Scientists, medical researchers and drug developers have made tremendous progress in providing tools that help fight the COVID-19 pandemic. Although vaccines and treatment options have been developed, the SARS-CoV-2 virus continues to be a global threat. There are two reasons for this.
First, even though this virus has a lower rate of mutation than other RNA viruses, it still can make mistakes when it replicates, giving rise to new mutations. These new mutations may give the virus certain advantages, such as evading detection, changing its transmissibility, altering the severity of the disease, or bypassing vaccine-mediated immunity.
Second, the vast number of infections across the globe provides ample opportunity for these new mutations to arise and spread. These are not just theoretical musings – many new strains with new mutations and different transmissibility have arisen in the first half of this year, including the B.1.1.7 and the B.1.617 variants of concern that have spread quickly across the globe (1). Thus, although we continue to make progress fighting the pandemic, researchers need to remain vigilant and monitor for the appearance of new mutations.
Sequence-Based Tools to Identify and Trace New Mutations in Strain Variants
The most effective way to identify and trace the spread of new mutations in strain variants is to use sequence-based tools that characterize the genomes that are circulating. Thermo Fisher Scientific has developed methods for strain identification on its Ion Torrent NGS platform, on the Applied Biosystems TaqMan RT-qPCR platform, and on the Applied Biosystems Genetic Analyzer capillary electrophoresis (CE) platform. Each of these different strategies provides slightly different advantages.
NGS, CE, RT-qPCR Solutions for SARS-CoV-2 Surveillance Research
In general, NGS-based approaches are ideal for unbiased discovery of new mutations in the whole genome, the CE approach is ideal for focused analysis of specific regions of the genome, and RT-qPCR approaches are ideal for highly specific detection of well-defined sequences and mutations. Thus, these three approaches have complementary strengths and each fulfils a need in the surveillance research community (for details, see the epidemiological research portal).
Sanger Sequencing Solution for SARS-CoV-2 Surveillance
To give investigators the flexibility needed to confirm any new mutation that might arise using gold-standard sequencing technology, we a developed a Sanger sequencing solution for sequencing any position in the SARS-CoV-2 genome.
Choose the Correct Primers for SARS-CoV-2 Sequencing
In a Sanger sequencing strategy, one of the hurdles that must be of correct primers. To help researchers identify suitable primers for SARS-CoV-2 sequencing, we first wet-lab tested all of the primers designed by the CDC (2) for successful sequencing using our protocol. We verified that these primers worked on the Wuhan reference RNA, and we confirmed that they correctly identified the mutations found in the B.1.17 strain.
Primers to Cover Any Region of the SARS-CoV-2 Genome
There were some primers that did not pass these tests – in those cases, we redesigned primers to cover the region and confirmed these redesigned primers passed the functional test. As a result of these efforts, we identified a set of primers that have been functionally tested to cover any region of the genome.
Web-Based Primer Selection Tools
Of course, in many cases it won’t be necessary to Sanger sequence the entire genome. To help identify primers that focus on a defined region or regions, we developed two web-based primer selection tools.
SARS-CoV-2 Sanger Primer Visualization App
One of these, the SARS-CoV-2 Sanger Primer Visualization App, was built using the publicly-available JBrowse framework (3). This tool allows users to see the relationship between the location of SARS-CoV-2 genes, mutations found in variants of concern, and the functionally tested primers (figure 1). Primers covering any position can be found by entering the coordinates into the appropriate field. The browser is pre-loaded with the mutations found in current VOCs (variants of concern) and Variants of Consequence as defined in outbreak.info. This site will be checked frequently (every other week) and new strains that are designated as VOC or variant of consequence will be added to the browser as needed.
SARS-CoV-2 Sanger Primer Pair Lookup
The other tool is the SARS-CoV-2 Sanger Primer Pair Lookup. This tool allows uses to find primers for any strain lineage, gene, amino acid change, or genomic coordinate by entering information into the appropriate box(es). The tool will also show the sequence of the functionally tested primers in our collection. And to facilitate use with our sequencing protocol, the primers can be shown with or without M13 tags. Once a primer or set of primers has been identified, the list can be exported to a preformatted Excel file that can be uploaded into into our primer bulk ordering site. As with the visualization tool, new variants will be added to the lookup app as needed.
Protocol Developed for Sequencing Specific Mutations in Variants
The protocol that makes use of these primers is the same that was previously developed for sequencing specific mutations found in variants and for sequencing the entire spike gene. Briefly, cDNA synthesis is performed on a sample containing viral RNA. Next, the cDNA is used in specific regions of target amplification using M13-tailed primers that cover the regions of interest. For this, the Applied Biosystems™ BigDye™ Direct Cycle Sequencing Kit and M13 sequence–tagged primer sets are used. The amplified sequences are then subjected to cycle sequencing using either M13-forward and M13-reverse primers provided in the BigDye Direct Cycle Sequencing Kit. Unincorporated nucleotides and primers are next removed using the Applied Biosystems™ BigDye XTerminator™ Purification Kit, and the sequences are read by standard capillary electrophoresis (CE). The sequences obtained can be read by any sequencing program, such as Applied Biosystems SeqA software. The protocol also contains details for using the primer look-up tools described above.
SARS-CoV-2 Variant Analysis Solutions
Once sequencing is complete, the results will have to be compared to a reference sequence, known mutations and strains to for positive identification. Although the sequences can be used to compare against known sequences using BLAST, in some cases a simpler and more definitive workflow is needed. We therefore developed options for SARS-CoV-2 variant analysis using Applied Biosystems Variant Reporter analysis and SeqScanner software packages. Analysis files containing the reference sequence, mutations found in VOCs, and simplified user guides can be downloaded and used for Variant Reporter and SeqScanner analysis on our Sanger for SARS-CoV-2 Research webpage. Although these user guides are not comprehensive, they are presented to allow users to begin to better understand their results. To take advantage of the complete features of the software packages, please see the appropriate user guides.
Sanger Sequencing Solutions for Viral Surveillance
Detecting and confirming mutations in newly arising SARS-CoV-2 strains are important strategies for controlling the spread of new viral lineages. Thermo Fisher Scientific continues to help researchers by developing and providing tools that help with viral surveillance needs.
- Adam, D. (2021) News: What scientists know about new, fast-spreading coronavirus variants. Nature 594, 19-20. doi: https://doi.org/10.1038/d41586-021-01390-4\
- Paden CR et al. (2020) Rapid, sensitive, full-genome sequencing of severe acute respiratory syndrome coronavirus 2. Emerg Infect Dis. 26(10):2401-2405. https://dx.doi.org/10.3201/eid2610.201800.
- Buels R et al. (2016) JBrowse: a dynamic web platform for genome visualization and analysis. Genome Biol. 17.1:66.