Viral Metagenomic Sequencing from Fecal Samples

Fecal samples were processed using a previously published method [22 (link),23 (link)]. Total RNA was extracted from enriched virus-like particles using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). The extracted RNA of all samples was pooled for library construction, followed by amplification using the REPLI-g Cell WGA & WTA Kit (150052; Qiagen, Hilden, Germany). Amplified DNA was randomly fragmented by ultrasound sonication (Covaris M220, Woburn, MA, USA) to produce 800 bp fragments, then sticky ends were repaired and adapters were added using T4 DNA polymerase (M4211, Promega, Madison, WI, USA), Klenow DNA Polymerase (KP810250, Epicentre, Woburn, MA, USA), and T4 polynucleotide kinase (EK0031, Thermo Scientific, Fermentas, GlenBurnie, MD, USA). Each viral sequencing library was prepared following the Illumina TruSeq DNA Preparation Protocol and was sequenced on the HiSeq4000 platform (Illumina, San Diego, CA, USA), with 150 bp paired ends. The library preparation and sequencing process was performed by BGI Tech (Shenzhen, China).