Submitted to: National Center for Biotechnology Information (NCBI)
Publication Type: Other
Publication Acceptance Date: 3/21/2016
Publication Date: N/A
Technical Abstract: The goal of this project was to examine the impact of microbial diversity in feed on gut communities of fish. To do so, tilapia larvae were fed three experimental diets incorporated with sludge produced under either aerobic, methanogenic or denitrifying conditions. Microbial diversity between different treatments and among feed and water samples was determined by high-throughput sequencing of 16S rRNA gene amplicons, detailed as follows. Barcoded amplicons from the V4 region of 16S rRNA genes were generated by polymerase chain reaction (PCR) using 515F and 806R primers in 50 microliter reactions. PCR was performed under the following cycling parameters: 98 degrees Celsius for 30 s, followed by 25 cycles consisting of denaturation at 98 degrees Celsius for 10 s, annealing at 56 degrees Celsius for 10 s, and elongation at 72 degrees Celsius for 10 s, and a final extension at 72 degrees Celsius for 7 min. PCR products were purified, separated on an agarose gel and bands visible at approximately 300 base pair (bp) were excised and purified. DNA concentrations were measured with a fluorometer. Using an Illumina HiSeq2000, 100 bp paired-end sequences were generated. Sequencing data is made available for the public at the NCBI SRA under the accession number SRP062676.