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

Abstract

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.

ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3′-Azido cDNAs

“…As we previously reported 13 , we found numerous intra-RNA recombination events in FHV ( Table S2 ). These correlate to defective-RNAs that accumulate during viral passaging in cell culture 18 .…”

“…Final cDNA library fragment sizes of 300-500 nts were selected and sequenced on an Illumina HiSeq for single-end 100 nt reads. This sequencing data (hereafter termed the ClickSeq data) was compared to our previous analyses 12; 13 that used the exact same RNA samples, but were prepared using the NEBNext RNAseq Library Prep Kit (hereafter termed the NEBNext data).…”

“…Artifactual recombination in NGS generates sequence chimeras that can be highly problematic in diverse areas of research including viral evolution and replication 13; 21 , bacterial recombination 22 , eukaryotic transcriptomics 23; 24 , mRNA splicing and detection of rare chromosomal rearrangements in oncogenic cells 25 . Artifactual chimeric DNA sequences also disrupt computational pipelines for the estimation of nucleotide polymorphisms frequency or species population diversity 21; 26; 27; 28; 29 and de novo genome assembly by generating incorrect edges in de Bruijn graphs 28; 30; 31; 32 .…”

“…Artifactual recombination is largely attributed to template-switching between homologous DNA/RNA fragments during the PCR steps of cDNA library synthesis 13; 33; 34; 35 and can account for over 10% of the output sequence reads 26; 29; 33; 36; 37 . This can be improved through careful optimization of reaction conditions 38 or emulsion PCR to physically segregate the individual DNA/RNA molecules 34; 39 .…”

Deep Sequencing of Virus-Derived Small Interfering RNAs and RNA from Viral Particles Shows Highly Similar Mutational Landscapes of a Plant Virus Population

“…Recently, some studies have shown that recombination events should be approximately as common as mutations in the evolution of RNA viruses (32,39,40). A recent study (41) illustrated this phenomenon in the case of tobacco etch virus, a member of the same (Potyvirus) genus as PVY.…”