The Research Progress of Single-Molecule Sequencing and Its Significance in Nucleic Acid Metrology

Abstract

Single-molecule sequencing technology, a novel method for gene sequencing, utilizes nano-sized materials to detect electrical and fluorescent signals. Compared to traditional Sanger sequencing and next-generation sequencing technologies, it offers significant advantages, including ultra-long read lengths, rapid sequencing, and the absence of amplification steps, making it widely applicable across various fields. By examining the development and components of single-molecule sequencing technology, it becomes clear that its unique characteristics provide new opportunities for advancing metrological traceability. Notably, its direct detection capabilities offer a novel approach to nucleic acid metrology. This paper provides a detailed overview of library construction, signal generation and detection, and data analysis methods in single-molecule sequencing and discusses its implications for nucleic acid metrology.

Keywords: nanopore; nucleic acid metrological traceability; single-molecule sequencing.

Figure 2

Flow chart of single-molecule sequencing technology. In signal detection, the red, green, yellow, and blue colors represent fluorescently labeled dNTPs, respectively. In library construction, purple represents the template strand, orange represents the coding strand, and blue repre-sents the adapter sequence.

Figure 1

Development of single-molecule sequencing technology.

Figure 3

Schematic diagram of the zero-mode waveguide hole structure.

Figure 4

Schematic diagram of the structural models of α-HL, MspA, and Phi29 biological nanopores.