Sanger Sequencing_SARS-CoV-2_research

Generate highly specific targeted results

With its greater than 99% accuracy and long-read capabilities, Sanger sequencing is the gold-standard sequencing technology. And to further enable your research, fragment analysis by capillary electrophoresis (CE) provides DNA sizing, relative quantitation, and genotyping information. With our simple workflows, Sanger sequencing and fragment analysis can be completed in less than one workday—from sample to answer—helping to make your research of SARS-CoV-2 (the coronavirus that causes COVID-19) fast and cost-effective.

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NEW Sequencing protocols for SARS-CoV-2 variant detection

Protocol for finding and using Sanger sequencing primers for any location in the SARS-CoV-2 genome, including new Variants of Concern 
Download protocol | Visualize SARS-CoV-2 genome and Order Primers | Search SARS-CoV-2 genome and Order Primers
Variant Analysis using Variant Reporter | Files for Variant Reporter (zip)
Variant Analysis using SeqScape | Files for SeqScape (zip)

Protocol to quickly confirm S-gene dropouts seen with the TaqPath COVID-19 assay by identifying if the S gene 69/70 deletion is present 
Download protocol | Order primers

Flexible protocol to confirm the presence of the highly transmissible SARS-CoV-2 B.1.1.7, B.1.351, B.1.1.28, B.1.427 or B.1.429 strain lineages in your sample.  Pick and choose primers pairs that best suit your needs.
Download protocol | Download primers sequences for B.1.1.7 | Download primers sequences for B.1.351 | Download primers sequences for B.1.1.28 | Download primers sequences for B.1.427 | Download primers sequences for B.1.429

Protocol to identify new variants in the Spike gene by sequencing the entire S-gene for vaccine development and epidemiological research.
Download protocol | Download primer sequences

Learn more about which protocol to choose ›

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Why use Sanger sequencing in your SARS-CoV-2 research?

Sanger sequencing can complement your laboratory’s techniques and lead to more efficient studies.
  • Since being developed in 1977, Sanger sequencing has been the most widely used DNA sequencing method for the past 40 years and remains in wide-spread use around the world
  • Sanger method remains in wide use for smaller-scale projects and for confirmation of NGS results
  • Smaller inquiries with more specific goals can benefit from more focused, less expensive laboratory procedures
The benefits of Sanger sequencing

Sanger sequencing is the gold standard for sequencing single genes, confirming gene variants, detecting repeat sequences, copy number variation, and single nucleotide changes. Sanger sequencing is perfect for:

  • Sequencing single genes and single nucleotide variants
  • Targeted sequencing of 100 amplicons or less
  • Sequencing up to 96 samples at a time, without barcoding
  • NGS confirmation
  • High GC-rich sequences
  • Microbial identification
  • Microsatellite or STR analysis
  • Plasmid sequencing

Learn more:
Sanger sequencing or NGS?

While NGS technologies are common in research labs due to higher throughput capabilities, Sanger sequencing offers a cost-effective solution for your research needs.  It does not require expensive equipment and can generate high quality data even for low viral titer samples that do not yield high genome coverage for some NGS technologies.

How can Sanger sequencing and fragment analysis be used in your SARS-CoV-2 research?

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As you work to understand SARS-CoV-2 to rapidly identify future treatment options and develop possible vaccine targets, our comprehensive portfolio of Sanger sequencing and fragment analysis research solutions is here to help advance your research.

In the context of an outbreak, where high numbers of samples need to be processed quickly and accurately, Sanger sequencing and fragment analysis can significantly help you to get answers quickly.

Virus identification

At the start of the SARS-CoV-2 outbreak, Sanger sequencing was used by several labs to confirm the causative pathogen as being SARS-CoV-2 1-4. Additionally, by analyzing these genomes and comparing to other known coronaviruses, it was determined that the virus originated in an animal reservoir that evolved from bats, with a likely intermediate animal host 5,6.

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Robust and cost-effective Sanger sequencing and fragment analysis enable you to:
  • Quickly identify and distinguish SARS-CoV-2 from other respiratory viruses 1-3
  • Confirm SARS-CoV-2 S gene 69/70 deletion variant (download protocol)
  • Confirm SARS-CoV-2 B.1.1.7 and B.1.351 strain lineages (download protocol)
  • Quickly sequence the entire SARS-CoV-2 spike (S) gene (download protocol)
  • Generate gap filling and assembly sequence to aid with full genome NGS virus sequencing 1-6
  • Confirm sequence variations identified from full genome NGS data 7
  • Utilize a nested PCR approach with full genome sequencing for samples with lower viral load 8

What else can Sanger sequencing and fragment analysis do?

