Nanopore Targeted Sequencing for the Accurate and Comprehensive Detection of SARS‐CoV‐2 and Other Respiratory Viruses

  1. Ming Wang
  2. Aisi Fu
  3. Ben Hu
  4. Yongqing Tong
  5. Ran Liu
  6. Zhen Liu
  7. Jiashuang Gu
  8. Bin Xiang
  9. Jianghao Liu
  10. Wen Jiang
  11. Gaigai Shen
  12. Wanxu Zhao
  13. Dong Men
  14. Zixin Deng
  15. Lilei Yu
  16. Wu Wei
  17. Yan Li
  18. Tiangang Liu

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Abstract

The ongoing global novel coronavirus pneumonia COVID‐19 outbreak has engendered numerous cases of infection and death. COVID‐19 diagnosis relies upon nucleic acid detection; however, currently recommended methods exhibit high false‐negative rates and are unable to identify other respiratory virus infections, thereby resulting in patient misdiagnosis and impeding epidemic containment. Combining the advantages of targeted amplification and long‐read, real‐time nanopore sequencing, herein, nanopore targeted sequencing (NTS) is developed to detect SARS‐CoV‐2 and other respiratory viruses simultaneously within 6–10 h, with a limit of detection of ten standard plasmid copies per reaction. Compared with its specificity for five common respiratory viruses, the specificity of NTS for SARS‐CoV‐2 reaches 100%. Parallel testing with approved real‐time reverse transcription‐polymerase chain reaction kits for SARS‐CoV‐2 and NTS using 61 nucleic acid samples from suspected COVID‐19 cases show that NTS identifies more infected patients (22/61) as positive, while also effectively monitoring for mutated nucleic acid sequences, categorizing types of SARS‐CoV‐2, and detecting other respiratory viruses in the test sample. NTS is thus suitable for COVID‐19 diagnosis; moreover, this platform can be further extended for diagnosing other viruses and pathogens.

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  1. Version published to 10.1002/smll.202002169
  2. ScreenIT

    SciScore for 10.1101/2020.03.04.20029538: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The study and use of all records were approved by the Ethics Committee of Hubei Provincial Renmin Hospital (
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Sequencing libraries were constructed using the 1D Ligation Kit (SQK-LSK109; Oxford Nanopore, UK) and sequenced using Oxford Nanopore MinION or GridION.
    MinION
    suggested: (MinION, RRID:SCR_017985)
    Nanopore sequencing data processing: Basecalling and quality assessment for MinION sequencing data were performed using high accuracy mode in Guppy (v. 3.1.5) software; for GridION, the process was conducted using MinKNOW (v. 3.6.5) integrated in the instrument.
    MinKNOW
    suggested: None
    All virus genomic sequences were downloaded from NCBI Refseq FTP and the SARS-CoV-2 genome sequence was added to the BLAST database subsequently.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Several limitations of the current NTS method should be noted. Because the designed amplified fragments are 300–950 bp in length, which constitute suitable lengths for detection by a nanopore platform as nucleic acid fragments < 200 bp cannot be readily detected19, 20, thereby, the sensitivity of NTS for detecting target COVID-19 fragments in highly degraded nucleic acids may be hampered. Additionally, although the turnaround time of NTS is longer than that of qPCR or other possible nucleic acid detection methods (e.g., SHERLOCK12), 6–10 h is considered acceptable for clinical use; moreover, NTS is already the fastest strategy based on sequencing methods to date and can detect variations directly from clinical samples. Whereas the detection throughput of NTS is not high at present, the NTS method can be integrated into widely used automated or semi-automated platforms to improve the detection throughput in the future21-23. In addition, because PCR is included in NTS, processes involving opening the lid of the PCR tubes may cause mutual contamination between samples24, 25. However, this situation also is inevitable in current nucleic acid detection methods (e.g., qPCR) or other nucleic acid detection schemes (e.g., SHERLOCK11, 12 or toehold switch biosensor9, 10) that also involve PCR. The introduction of integration systems or sealed devices such as microfluidics may avoid this situation26, 27. At present, our processes of sequencing data analysis and interpretation of result...

    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.

    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.

  3. Version published to 10.1101/2020.03.04.20029538 on medRxiv