Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis

  1. Rowena A. Bull
  2. Thiruni N. Adikari
  3. James M. Ferguson
  4. Jillian M. Hammond
  5. Igor Stevanovski
  6. Alicia G. Beukers
  7. Zin Naing
  8. Malinna Yeang
  9. Andrey Verich
  10. Hasindu Gamaarachchi
  11. Ki Wook Kim
  12. Fabio Luciani
  13. Sacha Stelzer-Braid
  14. John-Sebastian Eden
  15. William D. Rawlinson
  16. Sebastiaan J. van Hal
  17. Ira W. Deveson

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Abstract

Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, here we perform viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. We report that, despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives.

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  1. Version published to 10.1038/s41467-020-20075-6
  2. Version published to 10.1101/2020.08.04.236893v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.08.04.236893: (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.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Trimmed alignments were converted to pileup format using samtools mpileup (v1.9)38, with anomalous read pairs retained (--count-orphans), base alignment quality disabled (--no-BAQ) and all bases considered, regardless of PHRED quality (--min-BQ 0).
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    Up to twelve samples were multiplexed on a FLO-FLG001, FLO-MIN106D or FLO-PRO002 or flow-cell and sequenced on a GridION X5 or PromethION P24 device, respectively.
    PromethION
    suggested: (PromethION, RRID:SCR_017987)
    The RAMPART software package39 was used to monitor sequencing performance in real-time, with runs proceeding until a minimum ~200-fold coverage was achieved across all amplicons.
    RAMPART
    suggested: (Rampart, RRID:SCR_016742)
    Variant candidates identified by Illumina/ONT could then be considered concordant based on matching genome position, reference base and alternative base/s.
    Illumina/ONT
    suggested: None

    Results from OddPub: Thank you for sharing your code and 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.

    About SciScore

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  4. Version published to 10.1101/2020.08.04.236893v1 on bioRxiv