Synthetic Core + P. penneri +E. timonensis Mouse 4
Bacteria from frozen culture stocks were diluted in liquid media and introduced as a synthetic community into female germ-free C57BL/6 mice supplemented with 1.3% (w/v) L-carnitine. 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. DNA was extracted from cecal samples using the Macherey-Nagel NucleoSpin Tissue kit according to manufactures instructions for bacterial DNA isolation. Libraries were prepared from extracted gDNA 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 water bath sonicator then cleaned and concentrated using a Macherey-Nagel PCR Purification Column before further processing. Concentrated DNA was blunted and poly-A tailed. A-tailed molecules were then ligated to the specified barcoded Illumina adapter sequences.  Adapter bound DNA was size-selected on a 2% agarose gel by electrophoresis.  Purified fragments of the desired size were PCR amplified and again purified over a Macherey-Nagel PCR Purification Column.  Purified PCR products were given to the University of Wisconsin-Madison Biotechnology Center for quality assurance and library sequencing following the manufacturer's protocols for the Illumina MiSeq platform. Raw FASTQ files were provide by the Biotechnology Center for analysis after sequencing. Sequences were demultiplexed by 7bp barcodes present in the adapters. Barcodes required an exact match to be included in community analysis (sequences without a barcode match were excluded from the analysis). Sequences were aligned to the reference genomes of the 7 bacteria included in this study using Bowtie. Only reads which mapped uniquely to the reference genomes were used for abundance analysis. Raw counts were normalized based on the genome size of each organism included in the analysis to account for genome size bias. 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. Genomes used in the analysis include:  Bacteroides_caccae_ATCC_43185, Bacteroides_ovatus_ATCC_8483, Bacteroides_thetaiotaomicron_VPI-5482, Collinsella_aerofaciens_ATCC_25986, Eubacterium_rectale_ATCC_33656, Proteus_penneri_ATCC_35198, Emergencia_timonensis_SN18.
SUB9072861
gbu21