Third-generation sequencing (TGS) is a new generation of single-molecule sequencing technologies that aim to overcome the limitations of first- and second-generation sequencing (SGS) technologies. TGS technologies offer the potential for dramatically longer read lengths, shorter time to result, and lower overall cost. These technologies are designed to provide higher throughput, faster turnaround time, longer read lengths, higher consensus accuracy, smaller amounts of starting material, and lower cost compared to SGS technologies. First-generation sequencing, developed by Sanger in 1975, was the first method used to sequence DNA. It had a read length of approximately 800 bases and was limited by its low throughput and high cost. Second-generation sequencing, which emerged in 2005, improved upon SGS by achieving higher throughput and lower cost. However, it still had limitations, such as the need for PCR amplification, which could introduce errors and biases. TGS technologies, such as Pacific Biosciences' SMRT sequencing, use single-molecule real-time sequencing to directly observe DNA polymerase as it synthesizes a DNA strand. This allows for longer read lengths and higher accuracy. Other TGS technologies, such as nanopore sequencing, use the electrical properties of DNA molecules passing through a nanopore to detect individual bases. These technologies have the potential to provide longer read lengths and lower costs compared to SGS technologies. TGS technologies also offer new informatics opportunities, as they can provide longer reads that are more informative for genome assembly. However, they also present new challenges, such as the need to account for higher error rates and different error profiles compared to SGS technologies. Despite these challenges, TGS technologies have the potential to revolutionize genomics by providing more accurate and comprehensive sequencing data.Third-generation sequencing (TGS) is a new generation of single-molecule sequencing technologies that aim to overcome the limitations of first- and second-generation sequencing (SGS) technologies. TGS technologies offer the potential for dramatically longer read lengths, shorter time to result, and lower overall cost. These technologies are designed to provide higher throughput, faster turnaround time, longer read lengths, higher consensus accuracy, smaller amounts of starting material, and lower cost compared to SGS technologies. First-generation sequencing, developed by Sanger in 1975, was the first method used to sequence DNA. It had a read length of approximately 800 bases and was limited by its low throughput and high cost. Second-generation sequencing, which emerged in 2005, improved upon SGS by achieving higher throughput and lower cost. However, it still had limitations, such as the need for PCR amplification, which could introduce errors and biases. TGS technologies, such as Pacific Biosciences' SMRT sequencing, use single-molecule real-time sequencing to directly observe DNA polymerase as it synthesizes a DNA strand. This allows for longer read lengths and higher accuracy. Other TGS technologies, such as nanopore sequencing, use the electrical properties of DNA molecules passing through a nanopore to detect individual bases. These technologies have the potential to provide longer read lengths and lower costs compared to SGS technologies. TGS technologies also offer new informatics opportunities, as they can provide longer reads that are more informative for genome assembly. However, they also present new challenges, such as the need to account for higher error rates and different error profiles compared to SGS technologies. Despite these challenges, TGS technologies have the potential to revolutionize genomics by providing more accurate and comprehensive sequencing data.