TY - JOUR
T1 - Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping
AU - Credle, Joel J.
AU - Robinson, Matthew L.
AU - Gunn, Jonathan
AU - Monaco, Daniel
AU - Sie, Brandon
AU - Tchir, Alexandra
AU - Hardick, Justin
AU - Zheng, Xuwen
AU - Shaw-Saliba, Kathryn
AU - Rothman, Richard E.
AU - Eshleman, Susan H.
AU - Pekosz, Andrew
AU - Hansen, Kasper
AU - Mostafa, Heba
AU - Steinegger, Martin
AU - Larman, H. Benjamin
N1 - Publisher Copyright:
© 2021, The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.
PY - 2021/6
Y1 - 2021/6
N2 - There is an urgent and unprecedented need for sensitive and high-throughput molecular diagnostic tests to combat the SARS-CoV-2 pandemic. Here we present a generalized version of the RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) assay, called “capture RASL-seq” (cRASL-seq), which 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 purification or reverse transcription, steps that have introduced supply bottlenecks into standard assay workflows. Our simplified protocol 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 technology with the potential to help mitigate the current pandemic and prevent similar public health crises.
AB - There is an urgent and unprecedented need for sensitive and high-throughput molecular diagnostic tests to combat the SARS-CoV-2 pandemic. Here we present a generalized version of the RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) assay, called “capture RASL-seq” (cRASL-seq), which 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 purification or reverse transcription, steps that have introduced supply bottlenecks into standard assay workflows. Our simplified protocol 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 technology with the potential to help mitigate the current pandemic and prevent similar public health crises.
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U2 - 10.1038/s41379-020-00730-5
DO - 10.1038/s41379-020-00730-5
M3 - Article
C2 - 33536572
AN - SCOPUS:85100328997
SN - 0893-3952
VL - 34
SP - 1093
EP - 1103
JO - Modern Pathology
JF - Modern Pathology
IS - 6
ER -