About Sanger Sequencing

Sanger sequencing, also known as the “chain-termination” method, was developed by Frederick Sanger and his colleagues in 1977. This technology quickly became a popular method in molecular research labs for sequencing DNA fragments. The original technique of manual polyacrylamide gel sequencing with radiolabeled ddNTPs gradually became obsolete with the 1986 development of automated capillary electrophoresis DNA sequencing, which uses fluorescently labeled ddNTPs. Due to the high cost of instruments and reagents, as well as the need for greater efficiency, core labs at universities and specialized sequencing companies became the primary providers of automated sequencing.

Human Genome Project and Sanger sequencing

As the leading sequencing tool of its time, automated Sanger sequencing was instrumental in the Human Genome Project. This multinational collaboration, which spanned from 1990 to 2003, cost $2.7 billion to “complete.” The initial draft of the human genome had numerous gaps that have been filled over the years, though some still remain today.

Can NGS totally replace Sanger sequencing?

Today, the cost of Next-Generation Sequencing (NGS) has dropped significantly, and its high-throughput capabilities have led it to gradually replace Sanger sequencing in research and clinical applications where a large volume of sequencing is required. However, for sequencing a single gene or a few genes in a limited number of samples, such as for pathology testing and analyzing inherited genetic diseases, Sanger sequencing is still the more economical choice. Even in cases where NGS is used, the Sanger method is often employed to confirm the accuracy of the sequencing results. Sanger sequencing boasts an accuracy of 99.99%, while NGS has an accuracy of around 99%. In terms of precision, Sanger sequencing remains the gold standard.

Another important factor is assay sensitivity. NGS has a high sensitivity (detecting variants at 1% or lower) compared to Sanger sequencing (15% to 20%). However, the clinical significance of rare mutations detected by NGS but missed by Sanger sequencing is not always clear, as is the case with the HIV Drug Resistance (HIVDR) test. For this reason, along with the cost of new equipment, the analytical complexity, and the technical expertise required to adopt an NGS platform, many traditional HIVDR testing labs continue to use Sanger sequencing.

Sanger sequencing applications

The primary uses of Sanger sequencing are to determine, confirm, or identify mutations in DNA sequences. Today, it is routinely used in the following areas:

• Confirmation of sequences and identification of mutations in cloned genes or PCR products
• Verification of NGS sequencing results, including WGS and WES

While many research and testing labs have started to adapt NGS for the following applications, Sanger sequencing is still widely used and regarded as the gold standard. There is still significant work to be done before NGS can completely replace Sanger sequencing in these laboratory tests.

• Identifying mutations and analyzing gene-editing efficiency in CRISPR-generated mutant pools
• Detecting gene mutations in a whole gene, a few genes, or specific gene regions in clinical samples for disease or cancer diagnosis
• 16s rRNA gene sequencing for applications in clinical microbiology
• Mitochondrial DNA sequencing for genetic disease testing and human identification
• HLA (Human Leukocyte Antigen) typing to benefit organ transplantation
• HIV drug resistance testing to guide targeted therapy for HIV variants
• HPV genotyping to identify different subtypes

Read our blogs for more on Sanger sequencing applications.