An experimental strategy for preparing circular ssDNA virus genomes for next-generation sequencing

Abstract

The ability of begomoviruses to evolve rapidly threatens many crops and underscores the importance of detecting these viruses quickly and to understand their genome diversity. This study presents an improved protocol for the enhanced amplification and enrichment of begomovirus DNA for use in next generation sequencing of the viral genomes. An enhanced rolling circle amplification (RCA) method using EquiPhi29 polymerase was combined with size selection to generate a cost-effective, short-read sequencing method. This improved short-read sequencing produced at least 50 % of the reads mapping to the target viral reference genomes, African cassava mosaic virus and East African cassava mosaic virus. This study provided other insights into common misconceptions about RCA and lessons that could be learned from the sequencing of single-stranded DNA virus genomes. This protocol can be used to examine the viral DNA as it moves from host to vector, thus producing valuable information for viral DNA population studies, and would likely work well with other circular Rep-encoding ssDNA viruses (CRESS) DNA viruses.

Keywords: EquiPhi29; MiSeq; Phi29; Viral DNA sequencing; Whiteflies.