About Sanger Sequencing
Sanger sequencing, the “chain-termination” method was developed by Frederick Sanger and his colleague in 1977. The technology has quickly gained popularity among molecular research labs to sequence DNA fragments using one of the radiolabeled ddNTPs. The manual polyacrylamide sequencing gradually disappeared when automated capillary electrophoresis DNA sequencing using fluorescently labeled ddNTPs was developed in 1986. Core labs in universities and sequencing companies are the major source for the automated sequencing due to the high cost of instruments and dye reagents and better working efficiency.
Human genome project by Sanger sequencing
As the major sequencing tool, the automated Sanger sequencing was used to “complete” the Human Genome Project at an expense of $2.7 billion after 13 years (1990 to 2003) of multi-national collaboration. The draft of the human genome was left with many gaps to fill in over these years and even today there are still gaps to be completed.
Can NGS totally replace Sanger sequencing?
Today, the cost for NGS has significantly reduced and the high-throughput sequencing has gradually replaced Sanger sequencing when throughput is in demand from research to clinical applications. For sequencing of a single gene or a few genes in a limited number of samples for pathology testing and inherited genetic diseases analysis, Sanger sequencing is still the method of choice for economic sequencing. Even for the cases where NGS is chosen, Sanger sequencing method is often used to confirm sequencing accuracy. As reported, Sanger sequencing has 99.99% accuracy while NGS has 99% accuracy. In term of accuracy, Sanger sequencing is still the gold standard.
An important consideration for replacing Sanger sequencing by NGS is assay sensitivity. Although NGS has high sensitivity (1% or even lower) compared to Sanger sequencing (15 to 20%), rare mutations detected by NGS but missed by Sanger sequencing may not have clear clinical significance, as in the HIV Drug Resistance (HIVDR) test case. In this situation, many traditional HIVDR testing labs may stay with the traditional Sanger sequencing due to the cost of the new instrumentation, analytical complexity of the test and technical expertise requirement for adaptation of the NGS platform.
Sanger sequencing applications
The major purpose for Sanger sequencing lies in sequencing, confirming of sequences, or identification of mutated sequences. Today, Sanger sequencing is routinely used in the following areas:
· Confirmation of sequences and mutation identification in cloned genes or PCR products
· Confirmation of NGS sequencing results including WGS and WES
The following application areas are still mainly performed by Sanger sequencing, but many research labs and some testing labs have also adapted NGS in these applications. Nevertheless, Sanger sequencing is still viewed as the gold standard. There is still much work to do for NGS to totally replace Sanger sequencing for lab testing in these applications.
· Mutation identification and gene editing efficiency analysis in CRISPR-generated mutant pools
· Gene mutations in a whole gene, or a few genes or gene regions in clinical samples for disease or cancer diagnosis
· 16s rRNA gene sequencing for clinical microbiology lab applications
· Mitochondrial DNA sequencing for genetic disease testing and human identification
· HLA (Human Leukocyte Antigen) typing for the benefits of organ transplantation
· HIV drug resistance testing for targeted therapy of HIV variants
· HPV genotyping for identifying the different subtypes
Read our blogs for some of the Sanger sequencing applications.