The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations.
High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first Mi...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4352061?pdf=render |
| _version_ | 1818925414038372352 |
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| author | Fiona Fouhy Jennifer Deane Mary C Rea Órla O'Sullivan R Paul Ross Grace O'Callaghan Barry J Plant Catherine Stanton |
| author_facet | Fiona Fouhy Jennifer Deane Mary C Rea Órla O'Sullivan R Paul Ross Grace O'Callaghan Barry J Plant Catherine Stanton |
| author_sort | Fiona Fouhy |
| collection | DOAJ |
| description | High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed.Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed.No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples.Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota. |
| first_indexed | 2024-12-20T02:40:50Z |
| format | Article |
| id | doaj.art-ceacc059a97242debc1d4a834a9a4638 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-20T02:40:50Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-ceacc059a97242debc1d4a834a9a46382022-12-21T19:56:19ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01103e011935510.1371/journal.pone.0119355The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations.Fiona FouhyJennifer DeaneMary C ReaÓrla O'SullivanR Paul RossGrace O'CallaghanBarry J PlantCatherine StantonHigh-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed.Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed.No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples.Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.http://europepmc.org/articles/PMC4352061?pdf=render |
| spellingShingle | Fiona Fouhy Jennifer Deane Mary C Rea Órla O'Sullivan R Paul Ross Grace O'Callaghan Barry J Plant Catherine Stanton The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations. PLoS ONE |
| title | The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations. |
| title_full | The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations. |
| title_fullStr | The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations. |
| title_full_unstemmed | The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations. |
| title_short | The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations. |
| title_sort | effects of freezing on faecal microbiota as determined using miseq sequencing and culture based investigations |
| url | http://europepmc.org/articles/PMC4352061?pdf=render |
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