DNA sequencing, the process of determining the order of the nucleotide bases—adenine (A), cytosine (C), guanine (G), and thymine (T)—in a DNA molecule, is fundamental to modern biology and medicine. This process offers insights into heredity, disease mechanisms, and evolution. Two key technologies, Sanger sequencing and Illumina sequencing, represent different approaches to reading DNA, each with strengths suited for specific scientific goals.
The Foundations: Sanger Sequencing
Developed by Frederick Sanger in 1977, this method was the first widely adopted technology for DNA sequencing and remained the dominant approach for three decades. Its invention was a foundational moment in molecular biology that enabled the mapping of the human genome. Known as the “chain-termination method,” it works by synthesizing new strands of DNA from a template, using a process that identifies the final nucleotide added to each new strand.
The process uses modified molecules called dideoxynucleotides (ddNTPs). When a ddNTP is incorporated into a growing DNA chain, it halts further extension of that strand. By including a small amount of each of the four ddNTPs in the reaction, a collection of DNA fragments is generated. These fragments all end at different positions corresponding to a specific nucleotide and are then separated by size using capillary electrophoresis.
Each fragment is tagged with a fluorescent dye corresponding to its final base. As the fragments move through a capillary, a laser excites the dyes, and a detector reads the color of each fragment as it passes from shortest to longest. This process generates a chromatogram that reveals the DNA sequence. The method is known for its high per-read accuracy of 99.99%.
The Revolution: Illumina Sequencing
As a leading “Next-Generation Sequencing” (NGS) technology, Illumina changed the scale and speed of genomics. Instead of reading one long DNA strand, Illumina uses a method called “Sequencing by Synthesis” (SBS) to read millions of DNA fragments simultaneously in a parallel fashion. This approach greatly increased the amount of data that could be generated in a single experiment, accelerating research across all fields of biology.
The process begins by fragmenting the source DNA into shorter pieces. DNA sequences called adapters are attached to the ends of these fragments, allowing them to bind to a glass slide called a flow cell. Once anchored, the fragments undergo bridge amplification, where each fragment is copied many times to create a dense cluster of identical DNA molecules. This step generates the strong signal needed for detection.
With millions of clusters on the flow cell, the sequencing reaction begins. In each cycle, a single nucleotide with a removable fluorescent tag and a reversible terminator is added to every strand in every cluster. A high-resolution camera then captures an image of the flow cell, recording the color emitted from each cluster, which corresponds to the specific base incorporated. The fluorescent tag and terminator are chemically removed, preparing the strands for the next cycle of adding a base, imaging, and cleaving.
Key Distinctions Between Sanger and Illumina
The primary distinction is technological: Sanger uses the chain-termination method for single fragments, while Illumina uses sequencing-by-synthesis for millions of fragments at once. This difference directly impacts throughput and scale. Sanger is a low-throughput method suited for focused analysis, whereas Illumina’s parallel approach provides the high throughput needed for sequencing entire genomes.
Scale directly links to read length and cost. Sanger sequencing produces long, accurate reads of 500-1,000 base pairs. In contrast, Illumina generates much shorter reads, between 50 and 300 base pairs. While individual Illumina reads are shorter, the high volume of data allows for excellent overall accuracy when the reads are computationally aligned to form a consensus sequence. This high-volume approach makes the cost per base for Illumina much lower, enabling large-scale projects.
Data output and analysis also differ. Sanger provides a small number of long reads that are straightforward to analyze. Illumina produces large datasets of short reads that require complex bioinformatic analysis to assemble and interpret. While both methods are highly accurate, they have different error profiles that researchers must consider when designing experiments.
Applications: How Sanger and Illumina Drive Discovery
Sanger sequencing is best for small-scale projects requiring high precision. Its applications include sequencing individual DNA fragments, like those from PCR, and verifying engineered plasmids or viral genomes. Its high per-read accuracy also makes it ideal for validating findings from NGS studies, such as confirming a potential disease-causing mutation.
Illumina’s high throughput powers large-scale genomics. It is used for:
- Whole Genome Sequencing (WGS) of complex organisms.
- Exome sequencing to analyze protein-coding genes.
- RNA sequencing (transcriptomics) to study gene expression.
- Metagenomics to sequence DNA from entire microbial communities.
- Discovering genetic variations, like SNPs, across large populations.
The two technologies are not competitors but are often used in a complementary fashion. A researcher might use an Illumina platform to scan an entire genome for thousands of potential cancer-related mutations. Once a few high-priority candidates are identified, they will turn to Sanger sequencing to confirm the exact sequence of those specific genetic locations. This synergy allows scientists to combine the strengths of both discovery and validation.