Fecal samples were collected and stored at −80°C prior to analysis. The standard PowerSoil kit protocol was used to extract bacterial genomic DNA. Briefly, the frozen samples were thawed on ice and then pulverized with a pestle and mortar in liquid nitrogen, we added MoBio lysis buffer to these fecal samples and then mixed them, and after centrifuging, we placed the obtained supernatant into MoBio garnet-bead tubes containing MoBio buffer. Extracted V3–V5 regions of the 16S rRNA gene from these fecal samples were amplified by polymerase chain reaction with bar-coded universal primers containing linker sequences for pyrosequencing.21 (link) The 454 sequencing system (Hoffman-La Roche, Basel, Switzerland) was used.
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Fecal Microbiome Profiling Protocol
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Other organizations : Chongqing Medical University, Institute of Life Sciences
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Variable analysis
independent variables
- Fecal sample collection and storage at -80°C prior to analysis
- Bacterial genomic DNA extraction
- Amplification of V3-V5 regions of the 16S rRNA gene by PCR
- Pyrosequencing using the 454 sequencing system
- Use of the standard PowerSoil kit protocol for bacterial genomic DNA extraction
- Use of bar-coded universal primers containing linker sequences for PCR amplification of the 16S rRNA gene
- No positive or negative controls were explicitly mentioned in the provided information.
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