Nature Methods
- 5, 629 - 635 (2008)
Published online: 25 May 2008; | doi:10.1038/nmeth.1216
Efficient targeted transcript discovery via array-based normalization of RACE librariesSarah Djebali1, 9, Philipp Kapranov2, 9, Sylvain Foissac3, 9, Julien Lagarde1, 9, Alexandre Reymond4, 9, Catherine Ucla5, Carine Wyss5, Jorg Drenkow2, Erica Dumais2, Ryan R Murray6, Chenwei Lin6, David Szeto6, France Denoeud1, Miquel Calvo7, Adam Frankish8, Jennifer Harrow8, Periklis Makrythanasis5, Marc Vidal6, Kourosh Salehi-Ashtiani6, Stylianos E Antonarakis5, Thomas R Gingeras2 & Roderic Guigó1, 31
Grup de Recerca en Informàtica Biomèdica, Institut Municipal d'Investigació Mèdica/Universitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain. 2
Affymetrix, Inc., 3420 Central Expressway, Santa Clara, California 95051, USA. 3
Center for Genomic Regulation, Dr. Aiguader 88, 08003 Barcelona, Spain. 4
Center for Integrative Genomics, University of Lausanne, Genopole Building, 1015 Lausanne, Switzerland. 5
Department of Genetic Medicine and Development, University of Geneva Medical School, 1 rue Michel Servet, 1211 Geneva, Switzerland. 6
Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA. 7
Departament d'Estadística, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain. 8
Human and Vertebrate Analysis and Annotation Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1HH, UK. 9
These authors contributed equally to this work.
Correspondence should be addressed to Roderic Guigó roderic.guigo@crg.es Rapid amplification of cDNA ends (RACE) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. To improve sampling efficiency of human transcripts, we hybridized the products of the RACE reaction onto tiling arrays and used the detected exons to delineate a series of reverse-transcriptase (RT)-PCRs, through which the original RACE transcript population was segregated into simpler transcript populations. We independently cloned the products and sequenced randomly selected clones. This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.
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