S-Gene Dropout and S-Gene Sequencing
The COVID-19 global pandemic continues to disrupt people’s lives and countries’ economies. However, since the onset of the pandemic, we have gained enormous scientific insights on virus transmission and pathogenesis, allowing us to rapidly develop strategies for virus detection and surveillance. SARS-CoV-2, like other RNA viruses, is observed to have a high mutation rate that gives it an advantage to adapt under selective immune pressure. Several studies investigating the dynamics of SARS-CoV-2 mutation have revealed that the Spike gene and the Orf1ab region are prone to a large number of recurrent mutations [1, 2]. Such mutations may impact virus infectivity, transmissibility and susceptibility to antivirals or vaccines.
Routine Surveillance Sequencing Detects New Variants
Recently, routine surveillance sequencing by Network for Genomics Surveillance laboratories in South Africa detected a new lineage of the virus , first designated B.1.1.529. Infections attributed to the new variant were seen at an alarmingly increasing rate in the Gauteng province in South Africa , and it has since been found in more than a dozen countries, including Botswana, the United Kingdom, Germany, Belgium, the Czech Republic, Israel, Netherlands, Japan, Australia, Hong Kong, and the United States. B.1.1.529 contains more than 30 amino acid mutations in the Spike protein, the region of the virus that binds to host cells and is the main target of several currently-available vaccines. This variant also shares some of the mutations with the Delta and Alpha variants which were linked to heightened infectivity with immune evasion potential.
Omicron – SARS-CoV-2 Variant of Concern
The rapid spread and the large number of mutations in the Spike gene drove the WHO to warn its 194 member states that the new variant “may confer immune escape potential and possibly transmissibility advantage,” and as a result “the likelihood of potential further spread of [this variant] at the global level is high.” The WHO subsequently raised its status to a Variant of Concern on November 26, 2021, giving it the name “Omicron” .
Continuous Testing and Monitoring of SARS-CoV-2 Strains
To better understand and control the outbreak of the omicron variant, it is crucial to continuously monitor virus evolution through epidemiological surveillance. Thermo Fisher Scientific is the global leader in 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 mutation. If one of the probes fail, the presence of two others still indicates presence of virus.
Genome Sequencing of Omicron Variant Mutations
Interestingly, this redundancy has been used as a primary indicator of the detected strain. A year ago, it was found that one specific mutation found in the B.1.1.7 variant disrupted binding of the signal from the spike gene – this S-gene dropout coupled with robust N and orf1ab signal was an early indicator of the B.1.1.7 strain . Upon sequencing of the Omicron strain, a similar mutation that causes a similar S-gene drop out was found. Thus, “This assay can be used not only to successfully detect COVID-19 but… it can also be used as a proxy for the [Omicron] variant,” Mark Stevenson, chief operating officer at Thermo Fisher Scientific, said in an interview.
Proven Sequencing Methods and Tools to Detect Mutations
Often a deeper understanding of the genomic mutation landscape is required. Moreover, there is frequently a need to understand why a PCR test produces anomalous results, such as the S-gene dropout. To do this, researchers turn to Sanger sequencing by capillary electrophoresis, the trusted standard for targeted genome sequencing. Sanger sequencing is a relatively simple workflow that provides a high degree of accuracy, long-read capabilities, and simple data analysis. Thermo Fisher Scientific recently introduced tools for sequencing any region of the SARS-CoV-2 genome by Sanger sequencing. Two web-based tools were developed for choosing and ordering wet-lab tested Sanger sequencing primers. One of these provides a visual representation of the genome, along with locations of mutations characteristic of strain variants and primers covering those mutations.
The other is a look-up table, that allows researchers to input any mutation or nucleotide location to select the correct primer pair.
The flexibility of these tools have enabled researchers to rapidly find the sequencing primers needed for any SARS-CoV-2 variant, including the new Omicron variant. In addition, we simplified the interface by updating the visual browser and look-up tables so that primers covering the entire set of mutations characteristic of the the Omicron variant can be ordered at one time.
Urgent Need for Viral Sequence Surveillance Strategies
SARS-CoV-2 will continue to mutate and evolve. The emergence of new variants that could spread faster or hamper the vaccination efforts underscores the importance of rapid and continuous viral sequence surveillance strategies. It is not yet known whether the B.1.1.529 variant will render universal vaccination and non-pharmaceutical interventions ineffective. However, our best bet for staying ahead of SARS-CoV-2 virus depends on the rapid identification of new variants by surveilling infected individuals for new sequence changes in the viral genome.
Protocol for finding and using Sanger Sequencing primers for any location in the SARS-CoV-2 genome, including new Variants of Concern: Download Protocol
- van Dorp, L., et al., Emergence of genomic diversity and recurrent mutations in SARS-CoV-2. Infect Genet Evol, 2020. 83: p. 104351.
- Xiaofei, Y., L. Li, and Y. Kai, Research Square, 2021.
- Omicron Variant Report. Alaa Abdel Latif, Julia L. Mullen, Manar Alkuzweny, Ginger Tsueng, Marco Cano, Emily Haag, Jerry Zhou, Mark Zeller, Emory Hufbauer, Nate Matteson, Chunlei Wu, Kristian G. Andersen, Andrew I. Su, Karthik Gangavarapu, Laura D. Hughes, and the Center for Viral Systems Biology. outbreak.info. Accessed 29 November 2021.
- Callaway, E., Heavily mutated Omicron variant puts scientists on alert. Nature, 2021. doi
- 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.