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A Comparison of Whole Genome Sequencing of SARS-CoV-2 Using Amplicon-Based Sequencing, Random Hexamers, and Bait Capture.
Nasir, Jalees A; Kozak, Robert A; Aftanas, Patryk; Raphenya, Amogelang R; Smith, Kendrick M; Maguire, Finlay; Maan, Hassaan; Alruwaili, Muhannad; Banerjee, Arinjay; Mbareche, Hamza; Alcock, Brian P; Knox, Natalie C; Mossman, Karen; Wang, Bo; Hiscox, Julian A; McArthur, Andrew G; Mubareka, Samira.
  • Nasir JA; Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON L8S 4K1, Canada.
  • Kozak RA; Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4K1, Canada.
  • Aftanas P; Division of Microbiology, Department of Laboratory Medicine and Molecular Diagnostics, Sunnybrook Health Sciences Centre, Toronto, ON M4N 3M5, Canada.
  • Raphenya AR; Division of Microbiology, Department of Laboratory Medicine and Molecular Diagnostics, Sunnybrook Health Sciences Centre, Toronto, ON M4N 3M5, Canada.
  • Smith KM; Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON L8S 4K1, Canada.
  • Maguire F; Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4K1, Canada.
  • Maan H; Perimeter Institute for Theoretical Physics, Waterloo, ON N2L 2Y5, Canada.
  • Alruwaili M; Faculty of Computer Science, Dalhousie University, Halifax, NS B3H 4R2, Canada.
  • Banerjee A; Peter Munk Cardiac Centre, University Health Network, Toronto, ON M5G 2N2, Canada.
  • Mbareche H; Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L69 3BX, UK.
  • Alcock BP; Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON L8S 4K1, Canada.
  • Knox NC; Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON L8S 4K1, Canada.
  • Mossman K; McMaster Immunology Research Centre, McMaster University, Hamilton, ON L8S 4K1, Canada.
  • Wang B; Division of Microbiology, Department of Laboratory Medicine and Molecular Diagnostics, Sunnybrook Health Sciences Centre, Toronto, ON M4N 3M5, Canada.
  • Hiscox JA; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A1, Canada.
  • McArthur AG; Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON L8S 4K1, Canada.
  • Mubareka S; Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4K1, Canada.
Viruses ; 12(8)2020 08 15.
Article in English | MEDLINE | ID: covidwho-717763
ABSTRACT
Genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly important to monitor the transmission and adaptive evolution of the virus. The accessibility of high-throughput methods and polymerase chain reaction (PCR) has facilitated a growing ecosystem of protocols. Two differing protocols are tiling multiplex PCR and bait capture enrichment. Each method has advantages and disadvantages but a direct comparison with different viral RNA concentrations has not been performed to assess the performance of these approaches. Here we compare Liverpool amplification, ARTIC amplification, and bait capture using clinical diagnostics samples. All libraries were sequenced using an Illumina MiniSeq with data analyzed using a standardized bioinformatics workflow (SARS-CoV-2 Illumina GeNome Assembly Line; SIGNAL). One sample showed poor SARS-CoV-2 genome coverage and consensus, reflective of low viral RNA concentration. In contrast, the second sample had a higher viral RNA concentration, which yielded good genome coverage and consensus. ARTIC amplification showed the highest depth of coverage results for both samples, suggesting this protocol is effective for low concentrations. Liverpool amplification provided a more even read coverage of the SARS-CoV-2 genome, but at a lower depth of coverage. Bait capture enrichment of SARS-CoV-2 cDNA provided results on par with amplification. While only two clinical samples were examined in this comparative analysis, both the Liverpool and ARTIC amplification methods showed differing efficacy for high and low concentration samples. In addition, amplification-free bait capture enriched sequencing of cDNA is a viable method for generating a SARS-CoV-2 genome sequence and for identification of amplification artifacts.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Pneumonia, Viral / RNA, Viral / Coronavirus Infections / Betacoronavirus Type of study: Diagnostic study / Prognostic study / Randomized controlled trials Limits: Humans Language: English Year: 2020 Document Type: Article Affiliation country: V12080895

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Pneumonia, Viral / RNA, Viral / Coronavirus Infections / Betacoronavirus Type of study: Diagnostic study / Prognostic study / Randomized controlled trials Limits: Humans Language: English Year: 2020 Document Type: Article Affiliation country: V12080895