Enriching Viral DNA by Random-Primed RCA

Viral DNA was enriched by randomly primed RCA [23 (link), 37 (link), 41 (link)] using a commercial kit (TempliPhi™100 Amplification Kit, GE Healthcare) to enrich circular DNA according to the manufacturer’s instructions. The amplicons were electrophoresed in a 0.5% agarose gel and visualized on a UV light source. The RCA products were purified using a commercial kit (Purelink Quick PCR Purification Kit, Thermo Fisher Scientific®), and the quality and quantity of the DNA were assessed by spectrophotometry and fluorometry using NanoDrop™ (Thermo Fisher Scientific®) and Qubit™ (Thermo Fisher Scientific®), respectively. DNA fragment libraries were prepared with 50 ng of purified DNA using a Nextera XT DNA sample preparation kit (Illumina, USA) and sequenced using an Illumina MiSeq System (Illumina, USA) using an Illumina v2 reagent kit (2 × 150 paired end reads).