencing technologies have made it increasingly feasible to analyze large numbers of human-associated samples, the extraction of DNA from samples often remains a bottleneck in the process. Here we tested a direct PCR approach using the Extract-N-Amp Plant PCR Kit to accelerate the 16S rRNA gene-based analyses of human-associated bacterial communities, directly comparing this method to a more commonly-used approach whereby DNA is first extracted and purified from samples using a series of steps prior to PCR amplification. We used both approaches on replicate samples collected from each of five body habitats (tongue surface, feces, forehead skin, underarm skin, and forearm skin) from four individuals. With the exception of the tongue samples, there were few significant differences in the estimates of taxon richness or phylogenetic diversity obtained using the two approaches. Perhaps more importantly, there were no significant differences between the methods in their ability resolve body habitat differences or inter-individual differences in bacterial community composition and the estimates of the relative abundances of individual taxa were nearly identical with the two methods. Overall, the two methods gave very similar results and the direct PCR approach is clearly advantageous for many studies exploring the diversity and composition of human-associated bacterial communities given that large numbers of samples can be processed far more quickly and efficiently.", "link"=>"http://www.mendeley.com/research/direct-pcr-approach-accelerate-analyses-humanassociated-microbial-communities", "reader_count"=>142, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>9, "Researcher"=>44, "Student > Doctoral Student"=>8, "Student > Ph. D. Student"=>35, "Student > Postgraduate"=>6, "Student > Master"=>17, "Other"=>3, "Student > Bachelor"=>10, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>6}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>9, "Researcher"=>44, "Student > Doctoral Student"=>8, "Student > Ph. D. Student"=>35, "Student > Postgraduate"=>6, "Student > Master"=>17, "Other"=>3, "Student > Bachelor"=>10, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>6}, "reader_count_by_subject_area"=>{"Engineering"=>4, "Unspecified"=>3, "Environmental Science"=>7, "Biochemistry, Genetics and Molecular Biology"=>6, "Agricultural and Biological Sciences"=>102, "Medicine and Dentistry"=>7, "Chemistry"=>3, "Computer Science"=>2, "Immunology and Microbiology"=>5, "Earth and Planetary Sciences"=>2}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>4}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>7}, "Chemistry"=>{"Chemistry"=>3}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>5}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>102}, "Computer Science"=>{"Computer Science"=>2}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>6}, "Unspecified"=>{"Unspecified"=>3}, "Environmental Science"=>{"Environmental Science"=>7}}, "reader_count_by_country"=>{"Venezuela"=>1, "Canada"=>1, "Sweden"=>1, "United States"=>12, "Japan"=>2, "Mexico"=>2, "South Africa"=>1, "France"=>1, "Chile"=>1, "Germany"=>2, "India"=>1}, "group_count"=>6}

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  • {"files"=>["https://ndownloader.figshare.com/files/582343"], "description"=>"<p>R-values in bold indicate statistically significant differences (p<0.05).</p>", "links"=>[], "tags"=>["anosim", "comparing", "extraction", "bacterial"], "article_id"=>252841, "categories"=>["Biotechnology", "Genetics", "Microbiology", "Evolutionary Biology"], "users"=>["Gilberto E. Flores", "Jessica B. Henley", "Noah Fierer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044563.t002", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Results_of_ANOSIM_tests_comparing_the_affect_of_extraction_method_on_bacterial_community_composition_unweighted_UniFrac_of_each_body_habitat_from_each_individual_/252841", "title"=>"Results of ANOSIM tests comparing the affect of extraction method on bacterial community composition (unweighted UniFrac) of each body habitat from each individual.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-19 23:58:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/582085"], "description"=>"<p>Samples are grouped by body habitat and individuals.</p>", "links"=>[], "tags"=>["taxonomic", "habitats", "observed"], "article_id"=>252582, "categories"=>["Biotechnology", "Genetics", "Microbiology", "Evolutionary Biology"], "users"=>["Gilberto E. Flores", "Jessica B. Henley", "Noah Fierer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044563.g003", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Average_taxonomic_composition_of_the_various_body_habitats_observed_using_both_the_direct_and_standard_extraction_purification_protocols_/252582", "title"=>"Average taxonomic composition of the various body habitats observed using both the direct and standard extraction/purification protocols.