Methods Fecal Sample Collection Fecal samples were collected from eight, check details six-month old Yorkshire pigs from a large swine operation located in Northeastern Ohio, which housed more than 1,000 head of swine at the time of collection. Swine were weaned eight weeks after birth. Their diets consisted of a high-energy corn-soybean meal diet containing 14.00% crude protein, 0.63% lysine, 3.00% crude fat, 4.00% crude fiber, 0.55%- 0.70% calcium, 0.52% phosphorus, 0.35%-0.50% salt, 0.3 ppm selenium, 80 ppm zinc.
(Kalmbach Feeds, OH). In addition, swine were supplemented this website with feed grade antibiotics for improvement in growth performance. Antibiotics consisted of chlortetracycline and penicillin at the concentration of 20 g per ton of feed. Fecal samples were transported to the laboratory on ice within four hours of collection, and stored at -20°C until further processing. Fecal DNA was extracted with the FastDNA SPIN Kit (MP Biomedicals, Inc., Solon, OH) according to the manufacturer’s instructions using 0.25 g of each fecal sample. Total DNA was quantified using a NanoDrop® ND-1000 UV spectrophotometer (NanoDrop Technologies, Wilmington, DE). Pyrosequencing and Gene Annotation
A total of 24 μg (3 μg of each fecal DNA extract, n = 8) were pooled and sent for pyrosequencing CB-5083 concentration to 454 Life Sciences, where two different sequencing runs were performed. The first run was performed using Genome Sequencer GS20 platform while the Genome Sequencer FLX instrument was used for the second run. Each pig fecal metagenomic sequencing run was assembled de novo using the Newbler assembly software by 454 Life Sciences. The metagenomes used in this paper Farnesyltransferase are freely available from the SEED, JGI’s IMG/M, and NCBI Short Read Archive. The NCBI genome project ID and GOLD ID for swine fecal GS20 and FLX metagenomic sequencing runs generated
in this project are 39267 and Gm00197, respectively. Raw sequencing reads from both datasets were submitted to the Joint Genome Institute’s IMG/M-ER annotation pipeline using the proxygene method for gene annotation [4, 32]. Additionally, both metagenome runs were annotated using the “”Phylogenetic Analysis”" tool within the MG-RAST pipeline [33]. The BLASTn algorithm (e-value less than1 × 10-5 and a sequence match length greater than 50 nucleotides) was used to identify small subunit rRNA genes from RDP [34], SILVA SSU [35], and Greengenes databases [36]. Within the MG-RAST pipeline, the “”Metabolic Analysis”" tool was used to search sequences from pig fecal metagenomes against the SEED database using the BLASTx algorithm (e-value less than 1×10-5 and a sequence match length greater than 30 nucleotides) [37]. Comparative Metagenomics and Statistical Analyses Comparative metagenomics was performed using both the IMG/M and MG-RAST pipelines. GS20 and FLX pig metagenomic runs were compared to the current publicly available gut metagenomes within each of these databases.