Qsep400

Total genomic DNA was extracted from 27 samples using the TGuide S96 Magnetic Soil DNA Kit (Tiangen Biotech (Beijing) Co., Ltd.) according to manufacturer’s instructions. DNA was detected on a 1% agarose gel, and the concentration was determined using a spectrophotometer NanoDrop 2000 (NanoDrop Technologies, United States). The hypervariable region V3–V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F: 5′-ACTCCTACGGGAGGCAGCA-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′. Both the forward and reverse 16S primers were tailed with sample-specific Illumina index sequences to allow for deep sequencing. The PCR was performed in a total reaction volume of 10 μL: DNA template 5–50 ng, forward primer (10 μM) 0.3 μL, reverse primer (10 μM) 0.3 μL, KOD FX Neo Buffer 5 μL, dNTP (2 mM each) 2 μL, KOD FX Neo 0.2 μL, and finally ddH2O up to 20 μL. After with initial denaturation at 95°C for 5 min, followed by 20 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 40 s, and a final step at 72°C for 7 min. The amplified products were purified with Omega DNA purification kit (Omega Inc., Norcross, GA, United States) and quantified using Qsep-400 (BiOptic, Inc., New Taipei City, Taiwan, ROC). The amplicon library was paired-end sequenced (2 × 250) on an Illumina novaseq6000 (Beijing Biomarker Technologies Co., Ltd., Beijing, China).

RNA extracted from fish skin tissue was prepared for library construction. The RNA integrity was assessed by a Qsep400 (BiOptic, Taiwan) with quality number (RQN) values ≥ 7.0. A paired-end (PE) sequencing library was constructed using Illumina TruSeq RNA Library Prep Kit v2 (Illumina, USA), following the manufacturer’s protocol. The obtained library was sequenced using the Illumina NovaSeq 6000 platform (Illumina, USA) with 2 × 150 bp PE reads.

The target region PCR products (10 µl) were purified by adding VAHTS™ DNA Clean Beads at a 1:1 ratio, and then barcode indexing and Illumina adapters were added by Solexa PCR (20 µl reaction volumes), which used 5 µl of pooled PCR product, 2.5 µl of each index (forward and reverse), and 10 µl of 2× Q5 High-Fidelity Master Mix (NEB, USA) [19 (link)]. The PCR conditions were 98 °C for 30 s, followed by 10 cycles of 98 °C for 10 s, 65 °C for 30 s, and 72 °C for 30 s, and a final extension step of 5 min at 72 °C. Agarose gel electrophoresis was performed on a 1.8% (w/v) agarose gel, and the results were quantified by the ImageJ program. Each sample was mixed by aspirating 150 ng, and the mixed samples were purified before gel cutting using the Cycle Pure Kit and recovered by the Monarch DNA kit. The final gene library was evaluated on a Qsep400 (BiOptic, Taipei, Taiwan) for concentration and quality and then sequenced on an Illumina NovaSeq 6000. The 16S rRNA gene library construction and sequencing were completed by Biomarker Technologies Corporation (Beijing, China).