SUPPLEMENTAL METHODS:
Sample processing and next generation sequencing
For DNA extraction, 0.3g of stool were added to Mo Bio Powersoil® (Mo Bio, Carlsbad, CA) power bead tubes, vortexed for 10 minutes, centrifuged at 10,000 x g for 30 seconds; 200 µL of supernatant were processed using a bacterial card on the EZ1 Advanced XL automated extraction system (Qiagen, Valencia, CA). DNA was eluted in 50 µL and stored at -20°C.
In preparation for next generation sequencing, the V1-V2 hypervariable regions of the 16S ribosomal genes were amplified by PCR in a 25µL reaction that included 2µL of microbiome DNA and primers 8F (5’-AGAGTTTGA TCCTGGCTCAG) and 338R (5’-TGCTGCCTCCCGTAGGAGT), using the FastStart High Fidelity PCR System (Roche, Indianapolis, IN). Thermocycler conditions were 95˚C for 15 minutes; 30 cycles of 94˚C for 30 sec, 57˚C for 30 sec, 72˚C for 30 sec; 7 minute hold at 72˚C, and a final hold at 4˚C. PCR was performed in triplicate and products were pooled. PCR products were subsequently amplified following an Illumina procedure (http://res.illumina.com/documents/products%5Cappnotes%5Cappnote_16s_sequencing.pdf), using primers that include 5’ overhang adapter sequences. Products were cleaned using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA). Library preparation and sequencing was performed by the CDC Biotechnology Core using the Nextera XT Sample Preparation Kit and an Illumina MiSeq instrument (Illumina, San Diego, CA). ]
Next generation sequencing analysis
Sequencing generated 7.1 gigabases of 250 bp paired-end reads. Raw sequencing reads were assembled into 16S rRNA V1-V2 contigs using PANDAseq 2.6 (default settings),35 which corrects for base-call errors in the overlap region and discards low quality assemblies. Next generation sequencing yielded 1,008,018 (Range: 584,059-1,656,961) 16S V1-V2 contigs per sample with an average sequence length of 369 bp. Assembled DNA sequence data were processed using QIIME 1.8 suite scripts.36 DNA sequences sharing 97% similarity were clustered into OTUs using the UCLUST algorithm.19 The centroid sequence was selected as the representative sequence for all members within a single OTU. OTUs containing only one sequence (singletons) were discarded from the dataset. The centroid sequence of each accepted OTU was assigned to known taxa using the Greengenes database (v. 13-8),37 by means of the USEARCH component of UCLUST. Additionally, KRAKEN38 was used to align all sequences to the NCBI RefSeq database. KRONA39 and QIIME scripts were used to generate taxonomic distribution plots, for KRAKEN alignments and UCLUST clustering, respectively.
Using the QIIME 1.8 suite, all sequence data were rarefied (randomly selected) to 5,000 sequences per sample. Shannon Diversity Index9 and Observed Species were selected for alpha diversity analyses. Centroid sequences of each OTU were aligned with each other using PyNAST40 and these alignments were turned into an OTU phylogenetic tree using FastTree software.41 The inferred tree was utilized for beta-diversity community analyses (weighted UniFrac distance20). Beta-diversity analyses were visualized by principal coordinates plots using the cmdscale function in R statistical software (v. 3.0.2).
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