Transcript Profiling Analysis Through Paired-End Ditag (PET) Approach Coupled with Deep Sequencing Reveals Transcriptome Complexity in Yeast

Abstract

The identification of structural and functional elements encoded in a genome is a challenging task. Although the transcriptome of budding yeast has been extensively analyzed, the boundaries and untranslated regions of yeast genes remain elusive. To address this least-explored field of yeast genomics, we performed a transcript profiling analysis through paired-end ditag (PET) approach coupled with deep sequencing. With 562,133 PET sequences we accurately defined the boundaries and untranslated regions of 3,409 ORFs, suggesting many yeast genes have multiple transcription start sites (TSSs). We also identified 85 previously uncharacterized transcripts either in intergenic regions or from the opposite strand of reported genomic features. Furthermore, our data revealed the extensive 3′ end heterogeneity of yeast genes and identified a novel putative motif for polyadenylation. This study would serve as an invaluable resource for elucidating the regulation and evolution of yeast genes. Here we present a detailed protocol with minor modifications, which could be broadly applied to investigate transcripts from budding yeast to mammalian organisms.