Evaluation of methods for the extraction and purification of DNA from the human microbiome
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. In th...
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| Veröffentlicht in: | PloS one 2012-03, Vol.7 (3), p.e33865-e33865 |
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| creator | Yuan, Sanqing Cohen, Dora B Ravel, Jacques Abdo, Zaid Forney, Larry J |
| description | 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.
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.
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. |
| doi_str_mv | 10.1371/journal.pone.0033865 |
| format | Article |
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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.
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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0033865</identifier><identifier>PMID: 22457796</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Alcohol ; Bacteria ; Bacteria - classification ; Bacteria - genetics ; Biodiversity ; Bioinformatics ; Biology ; Catfish ; Communities ; Community structure ; Comparative analysis ; Cultivation ; Deoxyribonucleic acid ; DNA ; DNA, Bacterial - isolation & purification ; Edwardsiella ; Edwardsiella ictaluri ; Gardnerella vaginalis ; Gene sequencing ; Genes ; Genetic research ; Genetic testing ; Human body ; Humans ; Ictalurus punctatus ; Laboratories ; Medicine ; Methods ; Microorganisms ; Phenols ; Physics ; Purification ; Representations ; Reproducibility ; RNA ; rRNA 16S ; Shearing ; Species ; Statistical analysis ; Statistical methods ; Studies ; Taxonomy ; Variance analysis</subject><ispartof>PloS one, 2012-03, Vol.7 (3), p.e33865-e33865</ispartof><rights>COPYRIGHT 2012 Public Library of Science</rights><rights>2012 Yuan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Yuan et al. 2012</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c757t-80672d3e024db2283780f6c3b78b7dcd7c55199533397ca056feaf4a43d78b0d3</citedby><cites>FETCH-LOGICAL-c757t-80672d3e024db2283780f6c3b78b7dcd7c55199533397ca056feaf4a43d78b0d3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3311548/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3311548/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2095,2914,23846,27903,27904,53770,53772,79347,79348</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22457796$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Gilbert, Jack Anthony</contributor><creatorcontrib>Yuan, Sanqing</creatorcontrib><creatorcontrib>Cohen, Dora B</creatorcontrib><creatorcontrib>Ravel, Jacques</creatorcontrib><creatorcontrib>Abdo, Zaid</creatorcontrib><creatorcontrib>Forney, Larry J</creatorcontrib><title>Evaluation of methods for the extraction and purification of DNA from the human microbiome</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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.
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.
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.</description><subject>Alcohol</subject><subject>Bacteria</subject><subject>Bacteria - classification</subject><subject>Bacteria - genetics</subject><subject>Biodiversity</subject><subject>Bioinformatics</subject><subject>Biology</subject><subject>Catfish</subject><subject>Communities</subject><subject>Community structure</subject><subject>Comparative analysis</subject><subject>Cultivation</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA, Bacterial - isolation & purification</subject><subject>Edwardsiella</subject><subject>Edwardsiella ictaluri</subject><subject>Gardnerella vaginalis</subject><subject>Gene sequencing</subject><subject>Genes</subject><subject>Genetic research</subject><subject>Genetic testing</subject><subject>Human 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Anthony</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Evaluation of methods for the extraction and purification of DNA from the human microbiome</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2012-03-23</date><risdate>2012</risdate><volume>7</volume><issue>3</issue><spage>e33865</spage><epage>e33865</epage><pages>e33865-e33865</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>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.
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.
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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>22457796</pmid><doi>10.1371/journal.pone.0033865</doi><tpages>e33865</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Alcohol Bacteria Bacteria - classification Bacteria - genetics Biodiversity Bioinformatics Biology Catfish Communities Community structure Comparative analysis Cultivation Deoxyribonucleic acid DNA DNA, Bacterial - isolation & purification Edwardsiella Edwardsiella ictaluri Gardnerella vaginalis Gene sequencing Genes Genetic research Genetic testing Human body Humans Ictalurus punctatus Laboratories Medicine Methods Microorganisms Phenols Physics Purification Representations Reproducibility RNA rRNA 16S Shearing Species Statistical analysis Statistical methods Studies Taxonomy Variance analysis |
| title | Evaluation of methods for the extraction and purification of DNA from the human microbiome |
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