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-19 23:56:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/582037"], "description"=>"<p>Bars with asterisks denote comparisons that were statistically significant within an individual (t-test, one asterisks p≤0.05, two asterisks p≤0.01). Side brackets with asterisks denote comparisons that were statistically significant across all individuals (paired t-test, p≤0.01). Error bars are ± one standard deviation.</p>", "links"=>[], "tags"=>["phylogenetic", "observed", "communities", "pcr"], "article_id"=>252528, "categories"=>["Biotechnology", "Genetics", "Microbiology", "Evolutionary Biology"], "users"=>["Gilberto E. Flores", "Jessica B. Henley", "Noah Fierer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044563.g002", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Average_phylogenetic_diversity_observed_for_the_communities_of_each_body_habitat_of_each_individual_using_the_direct_PCR_dark_grey_and_standard_extraction_purification_light_grey_protocols_/252528", "title"=>"Average phylogenetic diversity observed for the communities of each body habitat of each individual using the direct PCR (dark grey) and standard extraction/purification (light grey) protocols.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-19 23:55:27"}
  • {"files"=>["https://ndownloader.figshare.com/files/581987"], "description"=>"<p>The diagram illustrates the workflow used for the mouth and skin samples (the fecal samples were processed in a slightly different manner). See the Methods text for more detailed descriptions of these two approaches.</p>", "links"=>[], "tags"=>["diagram", "comparing", "workflows", "dna", "pcr"], "article_id"=>252477, "categories"=>["Biotechnology", "Genetics", "Microbiology", "Evolutionary Biology"], "users"=>["Gilberto E. Flores", "Jessica B. Henley", "Noah Fierer", "Grace lymie James"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044563.g001", "stats"=>{"downloads"=>4, "page_views"=>440, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_diagram_comparing_the_workflows_used_for_the_standard_DNA_extraction_purification_approach_and_the_direct_PCR_approach_/252477", "title"=>"A diagram comparing the workflows used for the standard DNA extraction/purification approach and the direct PCR approach.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-19 23:53:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/582257"], "description"=>"<p>Bars with asterisks denote comparisons that were statistically significant within an individual (t-test, one asterisks p≤0.05, two asterisks p≤0.01). No statistical differences between methods across individuals were observed for any body habitats (paired t-test, p>0.05). Error bars are ± one standard deviation.</p>", "links"=>[], "tags"=>["unweighted", "unifrac", "distances", "replicate", "samples", "pcr"], "article_id"=>252750, "categories"=>["Biotechnology", "Genetics", "Microbiology", "Evolutionary Biology"], "users"=>["Gilberto E. Flores", "Jessica B. Henley", "Noah Fierer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044563.g005", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Variation_of_unweighted_UniFrac_distances_of_replicate_samples_using_the_direct_PCR_dark_grey_and_standard_extraction_purification_light_grey_protocols_/252750", "title"=>"Variation of unweighted UniFrac distances of replicate samples using the direct PCR (dark grey) and standard extraction/purification (light grey) protocols.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-19 23:57:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/582180"], "description"=>"<p>Letters A–D denote individual participants. Results of the ANOSIM testing for statistical differences between methods across individuals are shown for each body habitat. Note that individual C was not included in the underarm analysis as sequences were not obtained for the direct PCR samples.</p>", "links"=>[], "tags"=>["plots", "derived", "unweighted", "unifrac", "distances", "comparing", "communities", "observed", "pcr"], "article_id"=>252668, "categories"=>["Biotechnology", "Genetics", "Microbiology", "Evolutionary Biology"], "users"=>["Gilberto E. Flores", "Jessica B. Henley", "Noah Fierer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044563.g004", "stats"=>{"downloads"=>2, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PCoA_plots_derived_from_unweighted_UniFrac_distances_comparing_the_communities_observed_using_the_direct_PCR_black_circles_and_standard_extraction_purification_grey_circles_protocols_/252668", "title"=>"PCoA plots derived from unweighted UniFrac distances comparing the communities observed using the direct PCR (black circles) and standard extraction/purification (grey circles) protocols.