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

Area of Science:

• Plant virology
• Molecular biology
• Genomics

Background:

• Begomoviruses rapidly evolve, posing a significant threat to global crop production.
• Accurate and timely detection of begomoviruses is crucial for disease management and understanding viral genome diversity.
• Existing methods for begomovirus genome sequencing can be costly and time-consuming.

Purpose of the Study:

• To develop an improved protocol for enhanced amplification and enrichment of begomovirus DNA.
• To enable cost-effective, short-read next-generation sequencing of begomovirus genomes.
• To provide insights into the sequencing of single-stranded DNA viruses and address common misconceptions about rolling circle amplification (RCA).

Main Methods:

• An enhanced rolling circle amplification (RCA) method utilizing EquiPhi29 polymerase was developed.
• Size selection was incorporated to enrich for viral DNA fragments.
• The protocol was optimized for short-read sequencing platforms.

Main Results:

• The improved protocol achieved at least 50% of sequencing reads mapping to target viral reference genomes (African cassava mosaic virus and East African cassava mosaic virus).
• This method proved cost-effective for generating high-quality begomovirus genome data.
• The study identified common misconceptions regarding RCA and offered lessons for ssDNA virus genome sequencing.

Conclusions:

• The developed protocol significantly enhances the amplification and enrichment of begomovirus DNA for next-generation sequencing.
• This cost-effective method facilitates rapid detection and detailed analysis of begomovirus genome diversity.
• The protocol is applicable to studying viral DNA dynamics in host-vector interactions and can be adapted for other circular Rep-encoding ssDNA (CRESS) viruses.