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.
Original language | English |
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Article number | v12080895 |
Journal | Viruses |
Volume | 12 |
Issue number | 8 |
DOIs | |
Publication status | Published - Aug 2020 |
Bibliographical note
Funding Information:This research was funded by the Canadian Institutes of Health Research grant PJT-156214. Technical discussion from Jared Simpson (Ontario Institute for Cancer Research) was greatly appreciated. J.A.N. was supported by funds from the Comprehensive Antibiotic Resistance Database. B.P.A. and A.R.R. were supported by Canadian Institutes of Health Research (CIHR) funding (PJT-156214 to A.G.M.). Computer resources were supplied by Hewlett Packard Enterprise, Canada. K.M. is funded by CIHR and Natural Sciences and Engineering Research Council of Canada (NSERC). A.B. is funded by NSERC. F.M. is supported by a Donald Hill Family Fellowship in Computer Science. H.M. is supported by a postdoctoral fellowship from Fond de Recherche du Québec Nature et Technologie and is the recipient of the Lab Exchange Visitor Program Award from the Canadian Society for Virology. S.M. and R.A.K. are supported by the McLaughlin Centre and the Toronto COVID-19 Action Initiative from the University of Toronto. Methods development of an amplicon system for SARS-CoV-2 by J.A.H. and M.A. is funded by the US Food and Drug Administration.
Funding Information:
Acknowledgments: Technical discussion from Jared Simpson (Ontario Institute for Cancer Research) was greatly appreciated. J.A.N. was supported by funds from the Comprehensive Antibiotic Resistance Database. B.P.A. and A.R.R. were supported by Canadian Institutes of Health Research (CIHR) funding (PJT-156214 to A.G.M.). Computer resources were supplied by Hewlett Packard Enterprise, Canada. K.M. is funded by CIHR and Natural Sciences and Engineering Research Council of Canada (NSERC). A.B. is funded by NSERC. F.M. is supported by a Donald Hill Family Fellowship in Computer Science. H.M. is supported by a postdoctoral fellowship from Fond de Recherche du Québec Nature et Technologie and is the recipient of the Lab Exchange Visitor Program Award from the Canadian Society for Virology. S.M. and R.A.K. are supported by the McLaughlin Centre and the Toronto COVID-19 Action Initiative from the University of Toronto. Methods development of an amplicon system for SARS-CoV-2 by J.A.H. and M.A. is funded by the US Food and Drug Administration.
Publisher Copyright:
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
ASJC Scopus Subject Areas
- Infectious Diseases
- Virology