Sanger sequencing

Developed by Fred Sanger in 1975, Sanger sequencing was the first method of DNA sequencing. It was the method used for the ground-breaking Human Genome Project, completed in 2003. Other names for this technique are ‘chain-termination sequencing’ and ‘dideoxy sequencing’.

Sanger sequencing remains the most accurate form of DNA sequencing. It is still widely used in clinical laboratories for the following applications:

• Diagnostic sequencing of a single gene.
• Testing for a specific familial sequence variant. This can include:
  • predictive genomic testing in at-risk relatives (for example, for a familial BRCA1 variant conferring breast cancer risk);
  • carrier testing for parents, where a child has an autosomal recessive condition (for example, cystic fibrosis);
  • prenatal testing for known familial variants; and
  • segregation analysis, to aid the interpretation of the pathogenicity of a variant (for example, by establishing if a variant being investigated is present in an affected sibling as well as in the proband).
• To confirm variants that are identified by massively parallel sequencing (sometimes known as next-generation sequencing).
• To fill gaps in massively parallel sequencing data.

• Patient DNA is used as a template in a polymerase chain reaction (PCR).
• A mix of normal bases (dNTPs) and chain-terminating bases (ddNTPs) is used in the PCR reaction.
• When a chain-terminating base is randomly incorporated into a growing DNA chain, it cannot grow any further. This means that DNA fragments of different lengths are generated. Each fragment ends in a chain-terminating base.
• The DNA fragments are then separated by size using capillary electrophoresis.
• Each of the four chain-terminating bases (A/T/C/G) has a different fluorescent label. A laser is used to excite these fluorescently labelled bases at the end of each fragment.
• Shorter fragments come first in the sequence followed by increasingly longer fragments.
• The fluorescence of the base that terminated each length of fragment is recorded, and a chromatograph is generated showing which base is present at which position along the DNA fragment.
• The chromatogram is compared with a reference file to identify any variants.

Advantages

• Gold standard method for accurate detection of single nucleotide variants and small insertions/deletions.
• More flexible for testing for a specific familial variant than massively parallel sequencing.
• Cost effective where single samples need to be tested very urgently, so they cannot be batched up (for example, in prenatal testing or parental carrier testing during a pregnancy).
• Less reliant on computational tools than massively parallel sequencing.
• In some cases, longer fragments (up to approximately 1,000 base pairs) can be sequenced than in massively parallel sequencing.

Limitations

• Limited throughput.
• Not cost effective for sequencing many genes in parallel, or for sequencing the same region in many samples.
• May not detect mosaicism.
• Can require a larger amount of input DNA than massively parallel sequencing.

For a summary table comparing the advantages and disadvantages of the different approaches to gene sequencing (gene panel/WES/WGS), view our Knowledge Hub article, Different approaches to gene sequencing.

• Sanger sequencing is a fast and cost effective way of reading the sequence of small targeted regions of the genome.
• Sanger sequencing is the gold standard method for accurate detection of single nucleotide variants and small insertions/deletions.
• Sanger sequencing is widely used to test for known familial variants, for validation of results obtained through massively parallel sequencing and for some single gene sequencing.

For clinicans

• ThermoFisher Scientific: How Does Sanger Sequencing Work?