Rapid High-Throughput Whole-Genome Sequencing of SARS-CoV-2 by Using One-Step Reverse Transcription-PCR Amplification with an Integrated Microfluidic System and Next-Generation Sequencing

  1. Tao Li
  2. Hye Kyung Chung
  3. Papa K. Pireku
  4. Brett F. Beitzel
  5. Mark A. Sanborn
  6. Cynthia Y. Tang
  7. Richard D. Hammer
  8. Detlef Ritter
  9. Xiu-Feng Wan
  10. Irina Maljkovic Berry
  11. Jun Hang

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Abstract

The long-lasting global COVID-19 pandemic demands timely genomic investigation of SARS-CoV-2 viruses. Here, we report a simple and efficient workflow for whole-genome sequencing utilizing one-step reverse transcription-PCR (RT-PCR) amplification on a microfluidic platform, followed by MiSeq amplicon sequencing.

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  1. Version published to 10.1128/jcm.02784-20
  2. ScreenIT

    SciScore for 10.1101/2020.11.04.369165: (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

    Experimental Models: Organisms/Strains
    SentencesResources
    Serially diluted in vitro transcripts (IVT), corresponding to nucleotide region 15431 to 15530 of NC_045512.2 for strain Wuhan-Hu-1, were prepared and used as real-time RT-PCR standards for quantification of genome equivalent copy number (GE) of SARS-CoV-2 RNA.
    Wuhan-Hu-1
    suggested: None
    Software and Algorithms
    SentencesResources
    A minimum Phred base quality score of 35 and a minimum depth of coverage of 10 were utilized as the configuration parameters for this project.
    Phred
    suggested: (Phred, RRID:SCR_001017)
    Geneious R10 software, integrated genome viewer (IGV) (19), and MEGA version 7 (20) were used for quality control of ambiguous calls, insertions, deletions and primer-induced mutations.
    MEGA
    suggested: (Mega BLAST, RRID:SCR_011920)
    MEGA7 was used to align genomes using default parameters with Multiple Sequence Comparison by Log Expectation (MUSCLE) (21).
    MUSCLE
    suggested: (MUSCLE, RRID:SCR_011812)

    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: We detected the following sentences addressing limitations in the study:
    In consequence, Fluidigm amplification based whole genome sequencing has limitations in sequencing low titer samples. For RNA samples with 1000 GE/μl or lower concentration, multiplex RT-PCR based methods or SARS-CoV-2 hybridization-based enrichment method might be a more suitable choice for whole genome sequencing (26–28). The addition of a first-strand cDNA synthesis step prior to Fluidigm amplification, with a change of Fluidigm thermocycling program from RT-PCR to PCR, may help increase genome coverage for low titer samples. Many, if not all, COVID-19 specimens are tested with quantitative molecular tests and the Ct values or equivalent titer scores are readily available for deciding whether one-step Fluidigm amplification-based genome sequencing protocol is appropriate. Using this method, very few sequence assembly errors were observed throughout the tested SARS-CoV-2 sample genomes. These errors might be due to PCR, sequencing or basecalling algorithm errors, as well as due to normal fluctuations in the minor variant frequencies between the sample aliquots. When assembled sequences show potentially significant nucleotide alterations or indels, thorough examination of the data quality processing, primer trimming, curation of sequence assembly, and detailed laboratory records are needed. Whenever possible, running replicate samples, repeating the experiment entirely, or using other methods are important to validate genome variations and minor variants. In conclusion, our ...

    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.

  3. Version published to 10.1101/2020.11.04.369165v1 on bioRxiv