Primer ID Next-Generation Sequencing for the Analysis of a Broad Spectrum Antiviral Induced Transition Mutations and Errors Rates in a Coronavirus Genome

Shuntai Zhou; Collin S Hill; Michael U Clark; Timothy P Sheahan; Ralph Baric; Ronald Swanstrom

doi:10.21769/BioProtoc.3938

Primer ID Next-Generation Sequencing for the Analysis of a Broad Spectrum Antiviral Induced Transition Mutations and Errors Rates in a Coronavirus Genome

Bio Protoc. 2021 Mar 5;11(5):e3938. doi: 10.21769/BioProtoc.3938.

Authors

Shuntai Zhou¹, Collin S Hill¹, Michael U Clark¹, Timothy P Sheahan², Ralph Baric^{2

3}, Ronald Swanstrom^{1

4}

Affiliations

¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, USA.
² Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, USA.
³ Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, USA.
⁴ Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, USA.

Abstract

Next generations sequencing (NGS) has become an important tool in biomedical research. The Primer ID approach combined with the MiSeq platform overcomes the limitation of PCR errors and reveals the true sampling depth of population sequencing, making it an ideal tool to study mutagenic effects of potential broad-spectrum antivirals on RNA viruses. In this report we describe a protocol using Primer ID sequencing to study the mutations induced by antivirals in a coronavirus genome from an in vitro cell culture model and an in vivo mouse model. Viral RNA or total lung tissue RNA is tagged with Primer ID-containing cDNA primers during the initial reverse transcription step, followed by two rounds of PCR to amplify viral sequences and incorporate sequencing adaptors. Purified and pooled libraries are sequenced using the MiSeq platform. Sequencing data are processed using the template consensus sequence (TCS) web-app. The Primer ID approach provides an accurate sequencing protocol to measure mutation error rates in viral RNA genomes and host mRNA. Sequencing results suggested that β-D-N4-hydroxycytidine (NHC) greatly increased the transition substitution rate but not the transversion substitution rate in the viral RNA genomes, and cytosine (C) to uridine (U) was found as the most frequently seen mutation.

Keywords: Antivirals; Coronavirus; Mutation; Next generation sequencing; Primer ID.

Abstract

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