Metagenomic Shotgun Sequencing Protocol

All samples were prepared for the metagenomic shotgun sequencing according to previous protocols [6 (link)]. Briefly, total genomic DNA was extracted using QIAamp DNA Microbiome Kit (Qiagen, USA). After DNA extraction, 1 µg genomic DNA was randomly fragmented by Covaris, followed by purification by AxyPrep Mag PCR clean-up kit. The fragmented DNA was selected by Agencourt AMPure XP Medium kit to an average size of 200–400 bp. The fragments were end-repaired by End Repair Mix and purified afterward. The repaired DNAs were combined with A-Tailing Mix. Then the Illumina adaptors were ligated to the Adenylate 3’Ends DNA and followed by purification. The products were selected based on the insert size. Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix were performed to enrich the Adapter-ligated DNA fragments. After purification, the library was qualified by the Agilent 2100 bioanalyzer (Agilent, USA) and ABI StepOnePlus Real-time PCR System. Finally, the qualified libraries were sequenced on the Illumina Hiseq platform (BGI-Shenzhen, China).