The RNA viruses of enrichment of fecal samples were performed as mentioned above. RNA viruses in samples were extracted by using Magen RNA extraction kits (Magen, China), and the whole genome was amplified with Qiagen kit (REPLI-g Cell WGA & WTA Kit).
Sequencing libraries construction and sequencing method were identical as DNA virome. The bioinformatics analyses were similar as mentioned above. Specifically, Blast software (v2.9.0 +) was used to compare the unique contig with virus database, if the alignment similarity was ≥ 80%, the alignment length was ≥ 500 bp, and e ≤ 1e-5 was defined as virus sequence; if the alignment length was ≥ 100 bp, and e ≤ 1e-5 was defined as suspected virus sequence. The candidate viruses sequences were queried against the NCBI reference genomes using BWA-MEM, NT database using MegaBLAST (e-value cutoff 1E-10), BLASTn (e-value cutoff 1E-10), and NCBI NR database using BLASTx (e-value cutoff 1E-3). Sequences with significant hits were classified as novel viruses based on the taxonomy identity of the best BLAST hit [40 (link), 41 (link)]. Parts of the viruses’ contigs, such as Rotavirus, Gammacoronavirus and Deltacoronavirus were aligned using Blast software to compare their similarity with other references. MEGA 6.0 software was used to build the phylogenetic tree using N-J methods with 1000 bootstraps.
Sequencing libraries construction and sequencing method were identical as DNA virome. The bioinformatics analyses were similar as mentioned above. Specifically, Blast software (v2.9.0 +) was used to compare the unique contig with virus database, if the alignment similarity was ≥ 80%, the alignment length was ≥ 500 bp, and e ≤ 1e-5 was defined as virus sequence; if the alignment length was ≥ 100 bp, and e ≤ 1e-5 was defined as suspected virus sequence. The candidate viruses sequences were queried against the NCBI reference genomes using BWA-MEM, NT database using MegaBLAST (e-value cutoff 1E-10), BLASTn (e-value cutoff 1E-10), and NCBI NR database using BLASTx (e-value cutoff 1E-3). Sequences with significant hits were classified as novel viruses based on the taxonomy identity of the best BLAST hit [40 (link), 41 (link)]. Parts of the viruses’ contigs, such as Rotavirus, Gammacoronavirus and Deltacoronavirus were aligned using Blast software to compare their similarity with other references. MEGA 6.0 software was used to build the phylogenetic tree using N-J methods with 1000 bootstraps.
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