Pervasive generation of non-canonical subgenomic RNAs by SARS-CoV-2
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
Background
SARS-CoV-2, a positive-sense RNA virus in the family Coronaviridae , has caused a worldwide pandemic of coronavirus disease 2019 or COVID-19. Coronaviruses generate a tiered series of subgenomic RNAs (sgRNAs) through a process involving homology between transcriptional regulatory sequences (TRS) located after the leader sequence in the 5′ UTR (the TRS-L) and TRS located near the start of ORFs encoding structural and accessory proteins (TRS-B) near the 3′ end of the genome. In addition to the canonical sgRNAs generated by SARS-CoV-2, non-canonical sgRNAs (nc-sgRNAs) have been reported. However, the consistency of these nc-sgRNAs across viral isolates and infection conditions is unknown. The comprehensive definition of SARS-CoV-2 RNA products is a key step in understanding SARS-CoV-2 pathogenesis.
Methods
Here, we report an integrative analysis of eight independent SARS-CoV-2 transcriptomes generated using three sequencing strategies, five host systems, and seven viral isolates. Read-mapping to the SARS-CoV-2 genome was used to determine the 5′ and 3′ coordinates of all junctions in viral RNAs identified in these samples.
Results
Using junctional abundances, we show nc-sgRNAs make up as much as 33% of total sgRNAs in cell culture models of infection, are largely consistent in abundance across independent transcriptomes, and increase in abundance over time during infection. By assessing the homology between sequences flanking the 5′ and 3′ junction points, we show that nc-sgRNAs are not associated with TRS-like homology. By incorporating read coverage information, we find strong evidence for subgenomic RNAs that contain only 5′ regions of ORF1a. Finally, we show that non-canonical junctions change the landscape of viral open reading frames.
Conclusions
We identify canonical and non-canonical junctions in SARS-CoV-2 sgRNAs and show that these RNA products are consistently generated by many independent viral isolates and sequencing approaches. These analyses highlight the diverse transcriptional activity of SARS-CoV-2 and offer important insights into SARS-CoV-2 biology.
Article activity feed
-
-
-
SciScore for 10.1101/2020.04.28.066951: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources The exceptions are the transcriptomes from Finkel et al. and Emanuel et al. - because their read lengths were very short (60 and 72 bases in length respectively), mapping was conducted with STAR (31) using settings detailed in star.sh. STARsuggested: (STAR, RRID:SCR_015899)Analysis and visualization of junctions: The 5’ and 3’ coordinates of each junction were processed in R (32), and figures generated with ggplot2 (33). ggplot2suggested: (ggplot2, RRID:SCR_014601)(Command: … SciScore for 10.1101/2020.04.28.066951: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources The exceptions are the transcriptomes from Finkel et al. and Emanuel et al. - because their read lengths were very short (60 and 72 bases in length respectively), mapping was conducted with STAR (31) using settings detailed in star.sh. STARsuggested: (STAR, RRID:SCR_015899)Analysis and visualization of junctions: The 5’ and 3’ coordinates of each junction were processed in R (32), and figures generated with ggplot2 (33). ggplot2suggested: (ggplot2, RRID:SCR_014601)(Command: samtools depth -aa -d0 file.bam > file.cov). samtoolssuggested: (SAMTOOLS, RRID:SCR_002105)ORFs were called directly from each transcript using Prodigal (37), using parameters listed in prodigal.sh. Prodigalsuggested: (Prodigal, RRID:SCR_011936)DIAMOND parameters are described in diamond.sh and include an E-value threshold of 10. DIAMONDsuggested: (DIAMOND, RRID:SCR_009457)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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.
-