5′ Long serial analysis of gene expression (LongSAGE) and 3′ LongSAGE for transcriptome characterization and genome annotation

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Abstract
Complete genome annotation relies on precise identification of transcription units bounded by a transcription initiation site (TIS) and a polyadenylation site (PAS). To facilitate this process, we developed a set of two complementary methods, 5′ Long serial analysis of gene expression (LS) and 3′LS. These analyses are based on the original SAGE and LS methods coupled with full-length cDNA cloning, and enable the high-throughput extraction of the first and the last 20 bp of each transcript. We demonstrate that the mapping of 5′LS and 3′LS tags to the genome allows the localization of TIS and PAS. By using 537 tag pairs mapping to the region of known genes, we confirmed that >90% of the tag pairs appropriately assigned to the first and last exons. Moreover, by using tag sequences as primers for RT-PCRs, we were able to recover putative full-length transcripts in 81% of the attempts. This large-scale generation of transcript terminal tags is at least 20–40 times more efficient than full-length cDNA cloning and sequencing in the identification of complete transcription units. The apparent precision and deep coverage makes 5′LS and 3′LS an advanced approach for genome annotation through whole-transcriptome characterization.