key: cord-0736465-g5ihtgcg
authors: Allen, Richard A.; Williams, Christopher L.; Penrod, Yvonne; McCloskey, Cindy; Carpenter‐Azevedo, Kristin; Huard, Richard C.; King, Ewa; Terence Dunn, Samuel
title: A pyrosequencing protocol for rapid identification of SARS‐CoV‐2 variants
date: 2022-04-21
journal: J Med Virol
DOI: 10.1002/jmv.27770
sha: 4b58523cbd8dd94296dce7751d4d7ae1fa92cdd1
doc_id: 736465
cord_uid: g5ihtgcg

Next‐generation sequencing (NGS) is the primary method used to monitor the distribution and emergence of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) variants around the world; however, it is costly and time‐consuming to perform and is not widely available in low‐resourced geographical regions. Pyrosequencing has the potential to augment surveillance efforts by providing information on specific targeted mutations for rapid identification of circulating and emerging variants. The current study describes the development of a reverse transcription (RT)‐PCR‐pyrosequencing assay targeting >65 spike protein gene (S) mutations of SARS‐CoV‐2, which permits differentiation of commonly reported variants currently circulating in the United States with a high degree of confidence. Variants typed using the assay included B.1.1.7 (Alpha), B.1.1.529 (Omicron), B.1.351 (Beta), B.1.375, B.1.427/429 (Epsilon), B.1.525 (Eta), B.1.526.1 (Iota), B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.621 (Mu), P1 (Gamma), and B.1.1 variants, all of which were confirmed by the NGS data. An electronic typing tool was developed to aid in the identification of variants based on mutations detected by pyrosequencing. The assay could provide an important typing tool for rapid identification of candidate patients for monoclonal antibody therapies and a method to supplement SARS‐CoV‐2 surveillance efforts by identification of circulating variants and novel emerging lineages.

Several alternative technologies, less costly and complex to perform and with more efficient workflow, have the potential to augment SARS-CoV-2 variant screening efforts by providing information on specific targeted mutations for rapid identification of circulating and emerging variants. In addition, the urgent need for more real-time SARS-CoV-2 genotyping systems was recently realized by the lack of efficacy of certain monoclonal antibodies against specific variants. 3 Pyrosequencing technology is an attractive alternative sequencing-by-synthesis method to conventional Sanger-based sequencing that can be used to provide a rapid and accurate characterization of short DNA sequence variations. Since sequencing data are produced in real-time during synthesis rather than by electrophoretic separation of fluorescently labeled fragments postsynthesis, it is a much more rapid methodology and is less costly to perform; however, it is limited by the amount of sequence data generated in a single experiment (typically, 30−60 bases vs. 200−400 bases). One of two different pyrosequencing formats is adopted depending on the sequence to be analyzed. An allele quantification (AQ) genotyping assay dispenses nucleotides into reactions relative to a defined variant sequence and returns a percentage value of variant to normal nucleotide detected at the variable position. By contrast, a sequence analysis (SQA) assay can use either a cyclic dispensation of A, T, C, and G nucleotides to interrogate unknown sequences or a defined dispensation order of nucleotides to detect variant sequences without quantification.

This study describes a novel pyrosequencing protocol for rapid identification of a set of relevant sequence variations within the spike protein gene (S) that can be used to characterize SARS-CoV-2 variants currently circulating in the United States and potentially to screen for emerging variants. The protocol can be performed on residual RNA derived from specimens previously tested in various SARS-CoV-2 diagnostic tests, or on residual cDNA from those tests, and thus obviates the need for heightened biosafety containment during analysis.

The study was reviewed and approved by the University of Oklahoma Health Sciences Center Institutional Review Board (#14161).

RNA extracted from residual clinical specimens was obtained from Rhode Island Department of Health (RIDOH) Laboratories (Providence, RI) and from the OU Health Virology Laboratory (Oklahoma City, OK), following diagnostic testing using the TaqPath™ COVID-19 Combo Kit (Thermo Fisher Scientific). Original specimens were collected in various transport media from mid-January 2021 through mid-December 2021. Specimens were deidentifed before receipt at the OU Health Molecular Pathology Laboratory (Oklahoma City, OK). Variant determination from NGS data, C t values, and information regarding S gene target failure during diagnostic testing were provided with specimens, when available. In total, 49 specimens were obtained from RIDOH and 32 from OU Health. Specimens were purposely selected for analysis that were representative of the range of different variants detected at those facilities. RNA was stored at −80°C for up to 3 months before analysis by reverse transcriptase-PCR (RT-PCR) and pyrosequencing. Methods for acquisition, diagnostic testing, and sequencing of specimens at RIDOH are described elsewhere. 4 

Eight microliters of RNA was reverse-transcribed in a 25 µl reaction at 25°C for 10 min, then at 50°C for 60 min and 85°C for 5 min, followed by a 4°C hold, using random nonamers and Invitrogen SuperScript™ III Each 50 µl reaction was subjected to an initial denaturation of 95°C for 2 min followed by 45 cycles of 95°C for 20 s, 58°C for 20 s, and 72°C for 20 s followed by a final extension of 72°C for 2 min and 4°C hold. Reagent and thermocycling conditions were optimized and standardized across the four PCRs for ease of PCR setup and workflow.

