Shotgun Sequencing of the Human Transcriptome with ORF Expressed Sequence Tags

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 97; no. 7; pp. 3491 - 3496
• Main Authors: Neto, Emmanuel Dias, Correa, Ricardo Garcia, Verjovski-Almeida, Sergio, Marcelo R. S. Briones, Nagai, Maria Aparecida, da Silva, Wilson, Zago, Marco Antonio, Bordin, Silvana, Costa, Fernando Ferreira, Goldman, Gustavo Henrique, Carvalho, Alex F., Matsukuma, Adriana, Baia, Gilson S., Simpson, David H., Brunstein, Adriana, Paulo S. L. de Oliveira, Bucher, Philipp, Jongeneel, C. Victor, O'Hare, Michael J., Soares, Fernando, Brentani, Ricardo R., Luis F. L. Reis, de Souza, Sandro J., Andrew J. G. Simpson
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 28-03-2000; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences
• Subjects: Animals; Biological Sciences; Breast cancer; Breast neoplasms; Breast Neoplasms - genetics; Complementary DNA; Databases, Factual; DNA, Complementary; Expressed Sequence Tags; Genes; Genomes; Genomics; Humans; Libraries; Molecular Sequence Data; Open Reading Frames; Reverse Transcriptase Polymerase Chain Reaction; Sequencing; Transcription, Genetic; Transcriptomes; Tumors
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.97.7.3491

Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription-PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome.