Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis
Abstract Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fec...
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| Veröffentlicht in: | FEMS microbiology letters 2012-04, Vol.329 (2), p.193-197 |
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| creator | Bahl, Martin Iain Bergström, Anders Licht, Tine Rask |
| description | Abstract
Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology. |
| doi_str_mv | 10.1111/j.1574-6968.2012.02523.x |
| format | Article |
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Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology.</description><identifier>ISSN: 0378-1097</identifier><identifier>EISSN: 1574-6968</identifier><identifier>DOI: 10.1111/j.1574-6968.2012.02523.x</identifier><identifier>PMID: 22325006</identifier><identifier>CODEN: FMLED7</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Publishing Ltd</publisher><subject>Bacteria - chemistry ; Bacteria - genetics ; Bacteria - isolation & purification ; Bacteroidetes ; Bacteroidetes - chemistry ; Bacteroidetes - genetics ; Bacteroidetes - isolation & purification ; Biological and medical sciences ; Biomarkers ; Communities ; Community composition ; Composition ; Cryopreservation - methods ; Deoxyribonucleic acid ; DNA ; DNA, Bacterial - analysis ; DNA, Bacterial - isolation & purification ; Feces ; Feces - microbiology ; Firmicutes ; Freezing ; Fundamental and applied biological sciences. Psychology ; Genetic testing ; gut microbiology ; Intestinal microflora ; Microbiology ; Microbiota ; Polymerase chain reaction ; qPCR ; Real-Time Polymerase Chain Reaction - methods ; Real-Time Polymerase Chain Reaction - standards ; Reproducibility of Results ; RNA, Ribosomal, 16S - genetics ; rRNA 16S ; Specimen Handling - methods ; Storage conditions</subject><ispartof>FEMS microbiology letters, 2012-04, Vol.329 (2), p.193-197</ispartof><rights>2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved 2012</rights><rights>2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved</rights><rights>2015 INIST-CNRS</rights><rights>2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c6193-5edd7f3e26e6ad967d369f7df5dbc7280a5ea188e7ddbf5a9fb5364921ee09c53</citedby><cites>FETCH-LOGICAL-c6193-5edd7f3e26e6ad967d369f7df5dbc7280a5ea188e7ddbf5a9fb5364921ee09c53</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2Fj.1574-6968.2012.02523.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2Fj.1574-6968.2012.02523.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,27903,27904,45553,45554</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=25660725$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22325006$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Bahl, Martin Iain</creatorcontrib><creatorcontrib>Bergström, Anders</creatorcontrib><creatorcontrib>Licht, Tine Rask</creatorcontrib><title>Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis</title><title>FEMS microbiology letters</title><addtitle>FEMS Microbiol Lett</addtitle><description>Abstract
Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology.</description><subject>Bacteria - chemistry</subject><subject>Bacteria - genetics</subject><subject>Bacteria - isolation & purification</subject><subject>Bacteroidetes</subject><subject>Bacteroidetes - chemistry</subject><subject>Bacteroidetes - genetics</subject><subject>Bacteroidetes - isolation & purification</subject><subject>Biological and medical sciences</subject><subject>Biomarkers</subject><subject>Communities</subject><subject>Community composition</subject><subject>Composition</subject><subject>Cryopreservation - methods</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA, Bacterial - analysis</subject><subject>DNA, Bacterial - isolation & purification</subject><subject>Feces</subject><subject>Feces - microbiology</subject><subject>Firmicutes</subject><subject>Freezing</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genetic testing</subject><subject>gut microbiology</subject><subject>Intestinal microflora</subject><subject>Microbiology</subject><subject>Microbiota</subject><subject>Polymerase chain reaction</subject><subject>qPCR</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>Real-Time Polymerase Chain Reaction - standards</subject><subject>Reproducibility of Results</subject><subject>RNA, Ribosomal, 16S - genetics</subject><subject>rRNA 16S</subject><subject>Specimen Handling - methods</subject><subject>Storage conditions</subject><issn>0378-1097</issn><issn>1574-6968</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNqNkV2PEyEUhonRuN3Vv2BIjNGbGRkoMFx4sVu3alI_YvSa0OGgNPPRBcZt_RP-ZRlb10SjkRsIPO_hwIMQrkhZ5fF0U1ZczguhRF1SUtGSUE5ZubuFZjcHt9GMMFkXFVHyBJ3GuCGEzCkRd9EJpYxyQsQMfVsGgK--_4QdNKbF0XTbFiLeBj8EnAb8_M05hl0Kpkl-6LFxmUsRp8-Alz50vhlTxjN4kQkIg7cwbQSTcTytM9ODxes9tsN1H1MA0-Gr0fTJpwx9Afxu8R6b3rT76OM9dMeZNsL943yGPi4vPyxeFqu3L14tzldFIyrFCg7WSseAChDGKiEtE8pJ67hdN5LWxHAwVV2DtHbtuFFuzZmYK1oBENVwdoYeH-puw3A1Qky687GBtjU9DGPUiipOaKVkJp_8k6wIJTUTlE9FH_6GboYx5JdFTRnhslaSzjNVH6gmDDEGcDp_dmfCPpfSk1690ZNFPVnUk179Q6_e5eiD4wXjugN7E_zpMwOPjoCJWacLpm98_MVxIYikU6fPDty1b2H_3w3o5evVtMp5dsgP4_Yv6eLP9r8DsGvRkw</recordid><startdate>201204</startdate><enddate>201204</enddate><creator>Bahl, Martin Iain</creator><creator>Bergström, Anders</creator><creator>Licht, Tine Rask</creator><general>Blackwell Publishing Ltd</general><general>Wiley-Blackwell</general><general>Oxford University Press</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>201204</creationdate><title>Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis</title><author>Bahl, Martin Iain ; Bergström, Anders ; Licht, Tine Rask</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c6193-5edd7f3e26e6ad967d369f7df5dbc7280a5ea188e7ddbf5a9fb5364921ee09c53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Bacteria - chemistry</topic><topic>Bacteria - genetics</topic><topic>Bacteria - isolation & purification</topic><topic>Bacteroidetes</topic><topic>Bacteroidetes - chemistry</topic><topic>Bacteroidetes - genetics</topic><topic>Bacteroidetes - isolation & purification</topic><topic>Biological and medical sciences</topic><topic>Biomarkers</topic><topic>Communities</topic><topic>Community composition</topic><topic>Composition</topic><topic>Cryopreservation - methods</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA, Bacterial - analysis</topic><topic>DNA, Bacterial - isolation & purification</topic><topic>Feces</topic><topic>Feces - microbiology</topic><topic>Firmicutes</topic><topic>Freezing</topic><topic>Fundamental and applied biological sciences. 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Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology.</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>22325006</pmid><doi>10.1111/j.1574-6968.2012.02523.x</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Bacteria - chemistry Bacteria - genetics Bacteria - isolation & purification Bacteroidetes Bacteroidetes - chemistry Bacteroidetes - genetics Bacteroidetes - isolation & purification Biological and medical sciences Biomarkers Communities Community composition Composition Cryopreservation - methods Deoxyribonucleic acid DNA DNA, Bacterial - analysis DNA, Bacterial - isolation & purification Feces Feces - microbiology Firmicutes Freezing Fundamental and applied biological sciences. Psychology Genetic testing gut microbiology Intestinal microflora Microbiology Microbiota Polymerase chain reaction qPCR Real-Time Polymerase Chain Reaction - methods Real-Time Polymerase Chain Reaction - standards Reproducibility of Results RNA, Ribosomal, 16S - genetics rRNA 16S Specimen Handling - methods Storage conditions |
| title | Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis |
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