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

Joel J. Credle, View ORCID ProfileMatthew L Robinson, Jonathan Gunn, Daniel Monaco, Brandon Sie, Alexandra Tchir, Justin Hardick, Xuwen Zheng, Kathryn Shaw-Saliba, Richard E. Rothman, Susan H. Eshleman, Andrew Pekosz, Kasper Hansen, Heba Mostafa, View ORCID ProfileMartin Steinegger, H. Benjamin Larman
doi: https://doi.org/10.1101/2020.06.03.130591
Joel J. Credle
1Institute for Cell Engineering, Immunology Division, Department of Pathology, Johns Hopkins University, Baltimore, MD, USA
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Matthew L Robinson
2Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
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Jonathan Gunn
1Institute for Cell Engineering, Immunology Division, Department of Pathology, Johns Hopkins University, Baltimore, MD, USA
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Daniel Monaco
1Institute for Cell Engineering, Immunology Division, Department of Pathology, Johns Hopkins University, Baltimore, MD, USA
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Brandon Sie
1Institute for Cell Engineering, Immunology Division, Department of Pathology, Johns Hopkins University, Baltimore, MD, USA
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Alexandra Tchir
1Institute for Cell Engineering, Immunology Division, Department of Pathology, Johns Hopkins University, Baltimore, MD, USA
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Justin Hardick
2Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
3Department of Emergency Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
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Xuwen Zheng
1Institute for Cell Engineering, Immunology Division, Department of Pathology, Johns Hopkins University, Baltimore, MD, USA
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Kathryn Shaw-Saliba
3Department of Emergency Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
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Richard E. Rothman
2Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
3Department of Emergency Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
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Susan H. Eshleman
4Division of Transfusion Medicine, Department of Pathology, Johns Hopkins University, Baltimore, MD, USA
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Andrew Pekosz
5W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
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Kasper Hansen
6Department of Biostatistics, Johns Hopkins University, Baltimore, MD, USA
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Heba Mostafa
7Division of Medical Microbiology, Department of Pathology, Johns Hopkins University, Baltimore, MD, USA
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Martin Steinegger
8Biological Sciences & Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea
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  • For correspondence: hlarman1@jhmi.edu martin.steinegger@snu.ac.kr
H. Benjamin Larman
1Institute for Cell Engineering, Immunology Division, Department of Pathology, Johns Hopkins University, Baltimore, MD, USA
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  • For correspondence: hlarman1@jhmi.edu martin.steinegger@snu.ac.kr
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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.

Competing Interest Statement

J.J.C. and H.B.L. are listed as inventors on a patent describing the cRASL-seq method. H.B.L. has founded a company to license and commercialize oligonucleotide probe ligation related technologies.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted June 03, 2020.
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Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping
Joel J. Credle, Matthew L Robinson, Jonathan Gunn, Daniel Monaco, Brandon Sie, Alexandra Tchir, Justin Hardick, Xuwen Zheng, Kathryn Shaw-Saliba, Richard E. Rothman, Susan H. Eshleman, Andrew Pekosz, Kasper Hansen, Heba Mostafa, Martin Steinegger, H. Benjamin Larman
bioRxiv 2020.06.03.130591; doi: https://doi.org/10.1101/2020.06.03.130591
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Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping
Joel J. Credle, Matthew L Robinson, Jonathan Gunn, Daniel Monaco, Brandon Sie, Alexandra Tchir, Justin Hardick, Xuwen Zheng, Kathryn Shaw-Saliba, Richard E. Rothman, Susan H. Eshleman, Andrew Pekosz, Kasper Hansen, Heba Mostafa, Martin Steinegger, H. Benjamin Larman
bioRxiv 2020.06.03.130591; doi: https://doi.org/10.1101/2020.06.03.130591

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