Enrichment and purification of virus-like particles (VLPs) Here we provide a full working protocol for analyzing human gut virome using physical enrichment, reverse transcription and random amplification, and eventually the state-of-art single-molecule real-time sequencing (SMRT) platform of Oxford Nanopore Technology (ONT). This workflow mainly contains four fragments: 1) Washing and filtration of fecal samples using PBS/Sterile and PVDF membrane; 2) Precipitation of VLPs; 3) Extraction, amplification and purification of viral nucleic acids; 4) Construction of library and ONT sequencing. First two steps were based on method of Thurber RV et al. (Laboratory procedures to generate viral metagenomes. Nat Protoc 2009;4(4):470-83. https://doi.org/10.1038/nprot.2009.10). The third step referred to Ge XY et al.( Metagenomic Analysis of Viruses from Bat Fecal Samples Reveals Many Novel Viruses in Insectivorous Bats in China. J Virol 2012;86(8):4620-30. https://doi.org/10.1128/Jvi.06671-11) and Froussard P (rPCR: a powerful tool for random amplification of whole RNA sequences. Genome Research 1993;2185-90. https://doi.org/10.1101/gr.2.3.185). The fourth step, PromethION library preparation, was performed according to the manufacturer¡¯s instructions for the barcoding cDNA/DNA and native DNA (SQK-LSK109 and EXP-NBD104) Citation: if you use this workflow, please cite: Jiabao Cao et al. Profiling of Human Gut Virome with Oxford Nanopore Technology, bioRxiv. doi: https://doi.org/10.1101/2020.02.03.933077