DNA sequencing determines the order of the four bases—adenine (A), thymine (T), cytosine (C), and guanine (G)—in a DNA molecule. This information is crucial for understanding genetics, diagnosing diseases, and advancing biotechnology. This article explores two prominent DNA sequencing technologies: Sanger sequencing and Next-Generation Sequencing (NGS).
What is Sanger Sequencing?
Sanger sequencing, also known as dideoxy chain termination sequencing, was developed in 1977. The method relies on the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) during DNA synthesis. A reaction mixture includes the DNA template, DNA polymerase, a primer, normal deoxynucleotides (dNTPs), and fluorescently labeled ddNTPs. When a ddNTP is incorporated, it terminates DNA strand elongation at that specific base.
This process generates DNA fragments of varying lengths, each ending with a specific ddNTP. These fragments are separated by size, typically using capillary electrophoresis, and fluorescent labels reveal the DNA sequence. Sanger sequencing was instrumental in the Human Genome Project and produces relatively long reads (500-1000 base pairs). Its limitations include low throughput and higher cost for large-scale projects.
What is Next-Generation Sequencing?
Next-Generation Sequencing (NGS) was introduced commercially around 2005 as a significant advancement in DNA sequencing. Its core principle is massively parallel sequencing, allowing it to sequence millions of DNA fragments simultaneously. This parallel approach increases throughput and reduces the cost per base compared to earlier methods.
The NGS workflow begins with preparing a DNA library, involving fragmentation and ligation of adapter sequences to the fragments. These prepared fragments are amplified, and millions are sequenced in parallel. NGS platforms generate shorter reads (50-600 base pairs). Computational methods then align and assemble these short reads into longer, contiguous sequences to reconstruct the original DNA.
Comparing Sanger and Next-Generation Sequencing
Sanger sequencing is not Next-Generation Sequencing; it is an earlier, foundational technology that paved the way for NGS. NGS was developed to overcome Sanger sequencing’s limitations, particularly in scale, speed, and cost for large genomic projects.
Sanger sequencing processes DNA fragments one at a time, making it a low-throughput method suitable for single reactions. Conversely, NGS performs millions of sequencing reactions in parallel, offering ultra-high throughput. The cost per base for large-scale projects is significantly lower with NGS, while Sanger is more expensive for such endeavors.
Sanger typically produces longer reads (500-1000 bp), while NGS generates shorter reads (50-600 bp). Both are highly accurate, but Sanger is often preferred for single-base validation. NGS handles more complex samples due to its deep sequencing capabilities, whereas Sanger often requires specific, purified DNA targets.
When is Each Method Used?
Sanger sequencing retains utility in modern molecular biology. It is routinely used for validating NGS results, confirming specific mutations or variant calls. Sanger sequencing is also ideal for small-scale projects, such as sequencing individual genes, PCR products, or plasmids, where its simplicity is advantageous. Its high accuracy makes it valuable for certain clinical diagnostics involving targeted, known mutations.
Next-Generation Sequencing is the preferred method for large-scale genomic studies due to its high throughput and cost-effectiveness. It is widely applied in whole genome sequencing (WGS) and exome sequencing (WES), which focuses on protein-coding regions. NGS is also used for RNA sequencing (RNA-Seq) to study gene expression, metagenomics for environmental samples, and clinical research for variant discovery and personalized medicine. Both technologies are complementary and play distinct roles in advancing biological and medical understanding.