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-19 23:57:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/305943", "https://ndownloader.figshare.com/files/305968", "https://ndownloader.figshare.com/files/305979", "https://ndownloader.figshare.com/files/306014", "https://ndownloader.figshare.com/files/306031"], "description"=>"<div><p>Since the composition of the human microbiome is highly variable both within and between individuals, researchers are increasingly reliant on high-throughput molecular approaches to identify linkages between the composition of these communities and human health. While new sequencing technologies have made it increasingly feasible to analyze large numbers of human-associated samples, the extraction of DNA from samples often remains a bottleneck in the process. Here we tested a direct PCR approach using the Extract-N-Amp Plant PCR Kit to accelerate the 16S rRNA gene-based analyses of human-associated bacterial communities, directly comparing this method to a more commonly-used approach whereby DNA is first extracted and purified from samples using a series of steps prior to PCR amplification. We used both approaches on replicate samples collected from each of five body habitats (tongue surface, feces, forehead skin, underarm skin, and forearm skin) from four individuals. With the exception of the tongue samples, there were few significant differences in the estimates of taxon richness or phylogenetic diversity obtained using the two approaches. Perhaps more importantly, there were no significant differences between the methods in their ability resolve body habitat differences or inter-individual differences in bacterial community composition and the estimates of the relative abundances of individual taxa were nearly identical with the two methods. Overall, the two methods gave very similar results and the direct PCR approach is clearly advantageous for many studies exploring the diversity and composition of human-associated bacterial communities given that large numbers of samples can be processed far more quickly and efficiently.</p> </div>", "links"=>[], "tags"=>["pcr", "analyses", "human-associated", "microbial", "communities"], "article_id"=>120362, "categories"=>["Biotechnology", "Genetics", "Microbiology", "Evolutionary Biology"], "users"=>["Gilberto E. Flores", "Jessica B. Henley", "Noah Fierer"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0044563.s001", "https://dx.doi.org/10.1371/journal.pone.0044563.s002", "https://dx.doi.org/10.1371/journal.pone.0044563.s003", "https://dx.doi.org/10.1371/journal.pone.0044563.s004", "https://dx.doi.org/10.1371/journal.pone.0044563.s005"], "stats"=>{"downloads"=>3, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_Direct_PCR_Approach_to_Accelerate_Analyses_of_Human_Associated_Microbial_Communities/120362", "title"=>"A Direct PCR Approach to Accelerate Analyses of Human-Associated Microbial Communities", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-01-19 19:47:55"}
  • {"files"=>["https://ndownloader.figshare.com/files/582374"], "description"=>"<p>R-values in bold indicate statistically significant differences (p<0.05).</p>", "links"=>[], "tags"=>["anosim", "comparing", "extraction", "bacterial"], "article_id"=>252870, "categories"=>["Biotechnology", "Genetics", "Microbiology", "Evolutionary Biology"], "users"=>["Gilberto E. Flores", "Jessica B. Henley", "Noah Fierer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044563.t003", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Results_of_ANOSIM_tests_comparing_the_affect_of_extraction_method_on_bacterial_community_structure_Bray_Curtis_of_each_body_habitat_from_each_individual_/252870", "title"=>"Results of ANOSIM tests comparing the affect of extraction method on bacterial community structure (Bray-Curtis) of each body habitat from each individual.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-19 23:58:42"}

PMC Usage Stats | Further Information

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  • {"unique-ip"=>"19", "full-text"=>"14", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2014", "month"=>"11"}
  • {"unique-ip"=>"9", "full-text"=>"7", "pdf"=>"5", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"12"}
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Relative Metric

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