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

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.

Accelerated SARS-CoV-2 intrahost evolution leading to distinct genotypes during chronic infection

“…To test this hypothesis, we used deep sequencing to quantify the number of unique intrahost single nucleotide variants (iSNVs, i.e., “mutations”) present at >3% within sample frequency in each sample ( Figure 4A-D ). To validate the iSNV frequencies that we generated from whole genome amplicon-based sequencing, we sequenced the spike gene of a subset of the samples using unique molecular index (UMI)-tagged primers that improves the accuracy of iSNV detection (Zhou et al, 2015, 2021). We found a high concordance between the iSNV frequencies measured from our whole genome amplicon-based and UMI sequencing (median [β]: 0.999) ( Figure S2 , Table S3 ).…”

“…Furthermore, we hypothesize that spike Q493K/R mutation could be important for chronic SARS-CoV-2 infections (Choi et al, 2020;Peacock et al, 2021;Wilkinson et al), even though neither became fixed in our study because they were on different genotypes. By validating the iSNV frequencies using a UMI-based sequencing approach (Primer ID) which helps to remove PCR artifacts (Zhou et al, 2015(Zhou et al, , 2021, our findings provide a robust assessment of intrahost evolutionary dynamics during chronic infection.…”

“…Primer ID sequencing using unique molecular identifiers UMI-guided deep sequencing was done using the previously published Primer ID nextgeneration sequencing protocol to sequence the SARS-CoV-2 viral genomes extracted from the specimens (Zhou et al, 2015(Zhou et al, , 2021. We used two sets of multiplexed UMI-tagged primers targeting SARS-CoV-2 ORF1ab (nsp12) and the spike gene.…”

Unique Molecular Identifiers and Multiplexing Amplicons Maximize the Utility of Deep Sequencing To Critically Assess Population Diversity in RNA Viruses

“…The principle of the MPID NGS approach has been previously described . In brief, we used Primer ID-tagged cDNA primers in the initial cDNA synthesis after viral RNA extraction to label each viral RNA template with a UMI.…”

“…The principle of the MPID NGS approach has been previously described. 29 In brief, we used Primer ID-tagged cDNA primers in the initial cDNA synthesis after viral RNA extraction to label each viral RNA template with a UMI. The multiplexing of the cDNA allowed us to sequence multiple regions of the viral genome in one reaction.…”