Direct RNA Sequencing for Complete Viral Genomes

ABSTRACTCurrent technologies for targeted characterization and manipulation of viral RNA primarily involve amplification or ultracentrifugation with isopycnic gradients of viral particles to decrease host RNA background. The former strategy is non-compatible for characterizing properties innate to RNA strands such as secondary structure, RNA-RNA interactions, and also for nanopore direct RNA sequencing involving the sequencing of native RNA strands. The latter strategy, ultracentrifugation, causes loss in genomic information due to its inability to retrieve unassembled viral RNA. To address this, we developed a novel application of current nucleic acid hybridization technologies for direct characterization of RNA. In particular, we modified a current enrichment protocol to capture whole viral native RNA genomes for downstream RNA assays to circumvent the abovementioned problems. This technique involves hybridization of biotinylated baits at 500 nucleotides (nt) intervals, stringent washes and release of free native RNA strands using DNase I treatment, with a turnaround time of about 6 h 15 min. RT-qPCR was used as the primary proof of concept that capture-based purification indeed removes host background. Subsequently, capture-based purification was applied to direct RNA sequencing as proof of concept that capture-based purification can be coupled with downstream RNA assays. We report that this protocol was able to successfully purify viral RNA by 561-791 fold. We also report that application of this protocol to direct RNA sequencing yielded a reduction in human host RNA background by 1580 fold, a 99.91% recovery of viral genome with at least 15x coverage, and a mean coverage across the genome of 120x. This report is, to the best of our knowledge, the first description of a capture-based purification method for assays that involve direct manipulation or characterisation of native RNA. This report also describes a successful application of capture-based purification as a direct RNA sequencing strategy that addresses certain limitations of current strategies in sequencing RNA viral genomes.

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Direct RNA sequencing reveals SARS-CoV-2 m6A sites and possible differential DRACH motif methylation among variants

10.1101/2021.08.24.457397 ◽

2021 ◽

Author(s):

João H. Campos ◽

Juliana T. Maricato ◽

Carla T. Braconi ◽

Fernando Antoneli ◽

Luiz Mario R. Janini ◽

...

Keyword(s):

Rna Sequencing ◽

Untranslated Region ◽

Proper Function ◽

Viral Genomes ◽

Chemical Treatments ◽

Positive Sense ◽

Genome Rnas ◽

Almost All ◽

Fourth Position ◽

Initial Exploration

ABSTRACTThe causative agent of COVID-19 pandemic, the SARS-CoV-2 coronavirus, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 100 modified bases that are essential for proper function. Among internal modified bases, the N6-methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced so far are in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because incorporation of dNTPs hides RNA base modifications. Here, in this perspective paper, we present an initial exploration of the Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3’-untranslated region. We identified 15 m6A methylated positions, of which, 6 are in ORF N. Also, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants we show that variants Beta and Eta have a fourth position C>U change in DRACH at 28,884b that could affect methylation. The Nanopore technology offers a unique opportunity for the study of viral epitranscriptomics. This technique is PCR-free and is not sequencing-by-synthesis, therefore, no PCR bias and synthesis errors are introduced. The modified bases are preserved and assessed directly with no need for chemical treatments or antibodies. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.

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Native RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen

10.1101/373522 ◽

2018 ◽

Cited By ~ 8

Author(s):

Daniel P. Depledge ◽

Kalanghad Puthankalam Srinivas ◽

Tomohiko Sadaoka ◽

Devin Bready ◽

Yasuko Mori ◽

...

Keyword(s):

Rna Sequencing ◽

Rna Splicing ◽

Transcription Initiation ◽

Productive Infection ◽

Rna Modifications ◽

Viral Pathogen ◽

Viral Genomes ◽

Alternative Transcription ◽

Nanopore Arrays ◽

Simplex Virus

ABSTRACTViral genomes exhibit a higher gene density and more diversified transcriptome than the host cell. Coding potential is maximized through the use of multiple reading frames, placement of genes on opposing strands, inefficient or modified use of termination signals, and the deployment of complex alternative splicing patterns. As a consequence, detailed characterization of viral transcriptomes by conventional methods can be challenging. Full length native RNA sequencing (nRNA-seq) using nanopore arrays offers an exciting alternative. Individual transcripts are sequenced directly, without the biases inherent to the recoding or amplification steps included in other sequencing methodologies. nRNA-seq simplifies the detection of variation brought about by RNA splicing, use of alternative transcription initiation and termination sites, and other RNA modifications. Here we use nRNA-seq to profile the herpes simplex virus type 1 transcriptome during early and late stages of productive infection of primary cells. We demonstrate the effectiveness of the approach and identify a novel class of intergenic transcripts, including an mRNA that accumulates late in infection that codes for a novel fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L.

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Going the Distance: Optimizing RNA-Seq Strategies for Transcriptomic Analysis of Complex Viral Genomes

Journal of Virology ◽

10.1128/jvi.01342-18 ◽

2018 ◽

Vol 93 (1) ◽

Cited By ~ 7

Author(s):

Daniel P. Depledge ◽

Ian Mohr ◽

Angus C. Wilson

Keyword(s):

Rna Sequencing ◽

Transcriptome Profiling ◽

Rna Seq ◽

Dna Viruses ◽

Viral Genomes ◽

Sequencing Technologies ◽

Long Read ◽

Abundance And Diversity ◽

Sequencing Platforms ◽

Rna And Dna

ABSTRACTTranscriptome profiling has become routine in studies of many biological processes. However, the favored approaches such as short-read Illumina RNA sequencing are giving way to long-read sequencing platforms better suited to interrogating the complex transcriptomes typical of many RNA and DNA viruses. Here, we provide a guide—tailored to molecular virologists—to the ins and outs of viral transcriptome sequencing and discuss the strengths and weaknesses of the major RNA sequencing technologies as tools to analyze the abundance and diversity of the viral transcripts made during infection.

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