Nanopore direct RNA sequencing reveals modification in full-length coronavirus genomes
30th November 2018 - BioRxiv
Sequence analyses of RNA virus genomes remain challenging due 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 that are generally referred to as quasispecies. Although standard (short-read) sequencing technologies allow to readily determine consensus sequences for these quasispecies, it is far more difficult to reconstruct large numbers of full-length haplotypes of (i) RNA virus genomes and (ii) subgenome-length (sg) RNAs comprised of noncontiguous genome regions that may be present in these virus populations. Here, we used a full-length, direct RNA sequencing (DRS) approach without any amplification step to characterize viral RNAs produced in cells infected with a human coronavirus representing one of the largest RNA virus genomes known to date.
Using DRS, we were able to map the longest (~26 kb) contiguous read to the viral reference genome. By combining Illumina and nanopore sequencing, a highly accurate consensus sequence of the human coronavirus (HCoV) 229E genome (27.3 kb) was reconstructed. Furthermore, 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 does not require 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 demonstrating the feasibility of intra-sample haplotype separation. We also show how supplementary short-read sequencing (Illumina) can be used to reduce the error rate of nanopore sequencing.
Even though a number of technical challenges remain to be addressed to fully exploit the potential of the nanopore technology, our work illustrates that direct RNA sequencing may significantly advance genomic studies of complex virus populations, including predictions on long range interactions in individual full-length viral RNA haplotypes.