- Third-Generation Sequencing (TGS) refers to a set of advanced DNA sequencing technologies that enable the direct reading of long DNA or RNA molecules in real time, without the need for amplification. These technologies represent a significant evolution from First-Generation (Sanger) and Second-Generation (Next-Generation Sequencing, NGS) platforms by addressing key limitations, such as short read lengths and complex sample preparation.
- TGS enables sequencing of much longer fragments—often tens of thousands of base pairs in a single read—making it a powerful tool for resolving complex genomic regions, structural variations, and full-length transcripts.
- The two most prominent third-generation sequencing platforms are Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). PacBio’s Single Molecule, Real-Time (SMRT) sequencing works by immobilizing a single DNA polymerase molecule at the bottom of a tiny well (called a zero-mode waveguide). As the polymerase incorporates fluorescently labeled nucleotides into a growing DNA strand, the emitted light is detected in real time, allowing the direct observation of DNA synthesis. This method generates long and accurate reads, especially with the newer HiFi (high-fidelity) mode, which achieves high base-call accuracy by circular consensus sequencing.
- Oxford Nanopore sequencing, on the other hand, relies on passing single-stranded DNA or RNA molecules through a biological nanopore embedded in a synthetic membrane. As the nucleotides traverse the pore, they disrupt an ionic current in a sequence-specific manner. This change in current is recorded and translated into nucleotide sequences using sophisticated algorithms. Nanopore sequencing stands out for its portability (e.g., the palm-sized MinION device), real-time data streaming, and ability to sequence ultra-long fragments—sometimes exceeding 2 megabases.
- One of the defining advantages of TGS is the long read length, which greatly simplifies genome assembly, especially in repetitive or structurally complex regions. This capability allows for more accurate detection of structural variants, such as insertions, deletions, duplications, and inversions, which are often missed by short-read platforms. In transcriptomics, TGS enables full-length isoform sequencing, capturing entire mRNA molecules without the need for computational assembly, thus providing deeper insight into alternative splicing and gene regulation.
- Additionally, TGS technologies allow for direct sequencing of RNA and modified bases, such as methylated cytosines, without requiring chemical conversion or enrichment steps. This opens new avenues for studying epigenetics, epitranscriptomics, and real-time dynamics of gene expression, which are difficult to achieve using other sequencing technologies.
- Despite these advantages, TGS platforms face certain challenges. Historically, their per-base error rates were higher than those of NGS, although accuracy has improved significantly with recent innovations, particularly with PacBio HiFi and ONT’s duplex sequencing. TGS also tends to be more expensive on a per-base basis and may require high-quality, high-molecular-weight DNA, which can be difficult to obtain from some samples. However, improvements in hardware, software, and chemistry are steadily overcoming these limitations.
