Sanger Sequencing – An Efficient Solution
When Resources are Limited but the Virus is Not
The COVID-19 pandemic is entering its second year. Although the development of vaccines generated an initial sense of optimism in some parts of the globe, other parts of the world, particularly in health resource-limited areas, continue to have problems characterizing and controlling outbreaks. Coupled with these logistical problems, the sheer number of infections virtually guarantees that new variants with different biological dynamics will continue to arise. Several variants have been described that have been responsible for surges in incidence (for examples, see refs 1,2). And recently, the novel Omicron variant was first described in South Africa in November (3), but its increased transmissibility has caused it to become the dominant strain wherever it has been found.
Multiple Tools Available for Determining Strains in Circulation
Sanger Sequencing for Rapid & Cost-Effective Variant Detection & Surveillance
One important part of understanding the scope of an outbreak is quickly determining which strain(s) are circulating. Researchers and public health authorities have multiple tools available for surveillance of circulating strains, including the Applied Biosystems™ TaqMan™ SARS-CoV-2 Mutation assays and the Ion AmpliSeq™ SARS-CoV-2 Insight Research Assay. However, to rapidly determine whether a variant is present with modest expense, some researchers have turned to Sanger sequencing.
S-Gene Sequence – Focus of Surveillance by Sanger Sequencing Methods
The SARS-CoV-2 Spike (S)-protein is needed for virus entry into cells, and is the primary target of neutralizing antibodies and vaccine formulations. Therefore, changes in S-gene sequence are predicted to have the greatest effect on transmissibility and response to interventions. For this reason, the Spike (S)-gene has been the focus of surveillance by Sanger sequencing methods. Several groups have recently published their methods and results for analyzing the S-gene by Sanger sequencing using Big Dye Terminator chemistry (4,5). Because the Spike gene is relatively large (3822nt), these groups used multiple sets of primers to span regions containing mutations of interest. Finally, G.Cabral et al. used a nested PCR approach to sequence the S-gene (6). The nested PCR step allowed the investigators to increase the amount of DNA available for sequencing, increasing sensitivity and allowing them to perform multiple queries of the same sample.
Sanger Sequencing Advantages
SARS-CoV-2 Genome Can Be Queried in a Single Amplicon
However, one of the advantages of Sanger sequencing is that, by judicious choice of sequencing primers, many potentially mutated sites of the SARS-CoV-2 genome can be queried in a single amplicon. For example, M.Bezerra and his colleagues focused on the region of the Spike protein between nucleotides 22792 to 23522 (7). This 730-nucleotide region covered the receptor binding domain (RBD) of the Spike gene, which is responsible for binding to the ACE2 receptor. Changes in this region can lead to different affinities for ACE2, affecting the transmissibility of the virus. They were able to identify characteristic mutations associated with several VOCs within this one amplicon. Similarly, C.Torres and her colleagues designed primers to query a single 970-bp of the spike protein, and used Sanger sequencing of this amplicon to identify the likely VOC in samples from Argentina (8).
Variant Surveillance With Sanger Sequencing
Simple, Low-Cost, Accessible
Strain Variant Information in a Rapidly Changing Pandemic
These investigators have shown that surveillance using the relatively low-cost and simple Sanger sequencing methods can provide strain variant information. This approach is particularly important in regions where accessibility to other screening tools is difficult, expensive, or unavailable. Dr. Wallau and his team summed it up nicely in their publication: “[this] simple methodology will allow a much broader network of laboratories to perform molecular surveillance of SARS-CoV-2 VOSs, reporting results in a shorter time frame and in larger amounts, which is of utmost importance in the context of rapid public health decisions in a fast evolving worldwide pandemic” (7).
Download: [PDF] Protocol for Sanger sequencing of the SARS-CoV-2 spike (S) gene
Visualize / Search SARS-CoV-2 Genome & Primers
- Kirby, T. New variant of SARS-CoV-2 in UK causes surge of COVID-19. Lancet Respir. Med. 9(2), e20-e21 (2021)
- Mallapaty, S. India’s massive COVID surge puzzles scientists. Nature 592(7856):667-668 (2021) https://doi.org.10.1038/d41586-021-1059-y
- 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, (available at https://outbreak.info/situation-reports/omicron?loc=ZAF&loc=GBR&loc=USA&selected).
- Sallas TS et al. Genomic surveillance of SARS-CoV-2 Spike gene by Sanger sequencing. PLoS One 17(1):e0262170 (2022) https://doi.org/10.1371/journal.pone/0262170
- Lim HJ et al. Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations. PLoS One 16(12):e0260850 https://doi.org/10/1371/journal.pone/0260850
- Cabral GB et al. Use of Sanger protocols to identify variants of concern, key mutations, and track evolution of SARS-CoV-2. Journal of Virological Methods 300:114422 (2022) doi: 1016/j.jviromet.2021/114422
- Bezerra MF et al. A Sanger-based approach for scaling up screening of SARS-COV-2 variants of interest and concern. Infection, Genetic and Evolution 92:104910 (2021) doi 10/1016/jmeegid.2021.104910
- Torres C et al. Cost-effective method to perform SARS-CoV-2 surveillance: detection of alpha, gamma, lambda, delta, epsilon and zeta in Argentina. Frontiers Medicine 8:755463 (2021) doi: 10:3389/fmed.2021.755463