Phylogenetic analysis of two wild rat-origin PCV2d isolates in China:WaterControl
In 2021, we collected tissue samples from wild rats (Rattus norvegicus) in Jiangsu province, China. Totally, 14 tissue samples were included in this study. Total nucleic acid was extracted from tissue samples using QIAamp Viral RNA Mini Kit according to manufacturer's protocol and pooled into two nucleic acid sample pools. The libraries were then constructed using Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the Illumina NovaSeq 6000 platform with 250 base paired-ends with dual barcoding. To exclude the possibility of contamination with viral nucleic, a blank control, sterile ddH2O (Sangon, Shanghai, China) was prepared and going through the entire library preparation process. For bioinformatics analysis, the generated 250 bp paired-end reads were debarcoded using vendor software from Illumina. Clonal reads were removed, and low sequencing quality tails were trimmed using Phred quality score 30 (Q30) as the threshold. The cleaned reads were de novo assembly using the Geneious Prime, version 2019.2.3. The assembled contigs and singlet sequences were then matched against the viral proteome database using BLAST. The contigs with significant similarity to known porcine circovirus 2 (PCV2) were then determined from the virome. We designed nested PCR primers for PCV2 screening and full-genome acquisition based on the assembled PCV2-related contigs and the best hits of them to nucleotide sequences in the NCBI databse.