Capillary electrophoresis is a flexible and powerful technology whose applications are as multitudinous as they are lifesaving, especially as part of a fragment analysis or Sanger sequencing protocol. In this interview, Dr. Ashwin B. Dalal, head of the Diagnostic Division of the Centre for DNA Fingerprinting and Diagnositics (CDFD) in Hyderabad, India, explains how his laboratory used Sanger sequencing and fragment analysis to build a new diagnostic protocol for COVID-19 using Applied Biosystems 3500XL and 3730XL Genetic Analyzers and GeneMapper software to better serve the needs of the Telangana and Andhra Pradesh states in India.
Tell us about your lab and the connection to SARS-CoV-2.
The genetic diagnostic laboratory at Centre for DNA Fingerprinting & Diagnostics (CDFD) offers comprehensive diagnosis of genetic diseases. The center also offers prenatal diagnosis for detection of chromosomal abnormalities, single-gene disorders and inborn errors of metabolism. In addition to providing genetic diagnostic services, the laboratory carries out research work on molecular pathophysiology and mapping of genetic disorders.
When the pandemic started and the number of cases started rising, the Telangana state government approached CDFD to start COVID testing. Since CDFD is equipped with all technologies that can be used for COVID testing, research and surveillance, the Indian Council of Medical Research recognized CDFD as a partner for COVID testing in Andhra Pradesh and Telangana, India. We have done testing for more than 40,000 samples for SARS-CoV-2 using RT-PCR.
How do you use Sanger sequencing and fragment analysis by capillary electrophoresis in your SARS-CoV-2 research?
We carried out experiments with Applied Biosystems Capillary Electrophoresis 3500XL and 3730XL instruments using the Fragment Analysis protocol and Applied Biosystems reagents. The experiment was performed using VIC-labelled primers (ORF, N, S gene of SARS-CoV-2 with XENO RNA spike in control) and results were analyzed using GeneMapper v6.0 software.
We were successful in detecting all positive and negative samples with 100% concordance with respect to the traditional real-time PCR method.
Why did you decide to use the fragment analysis protocol to detect the SARS-CoV-2 virus?
We had a 3500 instrument recently installed and we have been using ABI products for several clinical applications. When we heard about this protocol, we thought to utilize it as an alternate technology to qPCR for detection of the virus.
How would you describe the benefits of Sanger sequencing or fragment analysis in SARS-CoV-2 research?
We demonstrated that the workflow is simple to perform, easy to use and cost effective. Most importantly, it is possible to use one instrument (Genetic Analyzer – ABI 3500xl/ABI 3730xl) for genotyping, sequencing confirmation and identification of the virus. Further, in absence of RT-PCR probes for each variant of the virus arising due to new mutations, Sanger sequencing can be helpful to quickly detect the variants in population and hence can be helpful for epidemiological studies.
Do you have any tips to share with other researchers interested in using Sanger sequencing or fragment analysis in SARS-CoV-2 research?
This method is a valuable tool for detection of SARS-CoV-2 in laboratories that have a Genetic Analyzer and existing sequencing infrastructure. We are satisfied with the results and recommend its use in other laboratories.