Cressdnavirus Genome Recovery via PCR

There were 28 cressdnavirus-like contigs identified, and these were used to design abutting primer pairs (Supplementary Table S3) for the recovery of complete viral genomes by PCR. The specific primers pairs, together with KAPA HiFi HotStart DNA Polymerase (Kapa Biosystems, Wilmington, MA, USA), were used to screen and amplify the virus genomes and viral-like elements from each sample, using the thermal cycling protocol recommended by the manufacturer with an annealing temperature of 60 °C and 0.5 µL of the RCA product. The resulting amplicons were resolved on a 0.7% agarose gel, excised, purified, and cloned into pJET1.2 plasmid (ThermoFisher, Waltham, MA, USA). The recombinant plasmids were Sanger-sequenced by primer walking at Macrogen Inc. (Seoul, Korea), and contigs assembled using Geneious Prime [32 (link)].