RT-PCR/qPCR result confirmationRapid testing for multiple targets
Sanger sequencing is being used to confirm RT-PCR/qPCR results and provide confidence in distinguishing SARS-CoV-2 from other respiratory pathogens. In a simplified workflow, PCR product is purified and sequencing primers bind to known sequences in the target region, allowing the sequence to be extended using capillary electrophoresis (CE) 1,3.Multiplexed qPCR solutions test for small numbers of pathogens, and its relatively small capacity can limit throughput when large numbers of targets or pathogens need to be detected. Fragment analysis by CE can be used to test multiple pathogens associated with different syndromes in a single sample 9. For example, a respiratory multiplex panel can detect viral and bacterial pathogens to help rule in/out common pathogens as the cause of infection.

Learn about a simple method for detecting SARS-CoV-2 viral sequences through fragment analysis-based target multiplexing solutions:

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Epidemiological surveillance research

Low-cost Sanger sequencing is considered the gold standard sequencing method for confirming the sequence of specific genes, including those already sequenced through NGS. It can be used to:

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  • Sequence and confirm variants of different SARS-CoV-2 genes within clusters of symptomatic and asymptomatic samples 10-12 and characterize the transmission patterns and genetic evolution of this virus among different populations 13, 4 
  • Confirm the origin and cross-species transmission of different coronaviruses, which is important in understanding potential sources of zoonotic transmission 15-17
  • Carry out full virus genome sequencing for one-off samples in locations without access to NGS sequencing or for low virus titer samples 8,11

An understanding of the type and subtype of viruses is essential for effective crisis response. Surveillance also plays a role in understanding cross-species transmission of the virus and its spatial spread over evolutionary time and at human-wildlife interfaces.  Monitoring helps detect the presence of different variants of the virus obtained from samples with different degrees of symptoms, including asymptomatic samples.

A potential early warning system for SARS-CoV-2

Identification of SARS-CoV-2 in wastewater can provide near real-time information about virus spread and could be an important approach for monitoring the virus spread 18,19. This method, known as wastewater-based epidemiology (WBE), may be able to act as an early warning system for infectious disease outbreaks in general, and in the immediate future could help predict the second wave of SARS-CoV-2 infections.  Multiple researchers around the world have used Sanger sequencing to confirm the identify of SARS-CoV-2 in wastewater following viral identification by RT-PCR or qPCR 20-22.

Read more: SARS-COV-2 in Wastewater: A Potential Early Warning Method

A role for the pharyngeal microbiome in understanding variability of SARS-CoV-2 infection susceptibility

Research indicates that the pharyngeal microbiota (PM) may influence susceptibility to different respiratory viral infections 23,24 making it possible that it may be important in understanding the epidemiology of SARS-CoV-2.  Fragment analysis by CE can be used identify microbial signatures associated with different disease phenotypes 25. It relies on amplification of 16S-23S rRNA interspace (IS) regions in bacteria to produce PCR fragments that vary in length and frequency depending upon bacterial species. Each PCR fragment can be separated and detected on a genetic analyzer. The resulting fragment patterns are compared to a curated database to determine the species present in the sample 25.

Read more: Could Differences in Microbiota Explain Variability in SARS-CoV-2 Susceptibility?

Vaccine and therapeutic research

There is an urgent need to develop safe and specific anti-viral treatments and/or vaccines for SARS-CoV-2. Vaccines provide a long-term solution to reduce the risk of infection by training the immune system to respond quickly and effectively. Therefore, it is critical to select a vaccine approach and begin with basic lab research to identify the pathogen and antigen candidates that could cause an immune response.

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Sanger sequencing: the ideal approach

The simple, fast, cost-effective Sanger sequencing workflow is ideal at different steps of the vaccine development workflow:

  • In target discovery, it is being used to identify the virus and its variants
  • It can help with optimizing of the sequence of the mRNA vaccine to maximize protein expression 26
  • It can be used to sequence and characterize the neutralizing antibodies generated as a response to the virus, which could potentially have therapeutic or prophylactic utility 27-29

The importance of human cell line authenticity

Vaccine research is often performed with cultured human cells obtained from major repositories or fellow researchers. An estimated 15–20% of the time, cells used in experiments have been misidentified or cross-contaminated with another cell line. Although major repositories now authenticate cell line identity, many are calling for all researchers to test and authenticate cell line identity using standard genotyping techniques like Short Tandem Repeat (STR) genotyping with CE.

