Nanopore ReCappable sequencing maps SARS-CoV-2 5′ capping sites and provides new insights into the structure of sgRNAs
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Abstract
The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5′ cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.
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SciScore for 10.1101/2021.11.24.469860: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources For secondary (P2) virus stock, Vero E6 cells seeded into 25 cm2 tissue culture flasks were infected with 0.5 mL of P1 stored aliquot, and infected cells and supernatant were collected 48 hours post-infection and stored at −80° C. Vero E6suggested: NoneInfection experiments: Caco2 and CaLu3 cells were seeded on 25 cm2 tissue culture flasks until 80% confluency. CaLu3suggested: NoneResults from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer:…SciScore for 10.1101/2021.11.24.469860: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources For secondary (P2) virus stock, Vero E6 cells seeded into 25 cm2 tissue culture flasks were infected with 0.5 mL of P1 stored aliquot, and infected cells and supernatant were collected 48 hours post-infection and stored at −80° C. Vero E6suggested: NoneInfection experiments: Caco2 and CaLu3 cells were seeded on 25 cm2 tissue culture flasks until 80% confluency. CaLu3suggested: NoneResults from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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