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

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 3LS. 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 5LS and 3LS 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 5LS and 3LS an advanced approach for genome annotation through whole-transcriptome characterization.genome annotation ͉ full-length cDNA ͉ transcription analysis T he complete genome sequences of human and other model organisms (1-5) have raised questions as to the accuracy of previously used gene annotation algorithms. Current genome annotations are mostly based on the growing but still limited cDNA sequence databases. Computational gene prediction algorithms, even when trained based on current cDNA data sets, may not provide reliable ab initio predictions of new genes, and all predictions need to be validated by further experimental evidence (6, 7).Efforts in large scale full-length cDNA sequencing (8-11; reviewed in ref. 12) provide not only the complete sequences of expressed genes but also the ability to locate transcription initiation sites (TISs), polyadenylation sites (PASs), as well as splicing junctions. The localization of these functional sites related to transcripts in the context of the genome can greatly help to define the surrounding regulatory elements such as promoters. However, sequencing all possible full-length transcript clones is an expensive and cumbersome process. Despite current progress, complete disclosure of the transcriptome has yet to be achieved. It is clear, therefore, that the precise identification of all genes and their regulatory elements will require more comprehensive and facile technologies with greater throughput to provide the necessary empirical evidence.Serial analysis of gene expression (SAGE) (13, 14) and massively parallel signature sequencing (MPSS) (15) are high-throughput methods that use short tags (14-21 bp of internal transcript signatures) to count transcripts in transcriptomes. When mapped to assembled genome sequences, these short tags help to locate transcription units in the context of the genome. Because concatenation of these short tags and t...

Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses

“…introduced based on the Cap-trapper method (Carninci and Hayashizaki 1999) to retain 59 intact transcripts, followed by ''tagging'' restriction digestion and the standard LongSAGE method to generate CAGE tags (Shiraki et al 2003). Two other groups including us (Hashimoto et al 2004;Wei et al 2004) also independently developed similar approaches as 59LongSAGE to map TSS. In addition, we simultaneously developed the companion 39LongSAGE method, so as to map both 59 TSS and the exact 39 polyadenylation sites (PASs) to define the boundaries of expressed genes using two end tags (Wei et al 2004).…”

“…Two other groups including us (Hashimoto et al 2004;Wei et al 2004) also independently developed similar approaches as 59LongSAGE to map TSS. In addition, we simultaneously developed the companion 39LongSAGE method, so as to map both 59 TSS and the exact 39 polyadenylation sites (PASs) to define the boundaries of expressed genes using two end tags (Wei et al 2004). Expanding from such a capacity, we then developed the paired-end ditag (PET) method that covalently links the 59 tag and 39 tag of a DNA fragment into a ditag structure for sequencing analysis (Ng et al 2005), thus combining the benefits of the costeffective SAGE and the linkage information from paired-end sequencing.…”

Robust analysis of 5′-transcript ends: a high-throughput protocol for characterization of sequence diversity of transcription start sites

“…These modifications have also been successfully adopted in a human transcriptome analysis project in which over 30 million tags are sequenced from over 250 tissues 14 . Recently, the SAGE method was also modified to recover transcript information at 5¢ ends of mRNA [15][16][17] . MPSS was also developed for in-depth transcriptome analysis using a novel hybridization-based sequencing method 17 .…”

Caracterização de perfis transcricionais de folhas e da região cambial de Eucalyptus grandis usando o SAGE

“…A identificação de mais de um gene para uma única tag ocorre basicamente quando seqüências genômicas são utilizadas no tag mapping e a tag é identificada em regiões mais internas e não na região 3' da seqüência (WEI et al, 2004). Variações do método SAGE originalmente descrito vêm sendo desenvolvidas para facilitar a identificação precisa de genes através do tag mapping e para favorecer a caracterização de novos genes através do uso direto da própria seqüência da tag como primers (MATSUMURA et al, 2003, WEI et al, 2004, GOWDA et al, 2004. Inicialmente foi desenvolvido o SuperSAGE que se baseia no uso de uma enzima de restrição tipoIII para gerar tags maiores que 25 pb (MATSUMURA et al, 2003).…”

“…Apenas poucos pesquisadores empregaram o método SAGE ao estudo da expressão gênica em plantas superiores (MATSUMURA et al, 1999(MATSUMURA et al, , 2003LORENZ & DEAN, 2002;GIBBINGS et al, 2003;COEMANS et al, 2005, BAO et al, 2005, e os primeiros trabalhos realizados com esta metodologia na espécie modelo Arabidopsis thaliana foram publicados apenas recentemente (JUNG et al, 2003;FIZAMES et al, 2004 internas e não na região 3' da seqüência (WEI et al, 2004). Variações do método SAGE originalmente descrito vêm sendo desenvolvidas para facilitar a identificação precisa de genes através do tag mapping e para favorecer a caracterização de novos genes através do uso direto da própria seqüência da tag como primers (MATSUMURA et al, 2003, WEI et al, 2004, GOWDA et al, 2004.…”