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Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing

Chenyu Li, David N. Debruyne, Julia Spencer, Vidushi Kapoor, Lily Y. Liu, Bo Zhou, Utsav Pandey, Moiz Bootwalla, Dejerianne Ostrow, Dennis T Maglinte, David Ruble, Alex Ryutov, Lishuang Shen, Lucie Lee, Rounak Feigelman, Grayson Burdon, Jeffrey Liu, Alejandra Oliva, Adam Borcherding, Hongdong Tan, Alexander E. Urban, Xiaowu Gai, Jennifer Dien Bard, Guoying Liu, Zhitong Liu
doi: https://doi.org/10.1101/2020.03.12.988246
Chenyu Li
1Paragon Genomics Inc., Hayward, CA 94545 USA
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David N. Debruyne
1Paragon Genomics Inc., Hayward, CA 94545 USA
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Julia Spencer
1Paragon Genomics Inc., Hayward, CA 94545 USA
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Vidushi Kapoor
1Paragon Genomics Inc., Hayward, CA 94545 USA
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Lily Y. Liu
1Paragon Genomics Inc., Hayward, CA 94545 USA
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Bo Zhou
2Department of Psychiatry and Behavioral Sciences, Department of Genetics, Stanford University, CA 94305 USA
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Utsav Pandey
5Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA 90027
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Moiz Bootwalla
5Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA 90027
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Dejerianne Ostrow
5Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA 90027
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Dennis T Maglinte
5Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA 90027
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David Ruble
5Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA 90027
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Alex Ryutov
5Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA 90027
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Lishuang Shen
5Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA 90027
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Lucie Lee
1Paragon Genomics Inc., Hayward, CA 94545 USA
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Rounak Feigelman
1Paragon Genomics Inc., Hayward, CA 94545 USA
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Grayson Burdon
1Paragon Genomics Inc., Hayward, CA 94545 USA
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Jeffrey Liu
1Paragon Genomics Inc., Hayward, CA 94545 USA
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Alejandra Oliva
1Paragon Genomics Inc., Hayward, CA 94545 USA
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Adam Borcherding
3MGI, BGI-Shenzhen, Shenzhen 518083 China
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Hongdong Tan
3MGI, BGI-Shenzhen, Shenzhen 518083 China
4BGI-Shenzhen, Shenzhen 518083 China
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Alexander E. Urban
2Department of Psychiatry and Behavioral Sciences, Department of Genetics, Stanford University, CA 94305 USA
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Xiaowu Gai
5Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA 90027
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Jennifer Dien Bard
5Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, Los Angeles, CA 90027
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Guoying Liu
1Paragon Genomics Inc., Hayward, CA 94545 USA
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Zhitong Liu
1Paragon Genomics Inc., Hayward, CA 94545 USA
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  • For correspondence: zhitong@paragongenomics.com
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Abstract

Many detection methods have been used or reported for the diagnosis and/or surveillance of COVID-19. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its high sensitivity, typically claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by some RT-PCR methods, probably due to low viral load, timing of sampling, degradation of virus RNA in the sampling process, or possible mutations spanning the primer binding sites. Therefore, alternative and highly sensitive methods are imperative. With the goal of improving sensitivity and accommodating various application settings, we developed a multiplex-PCR-based method comprised of 343 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purified from 13 COVID-19 positive specimens, and detected mutations through next generation sequencing. Finally, we developed a multiplex-PCR-based metagenomic method in parallel, that required modest sequencing depth for uncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates.

Competing Interest Statement

The authors have declared no competing interest.

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-NC-ND 4.0 International license.
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Posted May 18, 2020.
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Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing
Chenyu Li, David N. Debruyne, Julia Spencer, Vidushi Kapoor, Lily Y. Liu, Bo Zhou, Utsav Pandey, Moiz Bootwalla, Dejerianne Ostrow, Dennis T Maglinte, David Ruble, Alex Ryutov, Lishuang Shen, Lucie Lee, Rounak Feigelman, Grayson Burdon, Jeffrey Liu, Alejandra Oliva, Adam Borcherding, Hongdong Tan, Alexander E. Urban, Xiaowu Gai, Jennifer Dien Bard, Guoying Liu, Zhitong Liu
bioRxiv 2020.03.12.988246; doi: https://doi.org/10.1101/2020.03.12.988246
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Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing
Chenyu Li, David N. Debruyne, Julia Spencer, Vidushi Kapoor, Lily Y. Liu, Bo Zhou, Utsav Pandey, Moiz Bootwalla, Dejerianne Ostrow, Dennis T Maglinte, David Ruble, Alex Ryutov, Lishuang Shen, Lucie Lee, Rounak Feigelman, Grayson Burdon, Jeffrey Liu, Alejandra Oliva, Adam Borcherding, Hongdong Tan, Alexander E. Urban, Xiaowu Gai, Jennifer Dien Bard, Guoying Liu, Zhitong Liu
bioRxiv 2020.03.12.988246; doi: https://doi.org/10.1101/2020.03.12.988246

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