Sanger Sequencing for SARS-CoV-2 Omicron Variant Surveillance

Mutation Detection and Variant Surveillance

The SARS-CoV-2 virus continues to evolve as the pandemic progresses into the third year since its onset in March 2020. SARS-CoV-2, like other RNA viruses, is notorious for its high mutation rate, in part due to the error-prone RNA-dependent RNA polymerase [1, 2]. In addition, partial immunity can add selective evolutionary pressure, leading to the emergence of new, more infectious or pathogenic variants [3]. Mutations that could impact virus infectivity, transmissibility and susceptibility to antivirals or vaccines are of great concern and therefore need to be monitored on an ongoing basis to contain their spread.

Figure 1: SARS-Cov-2 Spike gene domain organization. Source: The Spike of Concern—The Novel Variants of SARS-CoV-2 (https://www.mdpi.com/1999-4915/13/6/1002/htm#)

The new lineage, B.1.1.529, also known as Omicron, was first detected in South Africa’s Gauteng province in late November 2021, and shortly after, declared as a ‘variant of concern’ by the WHO on November 26 [4]. The variant is characterized by over 30 non-synonymous mutations, primarily in the ORF1ab and Spike region of the virus genome [5]. Of particular concern are the mutations in the receptor binding domain and the N-terminal domain of the Spike gene, both of which are involved directly or indirectly, in ACE2 interaction and virus entry into the cell (See figure 1 for Spike gene domain organization; pls check for copyright). Since it’s detection, South Africa has recorded 8,561 cases of Omicron as of December 1, up from 3,402 that were reported on November 26^th [6]. In parallel, more than 24 countries have reported the variant.

Thermo Fisher Scientific has been at the forefront of the fight against the pandemic by providing rapid SARS-CoV-2 detection assays. The TaqPath COVID-19 qPCR assay was developed to analyze three different regions of the SARS-CoV-2 genome (N, S and orf1ab) for a built-in redundancy for mutations. If one of the probes fail, the presence of two others still indicates presence of virus. Interestingly, the deletion at amino acid positions 69/70 in the Spike region, seen previously in the B.1.1.7 strain led to the dropout of the S gene detection by the TaqPath assay – this spike-gene dropout coupled with robust N and orf1a signal was used as an early indicator of the B.1.1.7 strain [7]. The same phenomenon has been observed for Omicron and is currently being used as a preliminary test [8] for the variant to be followed up by sequencing-based confirmation, should the S gene fail detection. Epidemiological surveillance by Sanger and Next Generation Sequencing (NGS) are the technologies of choice for targeted and comprehensive sequencing, respectively.

Sanger Sequencing to Identify Mutations

Confirm Data from Next-Generation Sequencing

Several whole-genome Sanger sequencing approaches have been developed and can be used for single-sample sequencing of the virus to identify mutations and to confirm data from NGS sequencing.

Confirmation of S-Gene Dropout and Other Mutation Signatures for Omicron

More targeted Sanger sequencing approaches with amplification of a short stretch of the S gene in the region of the deletion can be used to verify S gene dropout in the TaqPath COVID-19 assay. In addition, the characteristic insertion of 3 amino acids (EPE) in position 214 for the Omicron can be verified using this approach. Detecting the signature mutations in these potentially highly transmissible lineages is important, not only for confirming that the sample contains the particular lineage, but also for epidemiological tracking of how the lineage changes over time. Sanger sequencing of the genomic regions carrying the signature mutations may be useful for both of these purposes. We have therefore designed a set of primers^† and a Sanger sequencing protocol^‡ that can be used to detect mutations that are characteristic of the B.1.1.529 in addition to the B.1.1.7 and B.1.351 lineages.

The use of Applied Biosystems BigDye Direct Cycle Sequencing Kit and BigDye XTerminator Purification Kit chemistries streamlines the process, providing sequencing data in about four hours. The panel is designed to be flexible, allowing researchers to pick and choose primer pairs that best suit their research needs. Finally, each of the amplicons is several hundred bases long. Any novel mutations that differ from a reference strain may be identified by Sanger sequencing, giving clues to the evolution of the virus over time.

Sanger Sequencing Resources

Protocol for Sanger Sequencing Any Region of the SARS-CoV-2 Genome:
https://assets.thermofisher.com/TFS-Assets/GSD/Application-Notes/sars-cov-2-anywhere-protocol-app-note.pdf

Visual Browser:
https://jbrowse-nc045512-2.thermofisher.com/

Lookup Table (includes primers for the Omicron variant):
https://sars-cov2-sanger-primer-lookup.thermofisher.com/

Protocol for Sequencing the del69/70 Region of the S Gene:
https://assets.thermofisher.com/TFS-Assets/GSD/brochures/sequencing-sars-cov-2-s-gene-69-70del-protocol.pdf

Next-Generation Sequencing

Next-generation sequencing (NGS) can be used for the analysis and monitoring of the complete SARS-CoV-2 genome. The Ion AmpliSeq SARS-CoV-2 Research Assay is a targeted NGS solution that facilitates complete viral genome sequencing and variant detection of SARS-CoV-2. The intelligent design, with a majority of the viral genome covered by two amplicon pools provides exceptional protection against naturally occurring variation and ensures robust performance even as the virus rapidly mutates, making it usable in a wide variety of epidemiological applications for SARS-CoV-2 research. The assay is part of a fast, automated, and accurate targeted NGS workflow that enables coronavirus typing in under a day. This end-to-end research solution includes the assay and plug-in suite that was developed in collaboration with researchers at the forefront of the SARS-CoV-2 health crisis. Our complementary targeted NGS systems make SARS-CoV-2 epidemiology research accessible to any lab, regardless of your team’s current level of NGS experience.

If you have additional questions or would like to discuss your specific situation, please contact our technical support team at www.thermofisher.com/contactus.

Banerjee, A., K. Mossman, and N. Grandvaux, Molecular Determinants of SARS-CoV-2 Variants. Trends Microbiol, 2021. 29(10): p. 871-873.
Chrisman, B.S., et al., Indels in SARS-CoV-2 occur at template-switching hotspots. BioData Min, 2021. 14(1): p. 20.
Saad-Roy, C.M., et al., Epidemiological and evolutionary considerations of SARS-CoV-2 vaccine dosing regimes. Science, 2021. 372(6540): p. 363-370.
Callaway, E., Heavily mutated Omicron variant puts scientists on alert. Nature, 2021. 600(7887): p. 21.
https://outbreak.info/situation-reports/omicron; accessed on December 3, 2021
https://doi.org/10.1038/d41586-021-03614-z , accessed on December 3, 2021
Kidd, M., et al., S-Variant SARS-CoV-2 Lineage B1.1.7 Is Associated With Significantly Higher Viral Load in Samples Tested by TaqPath Polymerase Chain Reaction. J Infect Dis, 2021. 223(10): p. 1666-1670.
https://www.cdc.gov/coronavirus/2019-ncov/science/science-briefs/scientific-brief-omicron-variant.html, accessed on 03Dec2021