《2019 Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants》由会员分享，可在线阅读，更多相关《2019 Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants（11页珍藏版）》请在金锄头文库上搜索。
1、Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis Adrian Viehweger,1,2,5Sebastian Krautwurst,1,2,5Kevin Lamkiewicz,1,2 Ramakanth Madhugiri,3John Ziebuhr,2,3Martin Hlzer,1,2and Manja Marz1,2,4 1RNA Bioi
2、nformatics and High-Throughput Analysis, Friedrich Schiller University Jena, 07743 Jena, Germany;2European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany; 3Institute of Medical Virology, Justus Liebig University Gieen, 35390 Gieen, Germany; 4Leibniz Institute on
3、 AgingFritz Lipmann Institute, 07743 Jena, Germany 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
4、of closely related viruses, so-called “quasispecies.” Standard (short-read) sequencing technologies are ill-suit- ed 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, weused afull-length,
5、 directRNA 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 recons
6、tructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long readsthat did not require an assemblystep, wewere able to identify,in infected cells, diverseandnovelHCoV-229EsgRNAsthatremaintobecharacterized.Also,theDRSapproach,whichcirc
7、umventsreverse 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 byshowing the feasibilityof intra-sample haplotype separation. Eventhough several technical challenges remain to be a
8、ddressed 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. Supplemental material is availabl
9、e for this article. Coronaviruses (subfamily Coronavirinae, family Coronaviridae, or- der Nidovirales) are enveloped positive-sense (+) single-stranded (ss)RNAvirusesthatinfectavarietyofmammalianandavianhosts and are of significant medical and economic importance, as illus- trated by recent zoonotic
10、 transmissions from diverse animal hosts to humans (Vijay and Perlman 2016; Menachery et al. 2017). The genomesizes of coronaviruses (30 kb)exceedthose of most other RNAviruses.Coronavirusesuseaspecialmechanismcalleddiscon- tinuous extension of minus strands (Sawicki and Sawicki 1995, 1998) to produ
11、ce a nested set of 5- and 3-coterminal subgenomic (sg) mRNAs that carry a common 5leader sequence that is identi- cal to the 5-end of the viral genome (Zuniga et al. 2004; Sawicki et al. 2007). These sg mRNAs contain a different number of open reading frames (ORFs) that encode the viral structural p
12、roteins and several accessory proteins. With very few exceptions, only the 5-located ORF (which is absent from the next smaller sg mRNA) is translated into protein (Fig. 1). In HCoV-229Einfected cells, a total of seven major viral RNAs are produced. The viral genome is also referred to as mRNA 1 bec
13、ause it has an mRNA function. In its 5-terminal region, the genome RNA contains two large ORFs, 1a and 1b, that encode the viral replicase polyproteins 1a and 1ab. mRNAs 2, 4, 5, 6, and 7 are used to produce the S protein, accessory protein 4, E pro- tein, M protein, and N protein, respectively. The
14、 5-region of mRNA 3 contains a truncated fragment of ORF S, which is consid- ered defective. Although this sg RNA has been consistently identi- fied in HCoV-229Einfected cells, its mRNA function has been disputed, and there is currently no evidence that this RNA is trans- lated into protein (Schreib
15、er et al. 1989; Raabe et al. 1990; Thiel et al. 2003). Likemanyother +RNAviruses,coronaviruses show high rates of recombination (Lai 1992; Liao and Lai 1992; Furuya et al. 1993). In fact, the mechanism to produce 5leader-containing sg mRNAs represents a prime example for copy-choice RNA recombinatio
16、n that, in this particular case, is guided by complex RNARNA inter- actions involving the transcription-regulating sequence (TRS) core sequences and likely requires additional interactions of viral pro- teinswithspecificRNAsignals.Inothervirussystems,RNArecom- bination has been shown to generate “transcriptional units” that control the expression of individual components of the genome (Holmes 2009). The mechanisms involved in viral RNA recombi- nation are diverse and may even extend to nonreplic
