Storage and handling of human faecal samples affect the gut microbiome composition: A feasibility study
Human gut microbiome analysis through faecal sampling typically involves five stages: sample collection, storage, DNA extraction, next generation sequencing and bioinformatics analysis. Of these, the first three are considered irreversible. This feasibility study describes an assessment of methodolo...
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| Veröffentlicht in: | Journal of microbiological methods 2019-09, Vol.164, p.105668-105668, Article 105668 |
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| creator | Ezzy, Alan C. Hagstrom, Amanda D. George, Chris Hamlin, Adam S. Pereg, Lily Murphy, Aron J. Winter, Gal |
| description | Human gut microbiome analysis through faecal sampling typically involves five stages: sample collection, storage, DNA extraction, next generation sequencing and bioinformatics analysis. Of these, the first three are considered irreversible. This feasibility study describes an assessment of methodologies used for faecal DNA extraction and sample handling, using the parameters DNA yield, purity and resultant microbial profile. Six DNA extraction techniques, including commercially available kits and manual protocols were compared on human faecal samples (n = 3). Different extraction techniques produced significant variance in DNA yield (range 2.7–164 ng/mg faeces) and microbial diversity profiles, with considerable variation in phyla dominance (Firmicutes (P |
| doi_str_mv | 10.1016/j.mimet.2019.105668 |
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[Display omitted]
•DNA extraction method of human faecal samples greatly influence DNA yield and the resultant microbial diversity profile.•A lab prepared extraction for faecal DNA extraction yields comparable results to commercially available designated kits.•Faecal samples can be stored under 4º or in stabilizing buffers for up to five days with no significant variance in microbial diversity profile.</description><identifier>ISSN: 0167-7012</identifier><identifier>EISSN: 1872-8359</identifier><identifier>DOI: 10.1016/j.mimet.2019.105668</identifier><identifier>PMID: 31302202</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>16S rDNA ; DNA extraction ; Faecal sample storage ; Gut microbiome ; Gut microbiota ; Intestinal microbiota</subject><ispartof>Journal of microbiological methods, 2019-09, Vol.164, p.105668-105668, Article 105668</ispartof><rights>2019 Elsevier B.V.</rights><rights>Copyright © 2019 Elsevier B.V. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c359t-ecef2500cef0b625bd2712f7735f1d38208448c389bdd4b16de11bd4e4c5d1c83</citedby><cites>FETCH-LOGICAL-c359t-ecef2500cef0b625bd2712f7735f1d38208448c389bdd4b16de11bd4e4c5d1c83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.mimet.2019.105668$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,777,781,3537,27905,27906,45976</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31302202$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ezzy, Alan C.</creatorcontrib><creatorcontrib>Hagstrom, Amanda D.</creatorcontrib><creatorcontrib>George, Chris</creatorcontrib><creatorcontrib>Hamlin, Adam S.</creatorcontrib><creatorcontrib>Pereg, Lily</creatorcontrib><creatorcontrib>Murphy, Aron J.</creatorcontrib><creatorcontrib>Winter, Gal</creatorcontrib><title>Storage and handling of human faecal samples affect the gut microbiome composition: A feasibility study</title><title>Journal of microbiological methods</title><addtitle>J Microbiol Methods</addtitle><description>Human gut microbiome analysis through faecal sampling typically involves five stages: sample collection, storage, DNA extraction, next generation sequencing and bioinformatics analysis. Of these, the first three are considered irreversible. This feasibility study describes an assessment of methodologies used for faecal DNA extraction and sample handling, using the parameters DNA yield, purity and resultant microbial profile. Six DNA extraction techniques, including commercially available kits and manual protocols were compared on human faecal samples (n = 3). Different extraction techniques produced significant variance in DNA yield (range 2.7–164 ng/mg faeces) and microbial diversity profiles, with considerable variation in phyla dominance (Firmicutes (P < 0.001), Bacteroidetes (P = 0.003), Actinobacteria (P = 0.003), One-way ANOVA). The most effective method, with the highest DNA yield, was a simple and inexpensive extraction technique named MetaHIT. Using this method, DNA was extracted from separate faecal samples (n = 3) and had been aliquoted to seven storage conditions including three stabilizing buffers and three temperature conditions, for a period of 120-h, with storage at −80 °C as a control treatment. DNA yield and purity was not statistically different between the control and remaining treatments. 16S rDNA-based diversity profile was largely comparable across the treatments with only minor differences in genera between samples stored at room temperature in air and − 80 °C control. Overall these results suggest that the choice of DNA extraction method has a greater influence on the resultant microbial diversity profile than the short-term storage method.
