CIDER-Seq: An efficient tool to study plant-geminivirus recombinant eccDNAs

Genetic recombination is an important feature of begomoviruses, single stranded DNA virus belonging to family Geminiviridae. Extremely complex and widely distributed recombinations have been observed upon begomovirus infections [1]. Apart from virusvirus recombination, a recent study has revealed spontaneous formation of hybrid DNA minicircles composed of begomovirus and plant genomic DNA sequences [2]. We have recently developed CIDER-Seq (Circular DNA Enrichment sequencing), a pipeline for the unbiased enrichment and highly accurate long-read sequencing of extra-chromosomal circular DNA (eccDNA) molecules [3]. Here we demonstrate the utilization of CIDER-Seq to sequence and characterize recombinant plant-geminivirus eccDNAs. Arabidopsis plants, agroinoculated with cabbage leaf curl virus (CaLCuV), were subjected to modified rolling circle amplification-mediated enrichment of eccDNAs. SMRT (Single Molecule Real Time) libraries were then prepared and sequenced on PacBio Sequel platform. BLAST analysis indicated that almost 80% of the reads contained CaLCuV sequences. Analysis of these reads lead to the identification of 74 Arabidopsis-CaLCuV recombinants, among which 22% (16/74) belonged to CaLCuV DNA-A and 78% (58/74) belonged to CaLCuV DNA-B. The size of recombinants ranged from 624nt to 7,535nt, with an average recombinant size of 3,520nt. Overall 82% (61/74) of recombinant fragments contained chromosomal DNA, while 15% (11/74) and 3% (2/74) had chloroplast and mitochondrial DNA origins, respectively. These host sequences from recombinant fragments did not share significant similarities or patterns, except all being AT rich (~70%) and almost all lacking open reading frames (ORFs). However, in two recombinants we were able to identify complete ORFs coding for Sucrase/ferredoxin-like family protein and DNA double-strand break repair protein. Further experiments are required to characterize the frequency and the role of host-geminivirus recombination events during geminivirus infection.