Comparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling
The microbiota of the lower female genital tract plays an important role in women's health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal D...
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| creator | Shvartsman, Elinor Richmond, Meika E I Schellenberg, John J Lamont, Alana Perciani, Catia Russell, Justen N H Poliquin, Vanessa Burgener, Adam Jaoko, Walter Sandstrom, Paul MacDonald, Kelly S |
| description | The microbiota of the lower female genital tract plays an important role in women's health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn60 UT.
DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition.
DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples. |
| doi_str_mv | 10.1371/journal.pone.0262355 |
| format | Article |
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DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition.
DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0262355</identifier><identifier>PMID: 35025956</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Bacteria ; Bacteria - genetics ; Biology and Life Sciences ; Blood ; Comparative analysis ; Deoxyribonucleic acid ; DNA ; DNA - isolation & purification ; DNA sequencing ; DNA, Bacterial - genetics ; Ecology and Environmental Sciences ; Ethics ; Female ; Genetic aspects ; Genital tract ; Gram-positive bacteria ; Gynecology ; Health care ; Humans ; Immunology ; Infectious diseases ; Internal medicine ; Lysis ; Lysozyme ; Medical schools ; Medicine ; Medicine and Health Sciences ; Methods ; Microbiomes ; Microbiota ; Microbiota (Symbiotic organisms) ; Microbiota - genetics ; Microorganisms ; Nucleic Acid Amplification Techniques ; Obstetrics ; Polymerase chain reaction ; Polymerase Chain Reaction - methods ; Public health ; Purification ; Representations ; Reproducibility ; Research and analysis methods ; Ribosomal DNA ; RNA, Ribosomal, 16S - genetics ; Sample preparation ; Secretions ; Sequence Analysis, DNA - methods ; Species ; Species diversity ; Specimen Handling - methods ; Vagina ; Vagina - microbiology ; Womens health ; Yield</subject><ispartof>PloS one, 2022-01, Vol.17 (1), p.e0262355-e0262355</ispartof><rights>COPYRIGHT 2022 Public Library of Science</rights><rights>2022 Shvartsman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://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>2022 Shvartsman et al 2022 Shvartsman et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5005-a02d65fc848e85388a4b183e361be65c5c1bd9dec394d022a6e93b7f41cfb633</citedby><cites>FETCH-LOGICAL-c5005-a02d65fc848e85388a4b183e361be65c5c1bd9dec394d022a6e93b7f41cfb633</cites><orcidid>0000-0003-3970-1608 ; 0000-0001-6456-2836 ; 0000-0002-5275-0116 ; 0000-0003-3216-247X ; 0000-0003-0526-3902</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8758110/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8758110/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79569,79570</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/35025956$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Shvartsman, Elinor</creatorcontrib><creatorcontrib>Richmond, Meika E I</creatorcontrib><creatorcontrib>Schellenberg, John J</creatorcontrib><creatorcontrib>Lamont, Alana</creatorcontrib><creatorcontrib>Perciani, Catia</creatorcontrib><creatorcontrib>Russell, Justen N H</creatorcontrib><creatorcontrib>Poliquin, Vanessa</creatorcontrib><creatorcontrib>Burgener, Adam</creatorcontrib><creatorcontrib>Jaoko, Walter</creatorcontrib><creatorcontrib>Sandstrom, Paul</creatorcontrib><creatorcontrib>MacDonald, Kelly S</creatorcontrib><title>Comparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>The microbiota of the lower female genital tract plays an important role in women's health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn60 UT.
DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition.
DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples.</description><subject>Bacteria</subject><subject>Bacteria - genetics</subject><subject>Biology and Life Sciences</subject><subject>Blood</subject><subject>Comparative analysis</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA - isolation & purification</subject><subject>DNA sequencing</subject><subject>DNA, Bacterial - genetics</subject><subject>Ecology and Environmental Sciences</subject><subject>Ethics</subject><subject>Female</subject><subject>Genetic aspects</subject><subject>Genital tract</subject><subject>Gram-positive bacteria</subject><subject>Gynecology</subject><subject>Health care</subject><subject>Humans</subject><subject>Immunology</subject><subject>Infectious diseases</subject><subject>Internal medicine</subject><subject>Lysis</subject><subject>Lysozyme</subject><subject>Medical schools</subject><subject>Medicine</subject><subject>Medicine and Health Sciences</subject><subject>Methods</subject><subject>Microbiomes</subject><subject>Microbiota</subject><subject>Microbiota (Symbiotic organisms)</subject><subject>Microbiota - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Shvartsman, Elinor</au><au>Richmond, Meika E I</au><au>Schellenberg, John J</au><au>Lamont, Alana</au><au>Perciani, Catia</au><au>Russell, Justen N H</au><au>Poliquin, Vanessa</au><au>Burgener, Adam</au><au>Jaoko, Walter</au><au>Sandstrom, Paul</au><au>MacDonald, Kelly S</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Comparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2022-01-01</date><risdate>2022</risdate><volume>17</volume><issue>1</issue><spage>e0262355</spage><epage>e0262355</epage><pages>e0262355-e0262355</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>The microbiota of the lower female genital tract plays an important role in women's health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn60 UT.
DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition.
DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>35025956</pmid><doi>10.1371/journal.pone.0262355</doi><orcidid>https://orcid.org/0000-0003-3970-1608</orcidid><orcidid>https://orcid.org/0000-0001-6456-2836</orcidid><orcidid>https://orcid.org/0000-0002-5275-0116</orcidid><orcidid>https://orcid.org/0000-0003-3216-247X</orcidid><orcidid>https://orcid.org/0000-0003-0526-3902</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2022-01, Vol.17 (1), p.e0262355-e0262355 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_2619535956 |
| source | Public Library of Science (PLoS) Journals Open Access; MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Bacteria Bacteria - genetics Biology and Life Sciences Blood Comparative analysis Deoxyribonucleic acid DNA DNA - isolation & purification DNA sequencing DNA, Bacterial - genetics Ecology and Environmental Sciences Ethics Female Genetic aspects Genital tract Gram-positive bacteria Gynecology Health care Humans Immunology Infectious diseases Internal medicine Lysis Lysozyme Medical schools Medicine Medicine and Health Sciences Methods Microbiomes Microbiota Microbiota (Symbiotic organisms) Microbiota - genetics Microorganisms Nucleic Acid Amplification Techniques Obstetrics Polymerase chain reaction Polymerase Chain Reaction - methods Public health Purification Representations Reproducibility Research and analysis methods Ribosomal DNA RNA, Ribosomal, 16S - genetics Sample preparation Secretions Sequence Analysis, DNA - methods Species Species diversity Specimen Handling - methods Vagina Vagina - microbiology Womens health Yield |
| title | Comparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling |
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