Dataset Information

Direct nanopore RNA sequencing of umbra-like virus-infected plants reveals long non-coding RNAs, specific cleavage sites, D-RNAs, foldback RNAs, and temporal- and tissue-specific profiles.

ABSTRACT: The traditional view of plus (+)-strand RNA virus transcriptomes is that infected cells contain a limited variety of viral RNAs, such as full-length (+)-strand genomic RNA(s), (-)-strand replication intermediate(s), 3' co-terminal subgenomic RNA(s), and viral recombinant defective (D)-RNAs. To ascertain the full complement of viral RNAs associated with the simplest plant viruses, long-read direct RNA nanopore sequencing was used to perform transcriptomic analyses of two related umbra-like viruses: citrus yellow vein-associated virus (CY1) from citrus and CY2 from hemp. Analysis of different timepoints/tissues in CY1- and CY2-infected Nicotiana benthamiana plants and CY2-infected hemp revealed: (i) three 5' co-terminal RNAs of 281 nt, 442 nt and 671 nt, each generated by a different mechanism; (ii) D-RNA populations containing the 671 fragment at their 5'ends; (iii) many full-length genomic RNAs and D-RNAs with identical 3'end 61 nt truncations; (iv) virtually all (-)-strand reads missing 3 nt at their 3' termini; (v) (±) foldback RNAs comprising about one-third of all (-)-strand reads and (vi) a higher proportion of full-length gRNAs in roots than in leaves, suggesting that roots may be functioning as a gRNA reservoir. These findings suggest that viral transcriptomes are much more complex than previously thought.

SUBMITTER: Johnson PZ

PROVIDER: S-EPMC11327873 | biostudies-literature | 2024 Sep

REPOSITORIES: biostudies-literature

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Direct nanopore RNA sequencing of umbra-like virus-infected plants reveals long non-coding RNAs, specific cleavage sites, D-RNAs, foldback RNAs, and temporal- and tissue-specific profiles.

Johnson Philip Z PZ Needham Jason M JM Lim Natalie K NK Simon Anne E AE

NAR genomics and bioinformatics 20240816 3

The traditional view of plus (+)-strand RNA virus transcriptomes is that infected cells contain a limited variety of viral RNAs, such as full-length (+)-strand genomic RNA(s), (-)-strand replication intermediate(s), 3' co-terminal subgenomic RNA(s), and viral recombinant defective (D)-RNAs. To ascertain the full complement of viral RNAs associated with the simplest plant viruses, long-read direct RNA nanopore sequencing was used to perform transcriptomic analyses of two related umbra-like viruse ...[more]

PMID: 39157584

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Project description:Recent studies have disclosed the genome, transcriptome, and epigenetic compositions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the effect of viral infection on gene expression of the host cells. It has been demonstrated that, besides the major canonical transcripts, the viral genome also codes for noncanonical RNA molecules. While the structural characterizations have revealed a detailed transcriptomic architecture of the virus, the kinetic studies provided poor and often misleading results on the dynamics of both the viral and host transcripts due to the low temporal resolution of the infection event and the low virus/cell ratio (multiplicity of infection [MOI] = 0.1) applied for the infection. It has never been tested whether the alteration in the host gene expressions is caused by aging of the cells or by the viral infection. In this study, we used Oxford Nanopore's direct cDNA and direct RNA sequencing methods for the generation of a high-coverage, high temporal resolution transcriptomic dataset of SARS-CoV-2 and of the primate host cells, using a high infection titer (MOI = 5). Sixteen sampling time points ranging from 1 to 96 hours with a varying time resolution and 3 biological replicates were used in the experiment. In addition, for each infected sample, corresponding noninfected samples were employed. The raw reads were mapped to the viral and to the host reference genomes, resulting in 49,661,499 mapped reads (54,62 Gbs). The genome of the viral isolate was also sequenced and phylogenetically classified. This dataset can serve as a valuable resource for profiling the SARS-CoV-2 transcriptome dynamics, the virus-host interactions, and the RNA base modifications. Comparison of expression profiles of the host gene in the virally infected and in noninfected cells at different time points allows making a distinction between the effect of the aging of cells in culture and the viral infection. These data can provide useful information for potential novel gene annotations and can also be used for studying the currently available bioinformatics pipelines.

Project description:BackgroundLong-read sequencing platforms offered by Oxford Nanopore Technologies (ONT) allow native DNA containing epigenetic modifications to be directly sequenced, but can be limited by lower per-base accuracies. A key step post-sequencing is basecalling, the process of converting raw electrical signals produced by the sequencing device into nucleotide sequences. This is challenging as current basecallers are primarily based on mixtures of model species for training. Here we utilise both ONT PromethION and higher accuracy PacBio Sequel II HiFi sequencing on two plants, Phebalium stellatum and Xanthorrhoea johnsonii, to train species-specific basecaller models with the aim of improving per-base accuracy. We investigate sequencing accuracies achieved by ONT basecallers and assess accuracy gains by training single-species and species-specific basecaller models. We also evaluate accuracy gains from ONT's improved flowcells (R10.4, FLO-PRO112) and sequencing kits (SQK-LSK112). For the truth dataset for both model training and accuracy assessment, we developed highly accurate, contiguous diploid reference genomes with PacBio Sequel II HiFi reads.ResultsBasecalling with ONT Guppy 5 and 6 super-accurate gave almost identical results, attaining read accuracies of 91.96% and 94.15%. Guppy's plant-specific model gave highly mixed results, attaining read accuracies of 91.47% and 96.18%. Species-specific basecalling models improved read accuracy, attaining 93.24% and 95.16% read accuracies. R10.4 sequencing kits also improve sequencing accuracy, attaining read accuracies of 95.46% (super-accurate) and 96.87% (species-specific).ConclusionsThe use of a single mixed-species basecaller model, such as ONT Guppy super-accurate, may be reducing the accuracy of nanopore sequencing, due to conflicting genome biology within the training dataset and study species. Training of single-species and genome-specific basecaller models improves read accuracy. Studies that aim to do large-scale long-read genotyping would primarily benefit from training their own basecalling models. Such studies could use sequencing accuracy gains and improving bioinformatics tools to improve study outcomes.

Dataset Information

Direct nanopore RNA sequencing of umbra-like virus-infected plants reveals long non-coding RNAs, specific cleavage sites, D-RNAs, foldback RNAs, and temporal- and tissue-specific profiles.

Publications

Direct nanopore RNA sequencing of umbra-like virus-infected plants reveals long non-coding RNAs, specific cleavage sites, D-RNAs, foldback RNAs, and temporal- and tissue-specific profiles.

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