The article reviews the advancements, challenges, and opportunities in RNA sequencing (RNA-seq) technology. RNA-seq has revolutionized the characterization and quantification of transcriptomes, enabling a more comprehensive understanding of RNA transcripts. Recent developments include improvements in transcription start site mapping, strand-specific measurements, gene fusion detection, small RNA characterization, and alternative splicing event detection. These advancements have provided insights into the dynamic state of genomes, including DNA modifications and RNA changes. The authors discuss the limitations of current RNA-seq methods, such as the need for cDNA synthesis and the challenges in strand-specific analysis, and highlight emerging technologies like direct RNA sequencing (DRS) and methods for profiling low-quantity RNA samples. DRS, which eliminates the cDNA synthesis step, offers advantages in terms of reduced input RNA requirements and improved accuracy. Methods for profiling low-quantity RNA samples, such as amplification-free RNA-seq and hybridization-based approaches, are also discussed. The article concludes by emphasizing the potential of these technologies to advance research in various fields, including personalized medicine and clinical diagnostics.The article reviews the advancements, challenges, and opportunities in RNA sequencing (RNA-seq) technology. RNA-seq has revolutionized the characterization and quantification of transcriptomes, enabling a more comprehensive understanding of RNA transcripts. Recent developments include improvements in transcription start site mapping, strand-specific measurements, gene fusion detection, small RNA characterization, and alternative splicing event detection. These advancements have provided insights into the dynamic state of genomes, including DNA modifications and RNA changes. The authors discuss the limitations of current RNA-seq methods, such as the need for cDNA synthesis and the challenges in strand-specific analysis, and highlight emerging technologies like direct RNA sequencing (DRS) and methods for profiling low-quantity RNA samples. DRS, which eliminates the cDNA synthesis step, offers advantages in terms of reduced input RNA requirements and improved accuracy. Methods for profiling low-quantity RNA samples, such as amplification-free RNA-seq and hybridization-based approaches, are also discussed. The article concludes by emphasizing the potential of these technologies to advance research in various fields, including personalized medicine and clinical diagnostics.