An experimental strategy for preparing circular ssDNA virus genomes for next-generation sequencing.
J Virol Methods
; 300: 114405, 2022 02.
Article
em En
| MEDLINE
| ID: mdl-34896458
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA Circular
/
Genoma Viral
/
Vírus de DNA
Idioma:
En
Revista:
J Virol Methods
Ano de publicação:
2022
Tipo de documento:
Article