Nucleotide-Resolution Profiling of RNA Recombination in the Encapsidated Genome of a Eukaryotic RNAVirus by Next-Generation Sequencing

Routh, Andrew; Ordoukhanian, Phillip; Johnson, John E.

Next-Generation Sequencing has been used in numerous investigations to characterize andquantifythe genetic diversity of a virus samplethrough the mapping of polymorphisms and measurement of mutation frequencies.Next-Generation Sequencing has also been employed to identifyrecombinationevents occurring within the genomes of higher organisms, for example, detecting alternative RNA splicing events and oncogenic chromosomal rearrangements. Here, we combine these two approaches toprofile RNA recombination within the encapsidated genome of a eukaryotic RNA virus, Flock House Virus. We detect hundreds of thousands of recombination events, with single-nucleotide resolution, which result indiversity in the encapsidated genome rivaling that due to mismatch mutation. We detect previously identified Defective-RNAs as well as many other abundant and novel Defective-RNAs. Our approach is exceptionally sensitive, unbiased, and requires no prior knowledge beyond the virus genome sequence. RNA recombination is a powerful driving force behind the evolution and adaptation of RNA viruses. The strategy implemented here is widely applicable and provides a highly detailed description of the complex mutational landscape of the transmissible viral genome.