In recent decades, the capacity to decipher the sequence of DNA code has resulted in enormous advances in our understanding of biology and disease. As scientists’ sequencing tools have progressed, it has become even cheaper and easier to read DNA sequences. With lower prices has come an increase in demand: DNA sequencing is now a standard instrument in laboratories all around the world.
The concepts behind Sanger and next generation sequencing are similar in many ways. Both rely on capillary electrophoresis, for example. It is the differences between these two processes that stand out.
How Does Sanger DNA Sequencing Work?
The majority of sequencing techniques, including Sanger methods, are based on the natural process by which a cell copies its DNA. When a cell duplicates its DNA, it employs a specialized enzyme known as a polymerase.
The cell begins by unwinding its DNA into two distinct single strands. Next, polymerase attaches to a single-stranded DNA and fills up the gaps one nucleotide at a time. Then, as the nucleotides (dNTPs; A, T, G, or C) come on board, they fill in the single-stranded DNA to form a complete piece of double-stranded DNA. Therefore, creating a duplicate.
Fredric Sanger created the first generation sequencing technology, Sanger sequencing (SGS), in 1977. During in vitro DNA replication, DNA polymerase selectively incorporates chain-terminating dideoxynucleotides. The generating amplicons are then separated using capillary electrophoresis. In general, Sanger sequencing is a quick and low-cost sequencing approach for small-scale applications.
How Does Next Generation Sequencing Work?
The term next generation sequencing (NGS) refers to a collection of technologies established throughout the last decade. These modern methods provide quick and high-throughput DNA and RNA sequencing.
NGS is similar to Sanger sequencing in that it involves the sequencing of DNA fragments. However, it can manage the sequencing of millions of fragments in a single run. It works on more than one strand at a time and runs them parallel. NGS may sequence a full genome’s worth of DNA in a single experiment.
What Is the Difference?
The primary distinction between Sanger sequencing and NGS is volume. While the Sanger technique only sequences a single DNA fragment at a time, NGS is massively parallel and may sequence millions of fragments at the same time per run. This method allows for the simultaneous sequencing of hundreds to thousands of genes. As a result, NGS provides a better discovery capacity for detecting novel or uncommon variants with deep sequencing.
The Sanger sequencing technology has over 99 percent accuracy, which remains the “gold standard” for fundamental and clinical research applications. In reality, most clinical laboratories use Sanger sequencing to verify gene variations discovered using NGS. However, additional studies are appearing that confirm the accuracy of NGS approaches for variant identification while challenging the need for a more time-consuming and costly validation procedure.
Sanger is the practical choice for a low target number and simple data analysis. NGS is likely the better option when a higher sequencing depth is necessary. However, Sanger has low sensitivity and discovery, and NGS is less cost-effective.