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Nucleic Acids Research 2004 32(12):e94; doi:10.1093/nar/gnh095
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Published online 6 July 2004

Nucleic Acids Research, Vol. 32 No. 12 © Oxford University Press 2004; all rights reserved

Generation of longer 3' cDNA fragments from massively parallel signature sequencing tags

Ana Paula M. Silva, Jianjun Chen1, Dirce M. Carraro, San Ming Wang2 and Anamaria A. Camargo*

Laboratory of Molecular Biology and Genomics, Ludwig Institute for Cancer Research, São Paulo 01509-010, Brazil, 1 Department of Medicine, University of Chicago, Chicago, IL 60637, USA and 2 Functional Genomics Laboratory, ENH Research Institute, Northwestern University, Evanston, IL 60201, USA

* To whom correspondence should be addressed. Tel: +55 11 3388 3248; Fax: +55 11 3207 7001; Email: anamaria{at}compbio.ludwig.org.br

Received May 5, 2004; Revised and Accepted June 16, 2004

Massively Parallel Signature Sequencing (MPSS) is a powerful technique for genome-wide gene expression analysis, which, similar to SAGE, relies on the production of short tags proximal to the 3'end of transcripts. A single MPSS experiment can generate over 107 tags, providing a 10-fold coverage of the transcripts expressed in a human cell. A significant fraction of MPSS tags cannot be assigned to known transcripts (orphan tags) and are likely to be derived from transcripts expressed at very low levels (~1 copy per cell). In order to explore the potential of MPSS for the characterization of the human transcriptome, we have adapted the GLGI protocol (Generation of Longer cDNA fragments from SAGE tags for Gene Identification) to convert MPSS tags into their corresponding 3' cDNA fragments. GLGI-MPSS was applied to 83 orphan tags and 41 cDNA fragments were obtained. The analysis of these 41 fragments allowed the identification of novel transcripts, alternative tags generated from polymorphic and alternatively spliced transcripts, as well as the detection of artefactual MPSS tags. A systematic large-scale analysis of the genome by MPSS, in combination with the use of GLGI-MPSS protocol, will certainly provide a complementary approach to generate the complete catalog of human transcripts.


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