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

ABSTRACT: 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.

SUBMITTER: Salehi-Ashtiani K

PROVIDER: S-EPMC2743938 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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

Salehi-Ashtiani Kourosh K Yang Xinping X Derti Adnan A Tian Weidong W Hao Tong T Lin Chenwei C Makowski Kathryn K Shen Lei L Murray Ryan R RR Szeto David D Tusneem Nadeem N Smith Douglas R DR Cusick Michael E ME Hill David E DE Roth Frederick P FP Vidal Marc M

Nature methods 20080615 7

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 appl ...[more]

PMID: 18552854

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