The recent news that a highly transmissible SARS-CoV-2 lineage originally detected in the United Kingdom (UK) has spread to the United States (US) serves as a reminder that it remains critical to continue to investigate the current crisis, characterize the virus strains further, and monitor the virus spread at the population level in order to assess the effectiveness of containment strategies—including a vaccine.
The new SARS-CoV-2 lineage—B.1.1.7—poses a challenge to these efforts. According to the European Centre for Disease Control and Prevention (eCDC), “while it is known and expected that viruses constantly change through mutation leading to the emergence of new variants, preliminary analysis in the UK suggests that this variant is significantly more transmissible than previously circulating variants” [1].
The new lineage B.1.1.7 is characterized by 17 mutations that cause amino acid changes, 8 of which occur in the gene for the spike (S) protein. The Applied Biosystems TaqPath COVID-19 Combo Kit, which uses an RT-qPCR test to detect SARS-CoV-2 in multiple countries, contains an S gene target in one of these regions, where there is the deletion of amino acids 69 and 70 (69-70del). Because of the multi-target design of this assay, overall test sensitivity should not be impacted by the B.1.1.7 variant.
Learn more about the impact of the 69-70del mutation on the TaqPath COVID-19 assay
If a sample with a variant that has the 69-70del mutation is tested using the TaqPath COVID-19 assay, it will result in an S gene “dropout.” Because of this, the test may signal the presence of the 69-70del mutation and, potentially, the B.1.1.7 lineage (variant 501Y.V1).
Confirmation of the variant strain may be accomplished by sequencing. There are multiple options available for routine sequencing of SARS-CoV-2 clinical research isolates.
The information and products presented below are for research use only and not for use in diagnostic purposes.
Sanger 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 [2,3].
Confirmation of S gene dropout
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. For low–viral load samples, a preamplification step can be used to boost the Sanger sequencing signal. The primers needed for pre-amplification and PCR (Table 1) can be ordered from our custom oligo ordering web page. These primers can be used to sequence the deletion, by performing PCR amplification followed by Sanger sequencing using generic M13 sequence-tagged primers. When ordering through the custom oligo ordering web page, the a pre-populated Microsoft Excel list can be used for convenient bulk upload. A detailed protocol for confirming the S gene deletion by Sanger sequencing** is available and has been developed to work with purified RNA samples.
Table 1. Forward and reverse primer sequences used for amplification and sequencing. These sequences can be used to verify the S gene sequence. The M13 sequences used for Sanger are highlighted in red.
Other mutation signatures, other lineages
Although S gene dropout in the TaqPath COVID-19 assay could signal the presence of the B.1.1.7 lineage, there are mutations in other regions of the SARS-CoV-2 genome that provide a genomic signature for this lineage [4]. Another highly transmissible lineage, designated as B.1.351 (also known as variant 501Y.V2), was recently identified and, similar to B1.1.7 was also found to have multiple mutations in the S gene [5].
And a recent outbreak of a variant (B.1.1.28) in Manaus, Brazil, has infection dynamics that suggest it could also be highly transmissible [6].
Detecting the signature mutations in these highly transmissible lineages (for B.1.1.7 the spread is thought to be 56% more rapid than other variants) [7] 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 [5]. Sanger sequencing of the genomic regions carrying the signature mutations may be useful for both of these purposes. We have therefore designed a a set of primers† and a Sanger sequencing protocol‡ that can be used to detect mutations that are characteristic of 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.
Access the protocol for Sanger sequencing of SARS-CoV-2 B.1.1.7 and B.1.351 strain lineages
Additional list of custom oligos related to the B.1.1.7 and B.1.351 lineages can also be used for bulk ordering on the custom oligos page
Find out more about our Sanger sequencing solutions for SARS-CoV-2 research
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 amplicons, 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.
Find out more about our NGS solutions for SARS-CoV-2 research
If you have additional questions or would like to discuss your specific situation, please contact our technical support team at www.thermofisher.com/contactus.
Resources
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- * List for Sanger Sequencing Oligos for the SARS-CoV-2 S-gene Assay (Microsoft Excel .xls file)
Note: Depending on individual browser settings, please “allow downloads” or right-click to open the file in a new tab. - ** Detailed protocol for analyzing SARS-2-CoV S-gene sequences by Sanger sequencing (.pdf)
- † Additional list of custom oligos related to the B.1.1.7 and B.1.351 lineages (Microsoft Excel .xls file)
Note: Depending on individual browser settings, please “allow downloads” or right-click to open the file in a new tab. - ‡ Protocol for Sanger sequencing of SARS-CoV-2 B.1.1.7 and B.1.351 strain lineages (.pdf)
The function of the primer sequences described in this document are in the process of being verified.
- * List for Sanger Sequencing Oligos for the SARS-CoV-2 S-gene Assay (Microsoft Excel .xls file)
References
- https://www.ecdc.europa.eu/en/publications-data/threat-assessment-brief-rapid-increase-sars-cov-2-variant-united-kingdom
- Paden CR, Tao Y, Queen K, Zhang J, Li Y, Uehara A, et al. Rapid, Sensitive, Full-Genome Sequencing of Severe Acute Respiratory Syndrome Coronavirus 2. Emerg Infect Dis. 2020;26(10):2401-2405. https://dx.doi.org/10.3201/eid2610.201800
- http://weekly.chinacdc.cn/en/article/doi/10.46234/ccdcw2020.088
- Wise J et al. (2020) Covid-19: New coronavirus variant is identified in UK. BMJ Dec 16;371:m4857. doi: 10.1136/bmj.m4857.
- Tegally H et al. (2020) Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa. medRxiv doi: https://doi.org/10.1101/2020.12.21.20248640.
- Faria, N.R. (2021) Genomic characterisation of an emergent SARS-CoV-2 lineage in Manaus: preliminary findings. https://virological.org/t/genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-manaus-preliminary-findings/586
- Davies NG, Barnard RC, Jarvis CI et al. (2020) Estimated transmissibility and severity of novel SARS-CoV-2 Variant of Concern 202012/01 in England. CMMID. Preprint published online December 23, 2020. Updated December 31, 2020. doi:10.1101/2020.12.24.20248822.