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 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, so-called 'quasispecies'. Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (i) RNA virus genomes and (ii) subgenome-length (sg) RNAs comprised 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. Using DRS, we were able to map the longest (∼26 kb) contiguous read to the viral reference genome. By combining Illumina and nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV) 229E genome (27.3 kb). 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 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 demonstrating 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 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.

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

“…A similar observation showing a lower accuracy proximal to the 3' poly(A) tail has been observed previously due to the DNA adapter, which can partially explain poor accuracy at the 3' end [41,59]. agrees with what has been observed previously ( Figure 2) [44,45]. This is not surprising since the sequence adapter was ligated to the poly(A) tail on the 3' end and this is where sequencing began.…”

“…The U bases in the query sequence were adjusted to T automatically by the minimap program in order to map to the reference sequence which was DNA. The depth of coverage across the PRRSV genome was observed to be extremely uneven with higher coverage on the 3' end of the genome and gradually decreasing towards the 5' end, which agrees with what has been observed previously ( Figure 2) [44,45]. This is not surprising since the sequence adapter was ligated to the poly(A) tail on the 3' end and this is where sequencing began.…”

“…Whole genome sequencing is greatly needed to provide a more complete picture of the virus [67,68], which is now gradually becoming more feasible with the rapid development and innovation of new sequencing technologies [69,70]. Oxford Nanopore direct RNA sequencing (DRS) is revolutionary for sequencing RNA viral genomes, since it can sequence the RNA directly, allowing for detection of methylation sites and decreasing bias inherent in reverse transcription and PCR amplification of samples prior to sequencing, and it can generate long reads, allowing for the elucidation of recombination events [71].…”

“…Influenza virus was the first pathogen to be successfully sequenced in its native RNA format by direct RNA sequencing (DRS) using Oxford Nanopore MinION technology [44]. Since then, studies have been performed for other viruses, confirming the potential of MinION technology to aid in the detection of infectious viral agents [45,46]. PRRSV whole genome sequencing (WGS) has been carried out previously using traditional Sanger sequencing [10] and next-generation short-read sequencing platforms [47].…”

Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing

“…As ONT-based native RNA-seq holds the capacity to sequence full-length transcripts, to identify RNA base modifications and to detect molecular heterogeneity in a transcriptome, the technology found widespread attention 16 . Recently, the technology was exploited to sequence viral RNA genomes 17–20 to gain insights into viral and eukaryotic transcriptomes 19,21–23 and to detect RNA isoforms in eukaryotes 24,25 . However, prokaryotic transcriptomes have not been characterized on the genome-wide level by native RNA-seq approaches so far as prokaryotic RNAs lack a poly(A) tail which is required to capture the RNA and feed it into the nanopore.…”