Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping

  1. Joel J. Credle
  2. Matthew L Robinson
  3. Jonathan Gunn
  4. Daniel Monaco
  5. Brandon Sie
  6. Alexandra Tchir
  7. Justin Hardick
  8. Xuwen Zheng
  9. Kathryn Shaw-Saliba
  10. Richard E. Rothman
  11. Susan H. Eshleman
  12. Andrew Pekosz
  13. Kasper Hansen
  14. Heba Mostafa
  15. Martin Steinegger
  16. H. Benjamin Larman

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Abstract

The emergence of SARS-CoV-2 has caused the current COVID-19 pandemic with catastrophic societal impact. Because many individuals shed virus for days before symptom onset, and many show mild or no symptoms, an emergent and unprecedented need exists for development and deployment of sensitive and high throughput molecular diagnostic tests. RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) is a highly multiplexed technology for targeted analysis of polyadenylated mRNA, which incorporates sample barcoding for massively parallel analyses. Here we present a more generalized method, capture RASL-seq (“cRASL-seq”), which enables analysis of any targeted pathogen-(and/or host-) associated RNA molecules. cRASL-seq enables highly sensitive (down to ∼1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ∼10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid extraction or reverse transcription, steps that have caused testing bottlenecks associated with other assays. Our simplified workflow additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new surveillance technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.03.130591: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Nasopharyngeal swab specimens: NP swab specimens were collected from patients after informed written consent was obtained, under a protocol approved by the local governing human research protection committee.
    IRB: The Johns Hopkins Influenza Research and Surveillance (JH-CEIRS) program’s human subjects protocol was approved by the Johns Hopkins School of Medicine Institutional Review Board (IRB): IRB90001667 and NIH Division of Microbiology and Infectious Diseases: Protocol 15-0103
    Randomizationnot detected.
    BlindingUnextracted NP swab specimens (n=9) were de-identified, blinded and provided for further analysis.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    The Zika virus isolate was from a patient in Cali, Colombia, and was grown in Vero-E6 cells.
    Vero-E6
    suggested: None

    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:
    The cRASL-seq methodology is not without limitations. While we have demonstrated a sensitivity comparable to single-plex RT-qPCR, the limits of detection are governed by overall sequencing depth, which can be reduced by consumption of reads from highly abundant ligation products (due to a high load of a co-infecting virus for example). However, since the ligation products are all amplified with a high degree of uniformity, simple RNA spike-in or PCR spike-in sequences can be used to determine the lower limit of detection sensitivity for each reaction. Analysis of host transcripts can also be used to assess sample acquisition sufficiency, a known source of false negative test results.29 Another important concern for COVID-19 molecular diagnostics is the turnaround time. When NGS is used to read out the cRASL-seq assay, the testing turnaround time is unlikely to be less than ∼24 hours with currently available instrumentation. Per-sample sequencing cost considerations will favor analysis of large sample batches, which could further increase turnaround times. For large-scale regional and national level surveillance purposes however, an occasional one to two days of self-quarantine while awaiting test results may be acceptable, given the costs and limitations associated with alternative methods. Faster, non-NGS-based readouts of cRASL probe ligation products may also be developed. For example, isothermal amplification, followed by array or test-strip hybridization may prove more a...

    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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  2. Version published to 10.1101/2020.06.03.130591 on bioRxiv