Details

Autor(en) / Beteiligte

• Viehweger, Adrian
• Krautwurst, Sebastian
• Lamkiewicz, Kevin
• Madhugiri, Ramakanth
• Ziebuhr, John
• Hölzer, Martin
• Marz, Manja

Titel

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

Ist Teil von

• Genome research, 2019-09, Vol.29 (9), p.1545

Ort / Verlag

United States

Erscheinungsjahr

2019

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.