4) 30µg/L Acetone: Atrazine stock solution was dissolved in a 100% acetone solution. In order to assume continuity throughout the experiment this treatment was added to assure that any noticed effect on development was due solely to atrazine exposure.
The oyster groups spent a total of 3 hours submerged in 2 liters of treated water within each glass tank twice per week on Mondays and Thursdays for X# of weeks in order to mimic the effects of heavy rainfall found within the Chesapeake Bay area (USGS: Chesapeake Region Climate/Precipitation Data). In order to minimize any atrazine contamination of the respective holding tanks after the 3-hour exposure period, each group was washed using a constant stream of pressure-filtered water for three one-minute rinses. Oyster groups were then relocated to their respective 40-liter glass bio-cube tanks. The bio-cube tanks were set to have the same parameters as the large holding tank.
16S Sequencing
A microbiome library was constructed from the tissues of five groups of oysters according to the concentration used in treatments, 30ug atrazine, 10ug atrazine, 3ug atrazine, 30ug acetone, and control group reads were de novo assembled together. The differential expression of microbes was analyzed using the general assembly as a reference for mapping the reads from each condition. In this project, 408,495 pair-end reads were obtained for 16 samples in total, after pair-end reads merging and filtering, 302,751 clean tags were generated, an average of 18,922 clean tags for each sample. Amplicons were performed on a paired-end Illumina HiSeq2500 platform to generate 250bp paired-end raw reads, and then pretreated. Specific processing steps are as follows:
1) Paired-end reads were assigned to a sample by their unique barcode, and the barcode and primer sequence were then truncated.
2) Paired-end reads were merged using FLASH (V1.2.7,http://ccb.jhu.edu/software/FLASH/ ) ,a very fast and accurate analysis tool to merge pairs of reads when the original DNA fragments are shorter than twice of the reads length. The obtained splicing sequences were called raw tags.
3) Quality filtering was then performed on the raw tags under specific filtering conditions of Trimmomatic v0.33 (http://www.usadellab.org/cms/?page=trimmomatic) quality control process. After filtering, high-quality clean tags were obtained.
4) The tags were compared with the reference database (Gold database, http://drive5.com/uchime/uchime_download.html) using UCHIME algorithm (UCHIME Algorithm. (http://www.drive5.com/usearch/manual/uchime_algo.html) to detect chimeric sequences, and then the chimeric sequences were removed. The data output of the above steps is shown in Table 1.
Results