Bacterial DNA sequencing was performed as described previously (20 (link)). Briefly, DNA from stool was extracted using the DNeasy power soil DNA isolation kit (Qiagen). The quality and quantity of the DNA were confirmed using a Nanodrop 1000 (Thermo Fisher Scientific). The 16S rRNA gene V4 variable region was amplified using primer pair F515/R806. Sequencing was performed at MR DNA (www.mrdnalab.com ) on a MiSeq (Illumina) following the manufacturer's guidelines. Sequence data were processed using a proprietary analysis pipeline (MR DNA). Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97% similarity). Final OTUs were taxonomically classified using BLASTn against a curated GreenGenes database (21 (link)). Within-community diversity (α-diversity) was calculated using the QIIME software package (22 (link)). Analysis of α-diversity (Shannon index) was performed by a 1-factor ANOVA. β-diversity was measured by calculating the weighted UniFrac distances (23 (link)) using QIIME default scripts, and weighted UniFrac PCoA biplot was visualized using EMPeror (24 (link)).
Protocol detail
Bacterial 16S rRNA Sequencing Protocol
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Corresponding Organization : University of California, Los Angeles
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Variable analysis
independent variables
- Bacterial DNA sequencing was performed
- Quality and quantity of the DNA
- Operational taxonomic units (OTUs)
- Within-community diversity (α-diversity) measured by Shannon index
- β-diversity measured by weighted UniFrac distances
- DNA extraction using the DNeasy power soil DNA isolation kit (Qiagen)
- Amplification of the 16S rRNA gene V4 variable region using primer pair F515/R806
- Sequencing performed at MR DNA on a MiSeq (Illumina) following the manufacturer's guidelines
- Sequence data processing using a proprietary analysis pipeline (MR DNA)
- Taxonomic classification of OTUs using BLASTn against a curated GreenGenes database
- Analysis of α-diversity (Shannon index) performed by a 1-factor ANOVA
- Calculation of β-diversity using weighted UniFrac distances and QIIME default scripts
- Visualization of weighted UniFrac PCoA biplot using EMPeror
- Positive control: Not specified
- Negative control: Not specified
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