Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome
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{"title"=>"Evaluation of methods for the extraction and purification of DNA from the human microbiome", "type"=>"journal", "authors"=>[{"first_name"=>"Sanqing", "last_name"=>"Yuan", "scopus_author_id"=>"57199904994"}, {"first_name"=>"Dora B.", "last_name"=>"Cohen", "scopus_author_id"=>"55129570300"}, {"first_name"=>"Jacques", "last_name"=>"Ravel", "scopus_author_id"=>"7006695370"}, {"first_name"=>"Zaid", "last_name"=>"Abdo", "scopus_author_id"=>"8610706400"}, {"first_name"=>"Larry J.", "last_name"=>"Forney", "scopus_author_id"=>"34568978300"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pui"=>"364494680", "pmid"=>"22457796", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "doi"=>"10.1371/journal.pone.0033865", "sgr"=>"84858763397", "scopus"=>"2-s2.0-84858763397"}, "id"=>"b16825a6-96ff-3472-a788-3cca3e6500c6", "abstract"=>"BACKGROUND DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled. METHODOLOGY/PRINCIPAL FINDINGS In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used. CONCLUSIONS/SIGNIFICANCE Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.", "link"=>"http://www.mendeley.com/research/evaluation-methods-extraction-purification-dna-human-microbiome-9", "reader_count"=>477, "reader_count_by_academic_status"=>{"Unspecified"=>7, "Professor > Associate Professor"=>16, "Researcher"=>106, "Student > Doctoral Student"=>24, "Student > Ph. D. Student"=>116, "Student > Postgraduate"=>24, "Student > Master"=>77, "Other"=>14, "Student > Bachelor"=>74, "Lecturer"=>7, "Lecturer > Senior Lecturer"=>2, "Professor"=>10}, "reader_count_by_user_role"=>{"Unspecified"=>7, "Professor > Associate Professor"=>16, "Researcher"=>106, "Student > Doctoral Student"=>24, "Student > Ph. D. 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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/663177"], "description"=>"<p>Euclidean distances between observed and expected proportions were calculated for each of eight replicates of each method.</p>", "links"=>[], "tags"=>["euclidean", "distances", "observed"], "article_id"=>333659, "categories"=>["Molecular Biology", "Physics", "Biochemistry", "Infectious Diseases", "Biophysics", "Genetics", "Biotechnology", "Ecology"], "users"=>["Sanqing Yuan", "Dora B. Cohen", "Jacques Ravel", "Zaid Abdo", "Larry J. Forney"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0033865.g001", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Boxplot_of_Euclidean_distances_between_observed_and_expected_species_proportions_/333659", "title"=>"Boxplot of Euclidean distances between observed and expected species proportions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-03-23 01:00:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/339963", "https://ndownloader.figshare.com/files/340014", "https://ndownloader.figshare.com/files/340064"], "description"=>"<div><h3>Background</h3><p>DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled.</p> <h3>Methodology/Principal Findings</h3><p>In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used.</p> <h3>Conclusions/Significance</h3><p>Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.</p> </div>", "links"=>[], "tags"=>["methods", "extraction", "purification", "dna", "microbiome"], "article_id"=>127203, "categories"=>["Molecular Biology", "Cancer", "Physics", "Biochemistry", "Biophysics", "Genetics", "Biotechnology", "Ecology"], "users"=>["Sanqing Yuan", "Dora B. Cohen", "Jacques Ravel", "Zaid Abdo", "Larry J. Forney"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0033865.s001", "https://dx.doi.org/10.1371/journal.pone.0033865.s002", "https://dx.doi.org/10.1371/journal.pone.0033865.s003"], "stats"=>{"downloads"=>13, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Evaluation_of_Methods_for_the_Extraction_and_Purification_of_DNA_from_the_Human_Microbiome/127203", "title"=>"Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-03-23 02:00:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/663456"], "description"=>"a<p>Anaerobic strains were cultivated in GasPak anaerobic chamber (Becton Dickinson, Franklin Lakes, NJ) with Pack-Anaero sachet (MGC Inc., New York, NY).</p>b<p>Medium: LB, Luria-Bertani; TSB, Trypticase soy base; BHI, Brain heart infusion; MRS, De Man, Rogosa and Sharpe.</p>", "links"=>[], "tags"=>["strains", "cultivation"], "article_id"=>333940, "categories"=>["Molecular Biology", "Physics", "Biochemistry", "Infectious Diseases", "Biophysics", "Genetics", "Biotechnology", "Ecology"], "users"=>["Sanqing Yuan", "Dora B. Cohen", "Jacques Ravel", "Zaid Abdo", "Larry J. Forney"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0033865.t002", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Bacterial_strains_and_cultivation_condition_used_/333940", "title"=>"Bacterial strains and cultivation condition used.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-03-23 01:05:40"}
