LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding

  1. Jonathan L. Schmid-Burgk
  2. Ricarda M. Schmithausen
  3. David Li
  4. Ronja Hollstein
  5. Amir Ben-Shmuel
  6. Ofir Israeli
  7. Shay Weiss
  8. Nir Paran
  9. Gero Wilbring
  10. Jana Liebing
  11. David Feldman
  12. Mikołaj Słabicki
  13. Bärbel Lippke
  14. Esther Sib
  15. Jacob Borrajo
  16. Jonathan Strecker
  17. Julia Reinhardt
  18. Per Hoffmann
  19. Brian Cleary
  20. Michael Hölzel
  21. Markus M. Nöthen
  22. Martin Exner
  23. Kerstin U. Ludwig
  24. Aviv Regev
  25. Feng Zhang

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Abstract

The ongoing SARS-CoV-2 pandemic has already caused devastating losses. Exponential spread can be slowed by social distancing and population-wide isolation measures, but those place a tremendous burden on society, and, once lifted, exponential spread can re-emerge. Regular population-scale testing, combined with contact tracing and case isolation, should help break the cycle of transmission, but current detection strategies are not capable of such large-scale processing. Here we present a protocol for LAMP-Seq, a barcoded Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method that is highly scalable. Individual samples are stabilized, inactivated, and amplified in three isothermal heat steps, generating barcoded amplicons that can be pooled and analyzed en masse by sequencing. Using unique barcode combinations per sample from a compressed barcode space enables extensive pooling, potentially further reducing cost and simplifying logistics. We validated LAMP-Seq on 28 clinical samples, empirically optimized the protocol and barcode design, and performed initial safety evaluation. Relying on world-wide infrastructure for next-generation sequencing, and in the context of population-wide sample collection, LAMP-Seq could be scaled to analyze millions of samples per day.

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  1. Version published to 10.1101/2020.04.06.025635v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.04.06.025635: (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
    The virus was propagated and titrated on VERO-E6 cells (ATCC CRL-1586).
    VERO-E6
    suggested: None
    Vesicular stomatitis virus (VSV) serotype Indiana, kindly provided by Eran Bacharach (Tel-Aviv University, Israel), was propagated and titrated on Vero cells (ATCC CCL-81).
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    Code and Data Availability: The LAMP-Seq Inspector tool for processing raw LAMP-Seq data is available at: http://manuscript.lamp-seq.org/Inspector.htm. Python scripts for designing the error-correcting barcodes are available at: https://github.com/feldman4/dna-barcodes.
    Python
    suggested: (IPython, RRID:SCR_001658)
    Jupyter Notebooks for numerical simulations and MATLAB scripts for figure generation are available at: https://github.com/dbli2000/SARS-CoV2-Bloom-Filter.
    MATLAB
    suggested: (MATLAB, RRID:SCR_001622)

    Results from OddPub: Thank you for sharing your code.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    A potential limitation of the presented approach is that skewing of sample representation at the pooling stage may affect testing sensitivity. Although the LAMP reaction saturates in positive samples largely independent of template concentrations (Fig. 1E), thus equalizing the representation across positive samples in an advantageous manner, the reaction might also add random skewing to pooled samples when scaling to hundreds of thousands of samples; however, preliminary modeling suggests that pooling 100,000 samples per NextSeq run offers robust detection (Supplementary Note 1 and 2). LAMP-Seq requires low amounts of consumables with the exception of three proprietary enzymes and buffer compositions; however, these enzymes could be mass-produced using E. coli or replaced by open-source alternatives. The established LAMP-Seq protocol used cotton-woodswabs that are available in mass quantities for < 5 ct. each. The synthesis cost of the barcode primer library is low overall (5,000 USD total for 960 barcodes, < 10 ct. per sample), leaving point-of-test infrastructure, logistics, and robotics as putative cost driving items. Once successfully established, however, this infrastructure could rapidly counter future waves of viral spread or pandemic outbreaks. Of note, LAMP-Seq could uniquely allow multiplexing multiple targets (of different viruses) to enable scalable differential diagnostics. Broadly similar approaches of barcoded isothermal amplification methods have been independ...

    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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  3. Version published to 10.1101/2020.04.06.025635v1 on bioRxiv