An improved experimental pipeline for preparing circular ssDNA viruses for next-generation sequencing

Abstract

We present an optimized protocol for enhanced amplification and enrichment of viral DNA for Next Generation Sequencing of begomovirus genomes. The rapid ability of these viruses to evolve threatens many crops and underscores the importance of using next generation sequencing efficiently to detect and understand the diversity of these viruses. We combined enhanced rolling circle amplification (RCA) with EquiPhi29 polymerase and size selection to generate a cost-effective, short-read sequencing method. This optimized protocol produced short-read sequencing with at least 50% of the reads mapping to the viral reference genome. We provide other insights into common misconceptions about RCA and lessons we have learned from sequencing single-stranded DNA viruses. Our protocol can be used to examine viral DNA as it moves through the entire pathosystem from host to vector, providing valuable information for viral DNA population studies, and would likely work well with other CRESS DNA viruses.

Population diversity of cassava mosaic begomoviruses increases over the course of serial vegetative propagation

“…Frozen leaf tissue was ground, total DNA was extracted using the MagMax Plant DNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA), and DNA <6 Kb in size was selected using the Blue Pippin Prep system (model no. BDQ3010, Sage Science, Beverly MA) prior to library prep [ 50 ].…”

“…This process was repeated six times with a total of seven rounds including the initial inoculated plant. Total DNA was isolated but was not size-fractionated because earlier serial experiments showed that sequencing total DNA samples produce sufficient read coverage for viral diversity analysis [ 50 ].…”

“…Raw sequencing data were processed according to Aimone et al [50], and the workflow is available on Galaxy (ViralSeq, https:// cass avav irus evol ution. vcl.…”

Population diversity of cassava mosaic begomoviruses increases over the course of serial vegetative propagation

“…Sequencing libraries for Veg2 and Veg6 were generated using EquiPhi polymerase (Thermo Fisher Scientific, Waltham MA) for rolling circle amplification (RCA) and the Nextera XT kit (Illumina, San Diego CA) with unique dual index sequences for library preparation (50). The protocol was modified to include two RCA reactions for each sample.…”

“…Leaf tissue collection and propagation continued for a total of two rounds at each temperature following the above protocol. Frozen leaf tissue was ground, total DNA was extracted using the MagMax TM Plant DNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA), and DNA < 6 Kb in size was selected using the Blue Pippin Prep system (Model # BDQ3010, Sage Science, Beverly MA) prior to library prep (50).…”