Bacterial DNA extraction and 16S rRNA gene library preparation
DNA was extracted from fish hindgut content using a sucrose lysis buffer solution method previously described (Shahraki et al., 2019) and extracted DNA was subsequently stored at –20 °C, until further analysis. Additionally, the PCR conditions and 16S rRNA primer sets (1st and second PCR) were the same as those used in previously described methods (Sadeghi et al., 2021). Briefly, 16S rRNA variable regions of V5-V6 were amplified with a PCR cycle program of 95 °C for 3 min followed by 28 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 m, and a final step at 72 °C for 7 m. A second short-cycle PCR (7 cycles) using purified first PCR products ligated the adaptor and barcode (10 -12 bp) sequences to the amplicons as required for sample identification and sequencing. During the first and second PCR, nine samples failed amplification and 263 samples (195 gut samples, and 68 water samples) remained for the gel extraction. For each 96 well PCR plate, one negative control consisting of PCR mix (of first and second PCR) with ultra-pure water instead of DNA template was included. The pooled purified PCR amplicon mix (i.e., sequencing library) was sequenced on an ION S5 sequencing system.