Sanger Sequencing Solutions for SARS-CoV-2 Research

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

• Sanger sequencing guide
• Fragment analysis guide

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.

• 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.

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

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?

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

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:

• Applied Biosystems CLA Identifiler Plus and Direct kits are optimized to analyze 16 highly variant human STRs with amplification time of under three hours
  
• Applied Biosystems GeneMapper Software 6 and the cloud-based microsatellite analysis (MSA) software solutions facilitate analysis of STRs by making use of pre-established allelic ladders and sizing bin sets for the various STR alleles covered by the Identifiler kits

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)