Sanger sequencing and traditional next-generation sequencing (NGS) are similar in concept, . Both techniques involve using DNA polymerase to add fluorescent nucleotides one at a time onto a growing DNA or RNA template strand. The fluorescent tags allow the identification of each successively incorporated nucleotide.
Where these two sequencing methods differ is in each method’s sequencing volume. The Sanger method sequences DNA or RNA one strand at a time, while next-generation sequencing – initially called “massively-parallel sequencing” – can simultaneously sequence hundreds to thousands of genes at one time.
More recently, new NGS methodologies have emerged. Often referred to as third-generation sequencing, these techniques enable the analysis of much longer DNA and RNA fragments than possible using either Sanger or traditional NGS – delivering significant advantages for a range of applications, including genome assembly and the analysis of large genomic aberrations (e.g. structural variants and repeats). One such technique, nanopore sequencing, enables the analysis of any length of DNA/RNA fragment – from short (20 bp) to ultra long (>4 Mbp). Furthermore, as amplification is not required, nanopore sequencing eliminates amplification bias and allows direct analysis of base modifications (e.g. methylation) alongside the nucleotide sequence While Sanger sequencing is still used frequently in clinical research labs, the use of NGS in these labs is expanding rapidly through the facility to cost-efficiently analyze a greater number of targets and samples at higher sensitivity .