Metagenomic libraries were constructed utilizing the iNextEra library preparation protocol. The DNA was tagmented using Illumina bead-linked transposomes (Illumina DNA library prep, Illumina, Inc., San Diego, CA, USA) and 2X TMP buffer in a total volume of 6 L, comprising 3 L of 2X TMP buffer, 0.5 L of bead-linked transposome, and 2.5 L of DNA, followed by incubation at 53C for 30 minutes. After tagmentation, a limited-cycle PCR step finalized the Illumina-compatible adapters with dual-indexes utilizing custom iNextEra primers for sequencing of pooled libraries. Each 51.5 L of PCR master mix comprised 40.25 L HyClone water (Cytiva Life Sciences, Marlborough, MA, USA), 9.5 L of Q5 Reaction Buffer (New England Biolabs, Ipswich, MA, USA), 1.25 L of 10mM dNTPs (Roche Diagnostic Corporation, Indianapolis, IN, USA), and 0.5 L of Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA). Subsequently, the master mix was incorporated into 6 L of the tagmentation mix. In conclusion, 2.5 L of 5 M iNextEra5 index primer and 2.5 L of 5 M iNextEra7 index primer were incorporated into the PCR mix, resulting in unique index combinations within a total PCR volume of 62.5 L. The limited-cycle PCR was performed using a T100 thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The protocol included an initial strand repair at 72C for 3 minutes, followed by denaturation at 98C for 3 minutes. This was succeeded by 12 cycles of 45 seconds at 98C, 30 seconds at 62C, and 2 minutes at 72C, concluding with a final extension at 72C for 1 minute. Following PCR, 5 L of the PCR product was analyzed on an agarose gel to confirm amplification of each sample, resulting in a smear within the target size range of 300-700 bp. The libraries were quantified with Qubit Flex (Thermo Fisher Scientific, Waltham, MA, USA), normalized, pooled, and purified using AMPure XP beads (Beckman Coulter, Inc., Brea, CA, USA). The final pool was quantified and dispatched to Novogene (Novogene Corporation Inc., Sacramento, CA, USA) for the acquisition of 2150 bp paired-end reads utilizing the Illumina NovaSeq X plus sequencing system (Illumina, Inc., San Diego, CA, USA).