DNA sequencing is the process of deciphering the exact order of the chemical building blocks, known as bases, that make up a DNA molecule. This genetic code holds the instructions for all living organisms, making sequencing an important tool for understanding biological processes and diseases. The evolution of DNA sequencing technology has occurred in distinct “generations,” each representing a leap in how efficiently and comprehensively we can read genetic information.
Understanding DNA Sequencing Generations
The first generation of DNA sequencing was largely defined by Sanger sequencing, developed by Frederick Sanger in 1977. This method provided high accuracy for individual DNA fragments. However, it was limited by low throughput, sequencing only short DNA segments (300 to 1,000 base pairs), and was expensive for larger projects.
Second-generation sequencing, often called Next-Generation Sequencing (NGS), enabled massively parallel sequencing. Millions of short DNA fragments (100 to 600 base pairs) could be sequenced simultaneously, making large-scale genome projects more feasible and cost-effective. Despite advancements, NGS relies on fragmenting DNA and computationally reassembling these short reads. This can be challenging for complex or repetitive genome regions and may introduce biases due to required DNA amplification. These limitations spurred the development of third-generation sequencing technologies.
How Third Generation Sequencing Works
Third-generation sequencing technologies sequence individual DNA molecules directly, bypassing the need for initial PCR amplification. This direct approach eliminates biases introduced during amplification and simplifies sample preparation. A defining characteristic is their ability to generate long reads, ranging from thousands to millions of base pairs in a single continuous read.
These platforms also offer real-time data acquisition, meaning data is generated and analyzed as the process unfolds, providing immediate insights. Two prominent technologies are Pacific Biosciences (PacBio) Single Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore Technologies (ONT) sequencing. PacBio SMRT sequencing observes DNA polymerase activity in tiny wells called zero-mode waveguides (ZMWs), where fluorescently labeled nucleotides are incorporated into a growing DNA strand, emitting a detectable light signal. Oxford Nanopore sequencing passes individual DNA or RNA molecules through a tiny protein pore (nanopore) embedded in a membrane, detecting characteristic changes in electrical current as different bases traverse the pore.
What Third Generation Sequencing Enables
The unique features of third-generation sequencing, such as long reads, direct sequencing, and real-time data acquisition, have expanded its applications. Long reads are particularly useful for accurately assembling entire genomes, especially in regions with repetitive DNA sequences, large structural variations, and gene duplications that short-read technologies struggle to resolve. This improved assembly leads to a more complete and accurate understanding of genomic architecture.
Third-generation sequencing also allows for direct detection of epigenetic modifications, such as DNA methylation, without separate chemical conversion steps. These modifications, which influence gene expression, are identified because they alter the signal produced during sequencing, providing insights into gene regulation and disease mechanisms. These technologies are also effective for sequencing full-length RNA molecules, known as isoforms, offering a comprehensive view of gene expression and alternative splicing patterns.
The portability and real-time capabilities of some third-generation sequencers, like Oxford Nanopore’s MinION, enable rapid pathogen identification and outbreak tracking outside traditional laboratory settings. This has been beneficial for quick diagnosis and epidemiological surveillance during infectious disease outbreaks, as seen in the 2015 Ebola outbreak. In advanced cancer genomics, long-read sequencing helps identify complex genomic rearrangements and structural variants that contribute to cancer development, often missed by shorter reads. Beyond human health, these technologies are applied in agriculture for crop improvement, in environmental science for understanding complex microbial communities, and in biodiversity studies, providing a deeper understanding of diverse biological systems.