Ten microliters of amplified products from each of the four PCRs were sequenced separately on a PyroMark Q24 System (Qiagen) using T A B L E 1 PCR and pyrosequencing primer sequences and pyrosequencing nucleotide dispensations 

The quality of sequence data revealed in pyrograms using products from individual PCRs was very good and easily interpreted for the majority of specimens; typically, single-height peaks exceeded 70 relative light units (RLUS) in amplitude with little to no background "noise." A few specimens, presumably due to lower amounts and/or quality of viral RNA, produced consistently low-amplitude peaks (single-height peaks~10 RLUs); nevertheless, usually the sequence of these specimens was unambiguous and easily interpreted. One specimen produced exceptionally poor-quality pyrograms for all targets and another failed to provide any sequencing data, despite repeated attempts to amplify and sequence these specimens. All specimens had C t values for viral-targets below 28 cycles when initially tested using the TaqPath™ COVID-19 Combo Kit, and C t values of compromised specimens did not appear significantly elevated relative to other specimens; nevertheless, the poor performance of these specimens in the pyrosequencing assay is likely due to low amounts and/or quality of viral RNA. Mutations specifically targeted by the assays had defined nucleotide dispensations set to detect the corresponding variable sequences at those locations, and therefore, produced pyrogram patterns that were easily interpreted (Figures 2A, B and 3A, B) .

F I G U R E 1 Schematic representation of the location of forward and reverse PCR primers (arrows) used to amplify four regions of the S gene of SARS-CoV-2. PCR 1 and PCR 2 target sequences corresponding to the N-terminal domain (NTD) of the S protein and overlap by 11 bases. PCR 2 resides within the receptor-binding domain (RBD), and PCR 4 overlies the junction between the S1 and S2 subunits (S1/S2). Common S gene mutations reported in SARS-CoV-2 variants, detectable by pyrosequencing the PCR products, using sequencing primers or forward PCR primers as listed in Table 1 , are indicated in the respective boxes. Forty-two mutations detected in specimens analyzed in the current study appear in bold type. Other rare, novel mutations may be detected within targeted sequences but are not listed.

Occasionally, however, unexpected mutations appeared within Unfortunately, as encountered during the development of our assays, unanticipated mutations occasionally occurred in the S gene that led to reduced or failed PCR amplification and/or low quality or failed pyrosequencing reactions. Initially, some of these incidental mutations impacting assay performance were addressed by redesigning/ moving primers; however, subsequently, we were able to resolve many of the same problems by substituting primer sets within individual assays (e.g., sequencing with the reverse PCR primer), or even between assays (e.g., using a forward PCR primer from one PCR together with the reverse biotinylated primer of another PCR to amplify cDNA). Other consequences of these unexpected base changes in viral sequence are that pyrosequencing reactions become temporarily stalled due to a divergence in viral sequence from the set nucleotide dispensation order and truncation of expected sequences.

Usually, such changes were easily resolved by re-pyrosequencing PCR products using a modified dispensation order to accommodate the mutation and/or by extending the number of nucleotide dispensations after the mutation to ensure appropriate coverage of sequences. PCR-based assays. Modifications to the standard protocol to resolve occasional problem specimens (e.g., using the forward PCR primer to pyrosequence) can be rapidly performed using residual PCR products, which does not significantly increase processing times; alternatively, such specimens may be processed for NGS. Significant improvements to workflow and specimen throughput likely could be achieved with use of a more rapid thermal cycling platform, the Pyromark Q96 ID, PyroMark Q48 Autoprep, and/or an automated fluid-handling system. Multiplexing PCRs or redesigning PCR formats (e.g., the forward primer from PCR 1 and reverse primer from PCR 2 can be used to produce a single PCR product for subsequent pyrosequencing) also has the potential to conserve reagents and supplies and simplify workflow.

Some of the limitations of our assay have been discussed above and include the inability to monitor effectively for emerging lineages given the limited amount of genome coverage as compared to NGS, the occasional interference in assay performance from incidental variant sequences underlying primers, and the low throughput and high hands-on time. In addition, pyrosequencing technology platforms are not widely available in public health or clinical laboratories, which certainly will limit any wholesale application of the assay. King at RIDOH. S. Terence Dunn was responsible for the study concept and design, data analysis and writing of the manuscript. All authors critically reviewed the manuscript before submission.

We wish to express our gratitude to the staff of the OU Health Virology Laboratory, Oklahoma City, OK and the RIDOH Laboratories, Providence, RI for their efforts in the routine processing of diagnostic samples and generation of data used in this study.

The authors declare no conflicts of interest.

The data that support the findings of this study are available from the corresponding author upon reasonable request. 

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