We offer products to authenticate cell line identity:

The Role of Capillary Electrophoresis (CE) in Drug Development

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Emerging viruses pose a great challenge to global health care and economic systems. Given their pandemic potential, as witnessed in the case of the SARS-CoV-2 virus, rapid development and screening of drugs is needed to combat and contain the infection spread. 

Read Blog ›

How are other scientists using Sanger sequencing in their infectious disease research?

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“Sanger sequencing can accomplish what some other new platforms can’t, especially when you want to look at minor allele frequency haplotypes from contagious viral genes that might not get captured in some of the higher throughout platforms.”     

Dr. Lillian Seu
Center for Infectious Disease Control, Zambia

Read how Sanger sequencing was used to help detect drug resistance mutation in HIV research in Zambia

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“The high-precision and sensitivity of Sanger sequencing make it irreplaceable in confirming point mutations, determining the position of cloned DNA, sequencing short internal gaps, or missing data at the genome termini.” 

Dr. Iryna Goraichuk
USDA-ARS Exotic and Emerging Avian Viral Unit

Read how new avian viruses have been discovered with the help of Sanger sequencing

Get SARS-CoV-2 research results faster with a single-cartridge multi-application genetic analyzer

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  • Run both Sanger sequencing and fragment analysis samples on the same plate at the same time
  • Reduce setup time
  • Maximize benchtop space
  • Access, analyze, and share data anywhere, anytime with Connect

Learn more ›

Sanger sequencing workflow

Our simple and fast Sanger sequencing workflow can be completed in less than one workday, from sample to answer. We offer products that support many steps of our recommended workflow, from PCR amplification to data analysis.

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Fragment analysis workflow

From straightforward sample preparation to peak analysis, we offer a wide range of products and services to simplify each step of the fragment analysis workflow.

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References

Sanger sequencing and fragment analysis protocols are referenced in the publications below:

