Subgenomic RNAs as molecular indicators of asymptomatic SARS-CoV-2 infection

  1. Chee Hong Wong
  2. Chew Yee Ngan
  3. Rachel L. Goldfeder
  4. Jennifer Idol
  5. Chris Kuhlberg
  6. Rahul Maurya
  7. Kevin Kelly
  8. Gregory Omerza
  9. Nicholas Renzette
  10. Francine De Abreu
  11. Lei Li
  12. Frederick A. Browne
  13. Edison T. Liu
  14. Chia-Lin Wei

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Abstract

In coronaviridae such as SARS-CoV-2, subgenomic RNAs (sgRNA) are replicative intermediates, therefore, their abundance and structures could infer viral replication activity and severity of host infection. Here, we systematically characterized the sgRNA expression and their structural variation in 81 clinical specimens collected from symptomatic and asymptomatic individuals with a goal of assessing viral genomic signatures of disease severity. We demonstrated the highly coordinated and consistent expression of sgRNAs from individuals with robust infections that results in symptoms, and found their expression is significantly repressed in the asymptomatic infections, indicating that the ratio of sgRNAs to genomic RNA (sgRNA/gRNA) is highly correlated with the severity of the disease. Using long read sequencing technologies to characterize full-length sgRNA structures, we also observed widespread deletions in viral RNAs, and identified unique sets of deletions preferentially found primarily in symptomatic individuals, with many likely to confer changes in SARS-CoV-2 virulence and host responses. Furthermore, based on the sgRNA structures, the frequently occurred structural variants in SARS-CoV-2 genomes serves as a mechanism to further induce SARS-CoV-2 proteome complexity. Taken together, our results show that differential sgRNA expression and structural mutational burden both appear to be correlated with the clinical severity of SARS-CoV-2 infection. Longitudinally monitoring sgRNA expression and structural diversity could further guide treatment responses, testing strategies, and vaccine development.

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  1. ScreenIT

    SciScore for 10.1101/2021.02.06.430041: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablePatient age ranged from 18 to 97 years (median 67 years); 35 were male and 45 female (ST. 1).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Genomic RNA and canonical sgRNA abundance in Vero cell: DNBseq RNA sequencing data of SARS-CoV-2-infected Vero cell 21 was downloaded.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    They were aligned to the SAR-Cov2 reference (MN908947.3) with STAR 48 (v2.7.3a) with many switches to completely turn off the penalties of non-canonical eukaryotic splicing as documented 21: “--outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outSJfilterOverhangMin 12 12 12 12 --outSJfilterCountUniqueMin 1 1 1 1 -- outSJfilterCountTotalMin 1 1 1 1 --outSJfilterDistToOtherSJmin 0 0 0 0 -- outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --scoreGapNoncan −4 --scoreGapATAC −4 --chimOutType Junctions WithinBAM HardClip --chimScoreJunctionNonGTAG 0 --alignSJstitchMismatchNmax −1 −1 −1 −1 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000”.
    STAR
    suggested: (STAR, RRID:SCR_015899)
    Reads deduplication were performed with UMI-tools (v1.0.1) 49.
    UMI-tools
    suggested: (UMI-tools, RRID:SCR_017048)
    cDNA yield of 160ng-500ng were then underwent SMRTbell library preparation including a DNA damage repair, end repair and A-tailing and finally ligated with Overhang Barcoded Adaptors.
    SMRTbell
    suggested: None

    Results 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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2021.02.06.430041v1 on bioRxiv