  • {"files"=>["https://ndownloader.figshare.com/files/663236"], "description"=>"<p>To calculate grand proportions, the total counts of 16S rRNA gene reads of each species were calculated for eight replicates of each method. Then grand proportions were calculated based on total counts of 16S rRNA gene reads of each species per method. Grand proportions were used to calculate Euclidean distances between observed and grand proportions.</p>", "links"=>[], "tags"=>["euclidean", "distances", "observed"], "article_id"=>333723, "categories"=>["Molecular Biology", "Physics", "Biochemistry", "Infectious Diseases", "Biophysics", "Genetics", "Biotechnology", "Ecology"], "users"=>["Sanqing Yuan", "Dora B. Cohen", "Jacques Ravel", "Zaid Abdo", "Larry J. Forney"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0033865.g002", "stats"=>{"downloads"=>2, "page_views"=>71, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Boxplot_of_Euclidean_distances_between_observed_and_grand_proportions_/333723", "title"=>"Boxplot of Euclidean distances between observed and grand proportions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-03-23 01:02:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/663413"], "description"=>"a<p>DNA concentrations are means calculated using data from eight replicates.</p>b<p>Means with the same letter are not significantly different.</p>", "links"=>[], "tags"=>["dna", "yields", "strains", "extraction"], "article_id"=>333893, "categories"=>["Molecular Biology", "Physics", "Biochemistry", "Infectious Diseases", "Biophysics", "Genetics", "Biotechnology", "Ecology"], "users"=>["Sanqing Yuan", "Dora B. Cohen", "Jacques Ravel", "Zaid Abdo", "Larry J. Forney"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0033865.t003", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparison_of_DNA_yields_of_type_strains_obtained_using_six_DNA_extraction_methods_/333893", "title"=>"Comparison of DNA yields of type strains obtained using six DNA extraction methods.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-03-23 01:04:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/663300"], "description"=>"<p>The mean concentrations (columns) were calculated based on nine replicated extractions per sample per mode. Pair-wise comparisons of DNA concentrations between modes per sample were performed by using Wilcoxon rank sum test. Bonferroni correction was used for multiple testing. Letters at the top of columns indicate whether there is significantly difference between columns per sample. Means with the same letter are not significantly different.</p>", "links"=>[], "tags"=>["extractions", "enzymatic", "lysis"], "article_id"=>333782, "categories"=>["Molecular Biology", "Physics", "Biochemistry", "Infectious Diseases", "Biophysics", "Genetics", "Biotechnology", "Ecology"], "users"=>["Sanqing Yuan", "Dora B. Cohen", "Jacques Ravel", "Zaid Abdo", "Larry J. Forney"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0033865.g003", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_DNA_extractions_using_different_enzymatic_lysis_modes_/333782", "title"=>"DNA extractions using different enzymatic lysis modes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-03-23 01:03:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/663376"], "description"=>"a<p>If more than one possible copy numbers are available for one species in <i>rrn</i> database (<a href=\"http://ribosome.mmg.msu.edu/rrndb/index.php\" target=\"_blank\">http://ribosome.mmg.msu.edu/rrndb/index.php</a>), the larger one was chosen.</p>b<p>Average proportions and standard deviations are calculated based on eight replicates.</p>", "links"=>[], "tags"=>["rrna", "observed", "proportions", "16s", "reads"], "article_id"=>333857, "categories"=>["Molecular Biology", "Physics", "Biochemistry", "Infectious Diseases", "Biophysics", "Genetics", "Biotechnology", "Ecology"], "users"=>["Sanqing Yuan", "Dora B. Cohen", "Jacques Ravel", "Zaid Abdo", "Larry J. Forney"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0033865.t004", "stats"=>{"downloads"=>1, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_16S_rRNA_gene_copy_numbers_expected_and_observed_proportions_of_16S_rRNA_gene_sequence_reads_for_each_type_strain_/333857", "title"=>"16S rRNA gene copy numbers, expected and observed proportions of 16S rRNA gene sequence reads for each type strain.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-03-23 01:04:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/663501"], "description"=>"a<p>Cell lysis method: B, bead beating; E1, lysozyme; E2, mutanolysin; E3, lysostaphin; C, chemical.</p>b<p>Phenol-chloroform purification and isopropanol precipitation.</p>", "links"=>[], "tags"=>["dna", "extraction", "methods"], "article_id"=>333983, "categories"=>["Molecular Biology", "Physics", "Biochemistry", "Infectious Diseases", "Biophysics", "Genetics", "Biotechnology", "Ecology"], "users"=>["Sanqing Yuan", "Dora B. Cohen", "Jacques Ravel", "Zaid Abdo", "Larry J. Forney"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0033865.t001", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Features_of_the_six_DNA_extraction_methods_used_/333983", "title"=>"Features of the six DNA extraction methods used.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-03-23 01:06:23"}

PMC Usage Stats | Further Information

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Relative Metric

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