Long-read sequencing of SARS-CoV-2 reveals novel transcripts and a diverse complex transcriptome landscape

  1. Jennifer Li-Pook-Than
  2. Selene Banuelos
  3. Alexander Honkala
  4. Malaya K. Sahoo
  5. Benjamin A. Pinsky
  6. Michael P. Snyder

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Abstract

Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2 (COVID-19), is a positive single-stranded RNA virus with a 30 kb genome that is responsible for the current pandemic. To date, the genomes of global COVID-19 variants have been primarily characterized via short-read sequencing methods. Here, we devised a long-read RNA (IsoSeq) sequencing approach to characterize the COVID-19 transcript landscape and expression of its ∼27 coding regions. Our analysis identified novel COVID-19 transcripts including a) a short ∼65-70 nt 5’-UTR fused to various downstream ORFs encoding accessory proteins such as the envelope, ORF 8, and ORF 9 (nucleocapsid) proteins, that are relatively highly expressed, b) novel SNVs that are differentially expressed, whereby a subset are suggestive of partial RNA editing events, and c) SNVs at functional sites, whereby at least one is associated with a differentially expressed spike protein isoform. These previously uncharacterized COVID-19 isoforms, expressed genes, and gene variants were corroborated using ddPCR. Understanding this transcriptional complexity may help provide insight into the biology and pathogenicity of SARS-CoV-2 compared to other coronaviruses.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.05.434150: (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 variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Two SARS-CoV-2 RNA synthetic controls were utilized and run in parallel with the Pools A and B for this work: 1) a Twist Bioscience SARS-CoV-2 (MT199235 – USA/CA9/2020) synthetic control containing six non-overlapping 5 kb fragments and 2) SARS-CoV-2 RNAs isolated from Vero E6 mammalian cells transfected with a 30 kb cloned isolate (MN985325.1 −2019-nCoV/USA-WA1/2020) supplied by ATCC (ATCC® VR-1986D™).
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    The quality of the total RNA was tested using the Agilent Bioanalyzer 2100
    Agilent Bioanalyzer
    suggested: None
    StringTie v2.1.4 usage included long reads processing (-L) which also enforces -s 1.5 -g 0 (default:false) and reference annotation (-G) used for guiding the assembly process (GTF/GFF3).
    StringTie
    suggested: (StringTie , RRID:SCR_016323)

    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 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

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.03.05.434150v1 on bioRxiv