16S rRNA Amplicon Sequencing of Fish Microbiome

The 16 S rRNA amplification and high-throughput sequencing was performed by Molecular Research LP (MR DNA; Shallowater, TX, USA) following the protocol described by Vargas et al. 2023 [70 (link)]. Briefly, each sample of DNA was diluted to 25 ng/μL. The DNA of three fish per pond were mixed in equal volumes to form a pool. For each condition, two pools were generated. Each DNA pool was used as a template to amplify the V4 variable region using primers 515-F and 806-R [71 (link)] with a HotStarTaq Plus Master Mix Kit (Qiagen, Hilden, Germany). The PCR conditions were set up to 94 °C (3 min), 30 cycles of 94 °C (30 s), 53 °C (40 s), and 72 °C (60 s), with a final elongation to 72 °C (5 min). PCR products were analyzed in a 2% agarose gel, purified using Ampure XP beads. Illumina DNA library using purified PCR products was prepared with a TruSeq Nano kit. MiSeq reagent kit v3 on the Illumina MiSeq platform (2 × 300-bp paired ends [PE]) was used for high-throughput sequencing of amplicons following the manufacturer’s guidelines.