The study introduces a novel sequencing-based method called RNA-Seq, which uses high-throughput sequencing to map transcribed regions of the yeast genome. The method was applied to generate a high-resolution transcriptome map, revealing that 74.5% of the unique sequence of the yeast genome is transcribed. The researchers successfully mapped 5' and 3' UTR boundaries, confirmed known and predicted introns, and identified alternative initiation codons and upstream open reading frames (uORFs). They also found unexpected 3' end heterogeneity and many overlapping genes, as well as novel transcribed regions not identified by other methods. RNA-Seq was found to be at least as accurate as DNA microarrays for quantifying RNA expression levels and has a much larger dynamic range. The results suggest that the yeast transcriptome is more complex than previously appreciated and that RNA-Seq will be a valuable approach for high-resolution mapping of transcriptomes in various organisms.The study introduces a novel sequencing-based method called RNA-Seq, which uses high-throughput sequencing to map transcribed regions of the yeast genome. The method was applied to generate a high-resolution transcriptome map, revealing that 74.5% of the unique sequence of the yeast genome is transcribed. The researchers successfully mapped 5' and 3' UTR boundaries, confirmed known and predicted introns, and identified alternative initiation codons and upstream open reading frames (uORFs). They also found unexpected 3' end heterogeneity and many overlapping genes, as well as novel transcribed regions not identified by other methods. RNA-Seq was found to be at least as accurate as DNA microarrays for quantifying RNA expression levels and has a much larger dynamic range. The results suggest that the yeast transcriptome is more complex than previously appreciated and that RNA-Seq will be a valuable approach for high-resolution mapping of transcriptomes in various organisms.