Isoform discovery by targeted cloning, 'deep-well' pooling and parallel sequencing.
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.
Center for Cancer Systems Biology, Department of Cancer Biology, Dana Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA. kourosh_salehi-ashtiani@dfci.harvard.edu
Kourosh
K
Salehi-Ashtiani
Xinping
X
Yang
Adnan
A
Derti
Weidong
W
Tian
Tong
T
Hao
Chenwei
C
Lin
Kathryn
K
Makowski
Lei
L
Shen
Ryan R
RR
Murray
David
D
Szeto
Nadeem
N
Tusneem
Douglas R
DR
Smith
Michael E
ME
Cusick
David E
DE
Hill
Frederick P
FP
Roth
Marc
M
Vidal
D017398:Alternative Splicing; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D020224:Expressed Sequence Tags; D005260:Female; D023281:Genomics / Q000379:methods; D006801:Humans; D008297:Male; D016366:Open Reading Frames; D011247:Pregnancy; D020033:Protein Isoforms / Q000235:genetics*; D012313:RNA / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017421:Sequence Analysis / Q000379:methods*
D018076:DNA, Complementary; D020033:Protein Isoforms; D012313:RNA