Low TMA producing human fecal community transplanted into germ-free C57BL/6 female mice
Bacteria from frozen fecal slurries were diluted in 5-fold and 200ul                 used to colonize germ-free female C57BL/6 mice. Cells were therefore maintained over                 the duration of the experiment within mice until ex vivo sampling occurred.                 Immediately after collection, cecal samples were subjected to flash-freezing and                 stored at -80C until total community DNA extractions could be performed. Microbial                 DNA was extracted following MO BIO PowerSoil-htp 96 Well Soil DNA Isolation Kit                 procedure with an additional incubation at 65C for 10 min prior to plate shaking.                 The 16S rRNA V4 hypervariable region was amplified with barcoded primers (Caporaso                 et al. 2012) in triplicate using the 5 PRIME HotMasterMix (VWR). Products were                 quantified with Quant-iTTM PicoGreen dsDNA Assay Kit (Thermo Fisher) and samples                 were combined in equal amounts (~250 ng per sample) to be purified with the                 UltraClean PCR Clean-Up Kit (MO BIO). Pooled amplicons were sequenced on the                 Illumina HiSeq platform to generate single-end reads. Raw sequences were processed                 with the open source Quantitative Insights Into Microbial Ecology (QIIME) software                 package version 1.9.1 (Caporaso et al. 2010) to produce de-multiplexed and quality                 controlled sequences. Samples were binned into OTUs using the                 pick_closed_reference_otus.py script at 97% similarity using SUMACLUST (Kopylova et                 al. 2016) against a Greengenes reference database (McDonald et al.                 2012).