Virome in healthy pangolins revealed compatibility with multiple potentially zoonotic viruses

Each sample was subjected to RNA extraction using the AxyPrepTM Multisource Total RNA Miniprep Kit (Axygen, China) according to the manufacturer’s instructions and immediately transferred to a -80 °C freezer for storage. A sequencing library was constructed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. The library was subsequently sequenced on an Illumina Novaseq 6000 (PE150) sequencing platform (San Diego, CA, USA).For each library, adaptor sequences and low-quality bases were first removed from the raw sequencing reads by the Fastp program(Chen et al., 2018) (v0.20.0). The remaining reads were compared with the SILIVA database (www.arb-silva.de, version 138.1) and pangolin genomes (GCF_014570555.1_YNU_ManPten_2.0 and GCF_014570535.1_YNU_ManJav_2.0) using Bowtie2(Langmead and Salzberg, 2012) (version 2.2.5) to remove the reads associated with ribosomal RNA and host genomes, respectively. Unmapped reads were assembled de novo using MEGAHIT(Li et al., 2015; Li et al., 2016) (v1.2.9). The assembled contigs were compared against the NCBI non-redundant protein database (nr) using Diamond blastx(Buchfink et al., 2015) (v 0.9.7) to identify virus-associated contigs based on the top BLAST hits of each contig.