DNA was extracted following the MetaHIT protocol, as previously described [40]. The extracted DNA from all samples was amplified to reach the requirement for subsequent library construction by PicoPLEX WGA Kit (Rubicon) following the manufacturer’s protocol. The DNA concentration was quantified by Qubit (Invitrogen).

A 500 ng of input DNA was fragmented ultrasonically with Covaris E220 (Covaris, Brighton, UK), yielding 300 to 700 bp of fragments. Sheared DNA without size selection was purified with an AxygenTM AxyPrepTM Mag PCR Clean-Up Kit. An equal volume of beads was added to each sample, and DNA was eluted with 45 μL TE buffer. Twenty nanograms of purified DNA was used for end-repairing and A-tailing with a 2:2:1 mixture of T4 DNA polymerase (ENZYMATICSTM P708–1500), T4 polynucleotide kinase (ENZYMATICSTM Y904–1500), and Taq DNA polymerase (TAKARATM R500Z) which was heat-inactivated at 75 °C. Adaptors with specific barcodes (Ad153 2B) were ligated to the DNA fragments by T4 DNA ligase (ENZYMATICSTM L603-HC-1500) at 23 °C. After the ligation, PCR amplification was carried out. Fifty-five nanograms of purified PCR products was denatured at 95 °C and ligated by T4 DNA ligase (ENZYMATICSTM L603-HC-1500) at 37 °C to generate a single-strand circular DNA library. Sequencing was performed according to the BGISEQ-500 protocol (SOP AO) employing the paired-end whole-metagenome sequencing (WMS) mode, as described previously [61].

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