  1. Chan, JFW., et. al. (2020) A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet; 395: 514–23 (https://doi.org/10.1016/S0140-6736(20)30154-9)
  2. Zhu, N., et. al. (2020) A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med 382;8 (https://doi.org/10.1056/nejmoa2001017)
  3. Caly, L., et. al. (2020) Isolation and rapid sharing of the 2019 novel coronavirus (SARS‐CoV‐2) from the first patient diagnosed with COVID‐19 in Australia. Med J Aust 2020; 212 (10): 459-462. (https://doi.org/10.5694/mja2.50569)
  4. Lam, LT., et. al. (2020) “Whole-genome sequencing and de novo assembly of a 2019 novel Coronavirus (sars-cov-2) strain isolated in Vietnam. Preprint at bioRxiv doi: (https://doi.org/10.1101/2020.06.12.149377)
  5. Zhou, P., et. al. (2020) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature (579) (https://doi.org/10.1038/s41586-020-2012-7)
  6. Lu R,. et.al. (2020) Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet: 395: 565-574 (https://doi.org/10.1016/S0140-6736(20)30251-8)
  7. Artesi, M., et al. (2020) A recurrent mutation at position 26,340 of SARS-CoV-2 is associated with failure of 2 the E-gene qRT-PCR utilized in a commercial dual-target diagnostic assay. J. Clin. Microbiol. doi:10.1128/JCM.01598-20 (https://doi.org/10.1128/jcm.01598-20)
  8. Paden CR, et al., (2020). Rapid, sensitive, full-genome sequencing of severe acute respiratory syndrome coronavirus 2. Emerg Infect Dis. 26 (10) 3201. (https://doi.org/10.3201/eid2610.201800)
  9. Gomez, J., et al. (2020) “Capillary electrophoresis of PCR fragments with 5´-labelled primers for testing the SARS-Cov-2”. J. Virology 284 113937 (https://doi.org/10.1016/j.jviromet.2020.113937)
  10. Yuan, Y,. et al. (2020) “Molecular Epidemiology of SARS-CoV-2 Clusters Caused by Asymptomatic Cases in Anhui Province, China” Research Square preprint (https://dx.doi.org/10.21203/rs.3.rs-29833/v1)
  11. Holland, LA., et al. (2020) “An 81 nucleotide deletion in SARS-1 CoV-2 ORF7a identified from sentinel surveillance in Arizona (Jan-Mar 2020)” J. Virology (https://doi.org/10.1128/jvi.00711-20)
  12. Liu, Z., et al. (2020) “Identification of common deletions in 1 the spike protein of SARS-CoV-2”. J. Virology (https:// doi:10.1128/JVI.00790-20)
  13. Zhang, Z-B., et al. (2020) “Genomic surveillance of a resurgence of COVID-19 in Guangzhou, China”. Research Square preprint (https://doi.org/10.21203/rs.3.rs-35869/v1)
  14. Wang, Y., et al. (2020) “Intra-host Variation and 1 Evolutionary Dynamics of SARS-CoV-2 Population in COVID-19 Patients”. Preprint at bioRxiv doi: (https://doi.org/10.1101/2020.05.20.103549)
  15. Valitutto, M.T., et al. (2020) “Detection of novel coronaviruses in bats in Myanmar,” PLoS One 15(4): e0230802.(https://doi.org/10.1371/journal.pone.0230802)
  16. Latinne A., et al. (2020) Origin and cross-species transmission of bat coronaviruses in China. (bioRxiv preprint doi: https://doi.org/10.1101/2020.05.31.116061)
  17. Huong, N.Q., et al (2020) “Coronavirus testing indicates transmission risk increases along 2 wildlife supply chains for human consumption in Viet Nam, 2013-2014 (bioRxiv preprint doi: https://doi.org/10.1101/2020.06.05.098590.)
  18. Kitajima, M. et al. (2020) “SARS-CoV-2 in wastewater: State of the knowledge and research needs”. Science of the Total Environment 739,  139076. (https://doi.org/10.1016/j.scitotenv.2020.139076)
  19. Carducci Aet al., (2020) Making Waves: Coronavirus detection, presence and persistence in the water environment: State of the art and knowledge needs for public health. Water Res. 2020;179:115907. (https://doi.org/10.1016/j.watres.2020.115907)
  20. Ahmed W et al. (2020) “First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for wastewater surveillance of COVID-19 in the community”. Sci Total Environ DOI:(https://doi.org/10.1016/j.scitotenv.2020.138764)
  21. Wu, F.Q. et al. (2020) “SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed cases.” medRxiv preprint,doi: (https://doi.org/10.1101/2020.04.05.20051540)
  22. Nemudryi, A. et al, (2020) “Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater”, Preprint at medRxiv (https://doi.org/10.1101/2020.04.15.20066746)
  23. De Steenhuijsen Piters WAA, et al. (2016) “Nasopharyngeal microbiota, host transcriptome, and disease severity in children with respiratory syncytial virus infection”. Am J Respir Crit Care Med; 194: 1104–15. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114450/)
  24. Tsang TK, et al. (2019) “Association Between the Respiratory Microbiome and Susceptibility to Influenza Virus Infection.” Clin Infect Dis; 48109: 1–9. (https://pubmed.ncbi.nlm.nih.gov/31562814/)
  25. Budding A., et al., (2020)  “An Age Dependent Pharyngeal Microbiota Signature Associated with SARS-CoV-2 Infection (4/21/2020).” Preprint at (http://dx.doi.org/10.2139/ssrn.3582780)
  26. Zeng, C., et al. (2020) “Leveraging mRNAs sequences to express SARS-CoV-2 antigens in vivo” Preprint at bioRxiv doi: (https://doi.org/10.1101/2020.04.01.019877).
  27. Klimstra, W.B., et al. (2020) “SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected, hospitalized COVID-19 patients”. Preprint at bioRxiv doi:(https://doi.org/10.1101/2020.06.19.154930)
  28. Seydoux E., et al. (2020) “Characterization of neutralizing antibodies 1 from a SARS-CoV-2 infected individual”.  Preprint at bioRxiv doi: (https://doi.org/10.1101/2020.05.12.091298)
  29. Noy-Porat, T., et al. (2020) “Tiger team: a panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes”.  Preprint at bioRxiv doi: (https://doi.org/10.1101/2020.05.20.106609)
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