Third-generation sequencing (TGS), also known as long-read sequencing, is a technology used for DNA sequencing that provides significantly longer read lengths compared to previous generations of sequencing technologies. TGS methods offer several advantages, including the ability to sequence through repetitive regions of the genome, capture structural variations, and characterize epigenetic modifications. One of the most widely used TGS platforms is Pacific Biosciences’ Single Molecule, Real-Time (SMRT) sequencing.
Here is some detailed information about third-generation sequencing:
- Principle: Third-generation sequencing technologies employ different approaches but share the common principle of directly sequencing single DNA molecules. These methods typically do not rely on amplification or the need to break DNA into smaller fragments. Instead, TGS platforms can generate long reads, ranging from several thousand to tens of thousands of bases, enabling the sequencing of contiguous genomic regions.
- Pacific Biosciences (PacBio) SMRT Sequencing: The PacBio SMRT sequencing is a leading third-generation sequencing platform. It utilizes a real-time, single-molecule sequencing approach. This method involves the incorporation of fluorescently labeled nucleotides into DNA strands, and as each nucleotide is added to the growing strand, a fluorescence signal is emitted and detected. The signals are recorded over time, allowing the determination of DNA sequence by identifying the order of nucleotide additions.
- Oxford Nanopore Technologies (ONT): Another prominent TGS platform is provided by Oxford Nanopore Technologies. It employs nanopore-based sequencing, where DNA strands are passed through tiny nanopores, and changes in electrical conductivity are measured as the DNA molecules translocate through the nanopores. These changes are then translated into DNA sequence information.
- Advantages of Third-Generation Sequencing: a. Long reads: The ability to generate long reads allows for the assembly of complex genomes, accurate identification of structural variations, and more precise characterization of repetitive regions. b. Capturing structural variations: TGS platforms excel at detecting large structural variations, such as insertions, deletions, duplications, inversions, and translocations. These variations are crucial for understanding genetic diseases and genome evolution. c. Epigenetic modifications: TGS technologies can provide insights into DNA methylation patterns and other epigenetic modifications that play a significant role in gene expression regulation and disease development. d. Real-time sequencing: Some TGS platforms offer real-time sequencing, enabling immediate data analysis during the sequencing run. This feature facilitates rapid turnaround time for certain applications.
- Limitations of Third-Generation Sequencing: a. Error rates: TGS technologies, particularly early iterations, have relatively higher error rates compared to second-generation sequencing methods like Illumina sequencing. However, advancements in base calling algorithms and improvements in sequencing chemistries have significantly reduced error rates. b. Lower throughput: Third-generation sequencing platforms typically have lower throughput compared to second-generation sequencers. However, recent developments have increased the yield and throughput of TGS platforms, narrowing the gap. c. Higher cost: The cost per base of TGS is generally higher than second-generation sequencing methods. However, as the technology advances and becomes more widespread, costs are expected to decrease.
Third-generation sequencing has revolutionized genomics research by providing long-read capabilities, enabling the comprehensive analysis of complex genomes and structural variations. These technologies continue to evolve, offering enhanced sequencing accuracy, improved throughput, and reduced costs, making them indispensable tools in various fields, including genomics, genetics, and personalized medicine.