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.