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The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

ABSTRACT: Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

SUBMITTER: Camargo AA

PROVIDER: S-EPMC59775 | biostudies-literature | 2001 Oct

REPOSITORIES: biostudies-literature

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The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

Camargo A A AA Samaia H P HP Dias-Neto E E Simão D F DF Migotto I A IA Briones M R MR Costa F F FF Nagai M A MA Verjovski-Almeida S S Zago M A MA Andrade L E LE Carrer H H El-Dorry H F HF Espreafico E M EM Habr-Gama A A Giannella-Neto D D Goldman G H GH Gruber A A Hackel C C Kimura E T ET Maciel R M RM Marie S K SK Martins E A EA Nobrega M P MP Paco-Larson M L ML Pardini M I MI Pereira G G GG Pesquero J B JB Rodrigues V V Rogatto S R SR da Silva I D ID Sogayar M C MC Sonati M F MF Tajara E H EH Valentini S R SR Alberto F L FL Amaral M E ME Aneas I I Arnaldi L A LA de Assis A M AM Bengtson M H MH Bergamo N A NA Bombonato V V de Camargo M E ME Canevari R A RA Carraro D M DM Cerutti J M JM Correa M L ML Correa R F RF Costa M C MC Curcio C C Hokama P O PO Ferreira A J AJ Furuzawa G K GK Gushiken T T Ho P L PL Kimura E E Krieger J E JE Leite L C LC Majumder P P Marins M M Marques E R ER Melo A S AS Melo M B MB Mestriner C A CA Miracca E C EC Miranda D C DC Nascimento A L AL Nobrega F G FG Ojopi E P EP Pandolfi J R JR Pessoa L G LG Prevedel A C AC Rahal P P Rainho C A CA Reis E M EM Ribeiro M L ML da Ros N N de Sa R G RG Sales M M MM Sant'anna S C SC dos Santos M L ML da Silva A M AM da Silva N P NP Silva W A WA da Silveira R A RA Sousa J F JF Stecconi D D Tsukumo F F Valente V V Soares F F Soares F F Moreira E S ES Nunes D N DN Correa R G RG Zalcberg H H Carvalho A F AF Reis L F LF Brentani R R RR Simpson A J AJ de Souza S J SJ

Proceedings of the National Academy of Sciences of the United States of America 20011001 21

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of a ...[more]

PMID: 11593022

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Project description:BACKGROUND: We have applied a high-throughput pyrosequencing technology for transcriptome profiling of Caenorhabditis elegans in its first larval stage. Using this approach, we have generated a large amount of data for expressed sequence tags, which provides an opportunity for the discovery of putative novel transcripts and alternative splice variants that could be developmentally specific to the first larval stage. This work also demonstrates the successful and efficient application of a next generation sequencing methodology. RESULTS: We have generated over 30 million bases of novel expressed sequence tags from first larval stage worms utilizing high-throughput sequencing technology. We have shown that approximately 14% of the newly sequenced expressed sequence tags map completely or partially to genomic regions where there are no annotated genes or splice variants and therefore, imply that these are novel genetic structures. Expressed sequence tags, which map to intergenic (around 1000) and intronic regions (around 580), may represent novel transcribed regions, such as unannotated or unrecognized small protein-coding or non-protein-coding genes or splice variants. Expressed sequence tags, which map across intron-exon boundaries (around 300), indicate possible alternative splice sites, while expressed sequence tags, which map near the ends of known transcripts (around 600), suggest extension of the coding or untranslated regions. We have also discovered that intergenic and intronic expressed sequence tags, which are well conserved across different nematode species, are likely to represent non-coding RNAs. Lastly, we have incorporated available serial analysis of gene expression data generated from first larval stage worms, in order to predict novel transcripts that might be specifically or predominantly expressed in the first larval stage. CONCLUSION: We have demonstrated the use of a high-throughput sequencing methodology to efficiently produce a snap-shot of transcriptional activities occurring in the first larval stage of C. elegans development. Such application of this new sequencing technique allows for high-throughput, genome-wide experimental verification of known and novel transcripts. This study provides a more complete C. elegans transcriptome profile and, furthermore, gives insight into the evolutionary and biological complexity of this organism.

Dataset Information

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

Publications

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

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