Journal Article

Isoform discovery by targeted cloning, 'deep-well' pooling and parallel sequencing

Cusick, Michael E, Tian, Weidong, Salehi-Ashtiani, Kourosh, Makowski, Kathryn, Szeto, David, Roth, Frederick P, Vidal, Marc, Murray, Ryan R, Shen, Lei, Yang, Xinping, Hill, David E, Derti, Adnan, Lin, Chenwei, Hao, Tong, Smith, Douglas R, Tusneem, Nadeem

2008

Overview

The complete set of coding sequences, including all splice isoforms, is not known for any metazoan organism. Combination of a normalized pooling scheme and a new assembly algorithm with 454 sequencing yields a methodological pipeline for isoform discovery. The validated pipeline may now be applied genome-wide. Describing the 'ORFeome' of an organism, including all major isoforms, is essential for a system-level understanding of any species; however, conventional cloning and sequencing approaches are prohibitively costly and labor-intensive. We describe a potentially genome-wide methodology for efficiently capturing new coding isoforms using reverse transcriptase (RT)-PCR recombinational cloning, 'deep-well' pooling and a next-generation sequencing platform. This ORFeome discovery pipeline will be applicable to any eukaryotic species with a sequenced genome.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: Alternative Splicing / Animals / Bioinformatics / Biological Microscopy / Biological Techniques / Biomedical and Life Sciences / Biomedical Engineering/Biotechnology / brief-communication / Cloning / Cloning, Molecular - methods / DNA, Complementary - genetics / Eukaryotes / Expressed Sequence Tags / Female / Genetic recombination / Genomics / Genomics - methods / Humans / Life Sciences / Male / Methods / Nuclear transfer (Cloning) / Open Reading Frames / Polymerase chain reaction / Pregnancy / Protein Isoforms - genetics / Proteomics / Research methodology / Reverse Transcriptase Polymerase Chain Reaction / RNA - genetics / Sequence Analysis - methods