Sanger sequencing, named after its developer Frederick Sanger, is a method for determining the exact sequence of nucleotide bases in a DNA strand. It was considered the gold standard for detecting DNA variants from its invention in the 1970s until the advent of . To perform Sanger sequencing, DNA polymerase, buffers, primers, a nucleotide mix of the four DNA bases, and fluorescence-labelled chain-terminating nucleotides (dideoxy-nucleotides) are required. The incorporation of these chain-terminating (stop) nucleotides during the sequencing reaction halts DNA chain elongation, producing fluorescence-labelled fragments of varying lengths. These fragments are then separated using electrophoresis in a polyacrylamide gel within a sequencing device. By exciting the fluorescent labels with laser light, the corresponding fluorescence colour of the dideoxy-nucleotide can be detected, indicating which base was incorporated at the end of each fragment. Using specialised software, the sequence of bases in the DNA fragments can be read, and deviations from the reference sequence can be identified.

Today, Sanger sequencing is primarily used in situations where NGS reaches technical or bioinformatic limitations and cannot reliably resolve a genotype (e.g., pseudogene regions, repeat expansions, homopolymers, complex rearrangements, hybrid genes) or when specific, already known variants are being analysed as part of targeted diagnostics (e.g., segregation analyses, family studies, prenatal diagnostics). The advantages of Sanger sequencing lie in its quick turnaround time, as samples can be processed individually without the need to batch them for an NGS run. Additionally, Sanger sequencing generates minimal data volume, which is particularly beneficial in cytogenetic diagnostics, where a full NGS dataset is not necessary to evaluate a single variant.