The article reviews the advancements and applications of PacBio sequencing, a third-generation sequencing technology developed by Pacific BioSciences. PacBio sequencing offers longer read lengths compared to second-generation sequencing (SGS) technologies, making it suitable for addressing complex genomic and transcriptomic challenges. The highly contiguous de novo assemblies produced by PacBio can close gaps in reference genomes and characterize structural variations (SVs) in personal genomes. PacBio's ability to sequence through repetitive regions and detect mutations, particularly those associated with diseases, is highlighted. Additionally, PacBio transcriptome sequencing is advantageous for identifying gene isoforms and novel genes, due to its capability to sequence full-length transcripts. The technology also provides valuable information for detecting base modifications, such as methylation. Hybrid sequencing strategies that combine PacBio with SGS are discussed, as they leverage the complementary strengths of both technologies to overcome their limitations. The article also covers the mechanisms and performance of PacBio sequencing, including the use of SMRTbell templates, zero-mode waveguides, and polymerase-based replication. Despite its advantages, PacBio sequencing faces challenges such as lower throughput, higher error rates, and higher costs per base. The article concludes by discussing the applications of PacBio sequencing in genome, transcriptome, and epigenetics research, emphasizing its potential in studying complex diseases and advancing biomedical research.The article reviews the advancements and applications of PacBio sequencing, a third-generation sequencing technology developed by Pacific BioSciences. PacBio sequencing offers longer read lengths compared to second-generation sequencing (SGS) technologies, making it suitable for addressing complex genomic and transcriptomic challenges. The highly contiguous de novo assemblies produced by PacBio can close gaps in reference genomes and characterize structural variations (SVs) in personal genomes. PacBio's ability to sequence through repetitive regions and detect mutations, particularly those associated with diseases, is highlighted. Additionally, PacBio transcriptome sequencing is advantageous for identifying gene isoforms and novel genes, due to its capability to sequence full-length transcripts. The technology also provides valuable information for detecting base modifications, such as methylation. Hybrid sequencing strategies that combine PacBio with SGS are discussed, as they leverage the complementary strengths of both technologies to overcome their limitations. The article also covers the mechanisms and performance of PacBio sequencing, including the use of SMRTbell templates, zero-mode waveguides, and polymerase-based replication. Despite its advantages, PacBio sequencing faces challenges such as lower throughput, higher error rates, and higher costs per base. The article concludes by discussing the applications of PacBio sequencing in genome, transcriptome, and epigenetics research, emphasizing its potential in studying complex diseases and advancing biomedical research.