This review by Schadt, Turner, and Kasarskis discusses the emergence of third-generation sequencing (TGS) technologies, which are poised to revolutionize genomics and related fields. First- and second-generation sequencing (SGS) technologies have significantly advanced our understanding of genome sequences, methylomes, transcriptomes, and protein-DNA interactions, but they have limitations in certain applications. TGS technologies, such as single-molecule sequencing, offer longer read lengths, faster turnaround times, and lower costs, addressing these limitations. The review highlights the advantages of TGS, including higher throughput, faster sequencing, longer read lengths, higher consensus accuracy, and reduced sample requirements. It also discusses the challenges and potential of various TGS technologies, such as single-molecule real-time sequencing (SMRT), nanopore sequencing, and direct imaging techniques. The authors emphasize the need for new informatics tools to analyze the unique characteristics of TGS data, such as stochasticity and high data volume, to fully capitalize on the benefits of these advanced sequencing methods.This review by Schadt, Turner, and Kasarskis discusses the emergence of third-generation sequencing (TGS) technologies, which are poised to revolutionize genomics and related fields. First- and second-generation sequencing (SGS) technologies have significantly advanced our understanding of genome sequences, methylomes, transcriptomes, and protein-DNA interactions, but they have limitations in certain applications. TGS technologies, such as single-molecule sequencing, offer longer read lengths, faster turnaround times, and lower costs, addressing these limitations. The review highlights the advantages of TGS, including higher throughput, faster sequencing, longer read lengths, higher consensus accuracy, and reduced sample requirements. It also discusses the challenges and potential of various TGS technologies, such as single-molecule real-time sequencing (SMRT), nanopore sequencing, and direct imaging techniques. The authors emphasize the need for new informatics tools to analyze the unique characteristics of TGS data, such as stochasticity and high data volume, to fully capitalize on the benefits of these advanced sequencing methods.