title: MIMS Environmental/Metagenome sample from sediment metagenome73.2 m
design: The DNA concentration and purity were checked by 1% agarose gel electrophoresis, followed by Nanodrop for DNA concentration and purity measurement. Specific primers (V3-V4: 341F: CCTACGGGNGGCWGCAG 805R: GACTACHVGGGTATCTAATCC)7 equipped with barcodes were utilized to amplify amplicons from the V3-V4 region of the 16S rRNA genes via PCR. The Phusion High-Fidelity PCR Master Mix (New England Biolabs) was employed for all PCR reactions. The PCR products were validated through 1% agarose gel electrophoresis. The amplicons underwent purification using Beckman DNA Clean Beads and were quantified with a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Post amplification, the PCR products were combined in equal density ratios. The mixed PCR products were subsequently purified utilizing the Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were constructed using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) and indexed accordingly. The quality of the library was evaluated by a Qubit 2.0 fluorometer (Thermo Fisher Scientific, USA) and an Agilent 2100 Bioanalyzer system.