Genome replication by viral RNA-dependent RNA polymerases is affected by high mutation and recombination rates, and leads to populations of closely related viruses (quasispecies). Current short-read sequencing technologies make the reconstruction of large numbers of full-length haplotypes difficult for both RNA viruses genomes and subgenome-length (sg) RNAs composed of noncontiguous genome regions. The authors developed a full-length, direct RNA sequencing (DRS) approach using nanopores to characterise viral RNAs produced in cells infected with a human coronavirus. They were able to map the longest (26 kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, the authors were able to reconstruct a highly accurate consensus sequence of human coronavirus (HCoV)-229E (27.3 kb). In addition, by using long reads that did not require an assembly step, the authors could characterise the diverse and novel HCoV-229E sg RNAs present in infected cells. This approach circumvents reverse transcription and amplification of RNA, allowing for the detection of methylation sites in viral RNAs. Therefore the use of these techniques allows for haplotype-based viral quasispecies sequencing.
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