Synthetic community used to colonize germ-free C57BL/6 females
Bacteria from frozen culture stocks were introduced as a synthetic community (AC) into 7 week old female gnotobiotic C57BL/6 mice.  Cells were therefore maintained over the course of the experiment within mice until sampling occurred. Immediately after collection, samples were subjected to flash-freezing in ethanol chilled with dry ice and stored at -80 C until total community DNA extractions could be performed. DNA was extracted from intestinal samples using mechanical disruption (bead-beating with 0.1 mm zirconium beads) in phenol:chloroform:IAA in the same manner as described by McNulty et al. in a previous publication (PMID: 22030749).Libraries were prepared according to a slightly modified version of the protocol accompanying the Illumina Genomic DNA Sample Prep Kit. Briefly, total bacterial gDNA was sonicated in a BioRuptor XL water bath sonicator, cleaned up and concentrated using a Macherey-Nagel PCR Purification column. DNA was blunted and poly-A tailed. The A-tailed molecules were ligated to the relevant barcoded Illumina adapter sequence.  Adaptered-DNA was then size-selected by agarose gel electrophoresis.  Fragments of the appropriate size were PCR amplified and purified, after which the purified PCR products were give to the University of Wisconsin-Madison Biotechnology Center for quality assurance and library sequencing following the manufacturer's protocols.Libraries were prepared according to a slightly modified version of the protocol accompanying the Illumina Genomic DNA Sample Prep Kit. Briefly, total bacterial gDNA was sonicated in a BioRuptor XL water bath sonicator, cleaned up and concentrated using a Macherey-Nagel PCR Purification column. DNA was blunted and poly-A tailed. The A-tailed molecules were ligated to the relevant barcoded Illumina adapter sequence.  Adaptered-DNA was then size-selected by agarose gel electrophoresis.  Fragments of the appropriate size were PCR amplified and purified, after which the purified PCR products were give to the University of Wisconsin-Madison Biotechnology Center for quality assurance and library sequencing following the manufacturer's protocols. Demultiplexing: sequences were demultiplexed by 4 nt barcode, requiring an exact match (sequences without a barcode match were excluded from the analysis), Trimming: after demultiplexing, all sequences were trimmed to 35 bases to eliminate low-quality bases at the ends of each read and to allow for analysis using Bowtie, Mapping: sequences were aligned to the reference genomes of the 6 bacteria included in the study using Bowtie. Only reads which mapped uniquely to the reference genomes were used for abundance analysis, Normalization: raw counts were normalized based on the genome size of each organism included in the analysis, Summarization: the proportional representation of each organism in the analysis was determined by dividing its normalized counts within a sample by the total normalized counts for all organisms within that sample, Organisms used:  Bacteroides_caccae_ATCC_43185, Bacteroides_ovatus_ATCC_8483, Bacteroides_thetaiotaomicron_VPI-5482,  Collinsella_aerofaciens_ATCC_25986, Eubacterium_rectale_ATCC_33656, Escherichia_coli_MS_200-1.