This course provides a systematic overview of the three generations of high-throughput sequencing technology. The first-generation Sanger method is based on double-deoxy termination and electrophoretic separation; it offers high accuracy but low throughput. The second generation, represented by Illumina and MGI, employs simultaneous synthesis and sequencing along with reversible blocking technology. It uses chip-based cluster amplification, cyclic extension of fluorescently labeled bases, and laser scanning to read sequences, supporting parallel sequencing of multiple samples. Third-generation technologies include PacBio’s Single-Molecule Real-Time (SMRT) sequencing and ONT’s nanopore sequencing. The former monitors the polymerase synthesis process in a zero-mode waveguide pore to generate long reads with random errors; the latter utilizes a nanopore protein and a motor protein to identify bases via electrical signals, supporting ultra-long reads and direct detection of methylation. The course concludes with a comparison of the differences among the three generations of technologies in terms of throughput, read length, accuracy, and application scenarios, providing methodological guidance for genomics research.