454 titanium platform

Stool collection and microbiome profiling methods have been described in detail previously.^{E2 (link)} DNA extraction was performed on the stool samples and sequencing of the bacterial 16S V3 to V5 hypervariable regions was performed by pyrosequencing (Roche 454 Titanium platform) at the Genome Center (TGI) at Washington University in St Louis, Mo. Filtering, trimming, and chimera checking were performed as previously described.^{E3 (link),E4 (link)} Closed reference OTU classification was performed using QIIME.^{E5 (link)} Additional data processing was performed using Phyloseq (version 1.20.0).^E6 All samples had total read counts of at least 1000. Of 1107 OTUs detected, those absent 5% of samples or more were excluded, leaving 420 OTUs for analysis.
Fecal metabolomic profiling was performed at Metabolon, Inc (Research Triangle Park, NC) using ultraperformance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS), as described earlier.^E7 Of 887 identified metabolites, 148 xenobiotic metabolites and 38 metabolites with an interquartile range of 0 were excluded from analysis, leaving 701 metabolites. For each metabolite, missing values were replaced with half of the minimum value of that metabolite. Most metabolites were not normally distributed and all metabolite relative abundances were log₁₀-normalized and pareto-scaled.

The V3–V5 region of the 16S rRNA gene was amplified using primers 357F (5′-CCTACGGGAGGCAGCAG -3′) and 926R (5′-CCGTCAATTCMTTTRAGT -3′). Primers also contained an adaptor sequence and one of 96 tags unique to each sample. PCR was performed with the following conditions: 30 cycles of 95 °C at 2 min, 50 °C at 0.5 min, and 72 °C at 5 min. Amplicons were purified, pooled at equimolar concentrations, and pyrosequenced on the Roche 454 Titanium platform using a protocol developed by the Human Microbiome Project.^{13 (link)} The 16s rRNA gene data was submitted to the Sequence Read Archives (SRA) database.
Data processing and quality control (QC) were performed according to standardized protocols developed by the Human Microbiome Project.^{13 (link)} In brief, samples were demultiplexed by sample barcode, allowing one mismatch per barcode. Reads were filtered to remove samples with average quality score <35 and/or read length less <200 nt. Chimeric sequences were removed using Chimera-Slayer. Following initial QC, samples with a read depth <1,000 were resequenced and reprocessed. Samples passing QC were then classified from the phylum to the genus level using the Ribosomal Database Project (RDP) Naive Bayesian Classifier (version 2.2, training set 6).^{14 (link)} Taxa assigned with <0.5 confidence were reassigned to the next higher taxonomic level in which the classification threshold was >0.5.

Sequencing of the bacterial 16S V3 to V5 hypervariable regions was performed using the Roche 454 Titanium platform. Filtering, trimming, and chimera checking were performed as previously described.^{14 (link),15 (link)} We used closed-reference operational taxonomic unit (OTU) classification in Qiime to group sequences according to sequence similarity and match to taxonomy.^{16 (link)} We excluded samples with total read counts <1000 and OTUs identified less than 10 times or in fewer than 10 subjects.