[Display omitted]
•DNA extraction method of human faecal samples greatly influence DNA yield and the resultant microbial diversity profile.•A lab prepared extraction for faecal DNA extraction yields comparable results to commercially available designated kits.•Faecal samples can be stored under 4º or in stabilizing buffers for up to five days with no significant variance in microbial diversity profile.</description><subject>16S rDNA</subject><subject>DNA extraction</subject><subject>Faecal sample storage</subject><subject>Gut microbiome</subject><subject>Gut microbiota</subject><subject>Intestinal microbiota</subject><issn>0167-7012</issn><issn>1872-8359</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><recordid>eNp9kMtKLDEQQIMoOlf9AkGydNNjHp3ujOBCxHuvILhQ1yGdVGYydDpjJy3M3xsddemmCopTr4PQGSVzSmhzuZ4HHyDPGaGLUhFNI_fQjMqWVZKLxT6aFaqtWkLZEfqT0poQKngtD9ERp5wwRtgMLZ9yHPUSsB4sXpXQ-2GJo8OrKegBOw1G9zjpsOkhYe0cmIzzCvByyjh4M8bOxwDYxLCJyWcfhyt8gx3o5Dvf-7zFKU92e4IOnO4TnH7lY_Ty9-759n_18Pjv_vbmoTLl5FyBAccEISWRrmGis6ylzLUtF45aLhmRdS0Nl4vO2rqjjQVKO1tDbYSlRvJjdLGbuxnj6wQpq-CTgb7XA8QpKcaEpKJpBSko36HliZRGcGoz-qDHraJEfRhWa_VpWH0YVjvDpev8a8HUBbA_Pd9KC3C9A6C8-eZhVMl4GAxYPxZ5ykb_64J3Bw2OnA</recordid><startdate>201909</startdate><enddate>201909</enddate><creator>Ezzy, Alan C.</creator><creator>Hagstrom, Amanda D.</creator><creator>George, Chris</creator><creator>Hamlin, Adam S.</creator><creator>Pereg, Lily</creator><creator>Murphy, Aron J.</creator><creator>Winter, Gal</creator><general>Elsevier B.V</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>201909</creationdate><title>Storage and handling of human faecal samples affect the gut microbiome composition: A feasibility study</title><author>Ezzy, Alan C. ; Hagstrom, Amanda D. ; George, Chris ; Hamlin, Adam S. ; Pereg, Lily ; Murphy, Aron J. ; Winter, Gal</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c359t-ecef2500cef0b625bd2712f7735f1d38208448c389bdd4b16de11bd4e4c5d1c83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>16S rDNA</topic><topic>DNA extraction</topic><topic>Faecal sample storage</topic><topic>Gut microbiome</topic><topic>Gut microbiota</topic><topic>Intestinal microbiota</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ezzy, Alan C.</creatorcontrib><creatorcontrib>Hagstrom, Amanda D.</creatorcontrib><creatorcontrib>George, Chris</creatorcontrib><creatorcontrib>Hamlin, Adam S.</creatorcontrib><creatorcontrib>Pereg, Lily</creatorcontrib><creatorcontrib>Murphy, Aron J.</creatorcontrib><creatorcontrib>Winter, Gal</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of microbiological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ezzy, Alan C.</au><au>Hagstrom, Amanda D.</au><au>George, Chris</au><au>Hamlin, Adam S.</au><au>Pereg, Lily</au><au>Murphy, Aron J.</au><au>Winter, Gal</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Storage and handling of human faecal samples affect the gut microbiome composition: A feasibility study</atitle><jtitle>Journal of microbiological methods</jtitle><addtitle>J Microbiol Methods</addtitle><date>2019-09</date><risdate>2019</risdate><volume>164</volume><spage>105668</spage><epage>105668</epage><pages>105668-105668</pages><artnum>105668</artnum><issn>0167-7012</issn><eissn>1872-8359</eissn><abstract>Human gut microbiome analysis through faecal sampling typically involves five stages: sample collection, storage, DNA extraction, next generation sequencing and bioinformatics analysis. Of these, the first three are considered irreversible. This feasibility study describes an assessment of methodologies used for faecal DNA extraction and sample handling, using the parameters DNA yield, purity and resultant microbial profile. Six DNA extraction techniques, including commercially available kits and manual protocols were compared on human faecal samples (n = 3). Different extraction techniques produced significant variance in DNA yield (range 2.7–164 ng/mg faeces) and microbial diversity profiles, with considerable variation in phyla dominance (Firmicutes (P < 0.001), Bacteroidetes (P = 0.003), Actinobacteria (P = 0.003), One-way ANOVA). The most effective method, with the highest DNA yield, was a simple and inexpensive extraction technique named MetaHIT. Using this method, DNA was extracted from separate faecal samples (n = 3) and had been aliquoted to seven storage conditions including three stabilizing buffers and three temperature conditions, for a period of 120-h, with storage at −80 °C as a control treatment. DNA yield and purity was not statistically different between the control and remaining treatments. 16S rDNA-based diversity profile was largely comparable across the treatments with only minor differences in genera between samples stored at room temperature in air and − 80 °C control. Overall these results suggest that the choice of DNA extraction method has a greater influence on the resultant microbial diversity profile than the short-term storage method.
[Display omitted]
•DNA extraction method of human faecal samples greatly influence DNA yield and the resultant microbial diversity profile.•A lab prepared extraction for faecal DNA extraction yields comparable results to commercially available designated kits.•Faecal samples can be stored under 4º or in stabilizing buffers for up to five days with no significant variance in microbial diversity profile.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>31302202</pmid><doi>10.1016/j.mimet.2019.105668</doi><tpages>1</tpages></addata></record> |
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| subjects | 16S rDNA DNA extraction Faecal sample storage Gut microbiome Gut microbiota Intestinal microbiota |
| title | Storage and handling of human faecal samples affect the gut microbiome composition: A feasibility study |
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