As technology evolves with each passing day, the iteration of tools based on technological advances becomes increasingly rapid. In the life sciences industry, sequencing technology has progressed from the initial Sanger sequencing to NGS, from high-throughput sequencing to single-molecule sequencing. Some people classify these as the first and second generations, while others refer to third, fourth, or even “2.5 generation”. But how are these so-called intergenerational relationships defined, and what are the criteria? Does each technology have its own strengths, or is there truly an “intergenerational relationship”?
What is the intergenerational division standard?
Currently, the industry-recognised unified division standards only distinguish “first generation” and “next generation,” used to separate Sanger sequencing from non-Sanger sequencing. These two types of technologies differ significantly in sequencing principles and throughput, yet share similarities. For example, whether it is Sanger dideoxy sequencing, edge-synthesis in high-throughput sequencing, or sequencing-by-synthesis methods, all rely on nucleotide polymerisation.
Distinguishing “second generation” from “third generation” sequencing
The term “third-generation sequencing” emerged in 2008–2009, referring mainly to new sequencing technologies that differ from large-scale parallel sequencing. Some scholars argue that single-molecule sequencing, real-time sequencing, and core methods distinguish these from prior methods, forming the defining characteristics of next-generation sequencing technologies. Typically, second-generation sequencing refers to high-throughput, large-scale parallel sequencing, while third-generation sequencing refers to single-molecule sequencing without DNA amplification.
However, some scholars suggest that these intergenerational divisions may be commercially motivated, as technological “generations” are often perceived as evolutionary upgrades. In fact, single-molecule sequencing was first described conceptually in 2003, while high-throughput sequencing reached the market in 2005. In 2008, Helicos BioSciences launched the first single-molecule sequencer, followed by Pacific Biosciences and Oxford Nanopore. Nevertheless, single-molecule sequencing required more complex technical systems, its development was slower than expected, and it has not yet reached the market scale of high-level sequencing technology. Helicos BioSciences went bankrupt in 2012, despite its technology aligning fully with current definitions of third-generation sequencing.
Conclusion
Sequencing technology continues to develop rapidly. The current intergenerational division lacks uniform industry standards. Both Sanger sequencing and non-Sanger sequencing (NGS/high-throughput sequencing) have driven the genomics revolution. Sanger sequencing contributed significantly to the Human Genome Project and remains routine in many laboratories. Non-Sanger sequencing dominates modern genome research and applications, reducing the economic barriers to widespread genome sequencing and supporting precision medicine and other fields. Single-molecule technology represents an important direction in sequencing development. With continued industry effort, it is beginning to emerge, though it will take time to mature. Each sequencing technology has distinct characteristics and suitable applications.
References
- Braslavsky I, Hebert B, Kartalov E, Quake SR. Sequence information can be obtained from single DNA molecules. Proc Natl Acad Sci USA, 2003, 100(7):3960–3964.
- Wangwei, 2019, http://blog.sina.com.cn/s/blog_bcb043950102zbn2.html
- Goodwin S, McPherson JD, McCombie WR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet, 2016, 17(6):333–351.
