Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

Abstract

Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

Deep splicing plasticity of the human adenovirus type 5 transcriptome drives virus evolution

“…This provides a direct and complete record of the exons present in any given mRNA without reverse transcription or amplification steps. dRNA-seq has recently been used to examine the transcriptome of human cells 9,10 , coronavirus 11 and herpes simplex virus 12 , revealing greater complexity than previously appreciated and underlining the power of this approach to improve our understanding of transcriptomes, even in well-studied systems.…”

Recent advances in the detection of base modifications using the Nanopore sequencer

“…They also measured polyA tail length using the polya option of Nanopolish and showed differences in polyA tail length both between genes and between transcripts of the same gene. Finally, Viehweger et al performed direct RNA-seq analysis of human coronavirus and detected m5C in the viral RNA using Tombo [56,57].…”

Pervasive generation of non-canonical subgenomic RNAs by SARS-CoV-2

“…The CoV-229E long-read dataset was generated by Viehweger et al [31]. In this experiment, Huh7 cells were infected with CoV-229E and RNA was collected 24 hours post infection.…”