For targeted metabarcoding sequencing, DNA fragments were amplified using primers targeting the 16S rDNA V4 region for bacteria (Additional file 2 : Table S1). PCR reaction was done for each sample under the following conditions [45 (link)]: 30 s at 98 °C; 35 cycles of denaturation at 98 °C for 10 s, annealing at 54 °C for 30 s, and extension at 72 °C for 45 s; final step 10 min at 72 °C. The 25 μL of PCR mixture contained 12.5 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs Inc., Beverly, MA), 2.5 μL of each primer (1 μM), and 50 ng of template DNA. The PCR products were electrophoresed and stained with ethidium bromide for visualization. The specific DNA bands on the gel were excised and purified using Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer’s recommendations and then sequencing was conducted using the Illumina NovaSeq 6000 Sequencer (Illumina, Inc., CA, USA).
For shotgun metagenomic sequencing, the extracted DNA samples were fractionated by ultrasound into approximately 350 bp fragments. The fragments were then used to construct the sequencing libraries using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA). The libraries were sequenced using the Illumina NovaSeq 6000 Sequencer (Illumina, Inc., CA, USA).
For shotgun metagenomic sequencing, the extracted DNA samples were fractionated by ultrasound into approximately 350 bp fragments. The fragments were then used to construct the sequencing libraries using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA). The libraries were sequenced using the Illumina NovaSeq 6000 Sequencer (Illumina, Inc., CA, USA).