16S Sequencing
Two separate sequencing events took place during this study. The first was conducted on specimens from the first stabilization and treatment periods. The second sequencing event was conducted in specimens from the second stabilization and treatment periods. The first sequencing event included five examined concentrations: 30 µg/L Atrazine, 10 µg/L Atrazine, 3µg/L Atrazine, 30µg/L Acetone and a control and included a total of 16 samples (six 30µg/L Acetone, three controls, three 30µg/L atrazine, two 10µg/L and two 3µg/L. Specimens in each group were sequenced both directly following the treatment periods and following a 30-day recovery period. The second sequencing event included six examined concentrations: 30 µg/L Atrazine, 20 µg/L Atrazine, 10 µg/L Atrazine, 3µg/L Atrazine, 30µg/L Acetone and a control and included a total of 24samples (four per sample). Amplicons were performed on a paired-end Illumina HiSeq2500 platform to generate 250bp paired-end raw reads, and then pretreated. Specific processing steps for both sequencing events were completed 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.
Statistical Analysis
To characterize microbial communities and to perform functional analyses, the following wrappers were employed: summarize_taxa_through_plots (to produce the taxonomical files and charts), alpha_rarefaction and beta_diversity_through_plots (to assess respectively the alpha- and beta-rarefaction diversity indices), principal_coordinates.py (to compare groups of samples based on phylogenetic distance metrics). To compare sampling treatments (Control versus all treatment concentrations) within each sampling season, ANOVA analyses have been performed at genus level (P-value < 0.005; FDR < 0.01) using Excel version 16.13.
Results
Diversity of microbial communities and taxonomic richness