An efficient and high-throughput approach for experimental validation of novel human gene predictions.
A highly automated RT-PCR-based approach has been established to validate novel human gene predictions with no prior experimental evidence of mRNA splicing (ab initio predictions). Ab initio gene predictions were selected for high-throughput validation using predicted protein classification, sequence similarity to other genomes, colocalization with an MPSS tag, or microarray expression. Initial microarray prioritization followed by RT-PCR validation was the most efficient combination, resulting in approximately 35% of the ab initio predictions being validated by RT-PCR. Of the 7252 novel genes that were prioritized and processed, 796 constituted real transcripts. In addition, high-throughput RACE successfully extended the 5' and/or 3' ends of >60% of RT-PCR-validated genes. Reevaluation of these transcripts produced 574 novel transcripts using RefSeq as a reference. RT-PCR sequencing in combination with RACE on ab initio gene predictions could be used to define the transcriptome across all species.
Brzoska
Pius M
PM
Applied Biosystems, 850 Lincoln Center Drive, Foster City, CA 94404, USA.
Brown
Clark
C
Cassel
Michael
M
Ceccardi
Toni
T
Di Francisco
Valentina
V
Dubman
Alex
A
Evans
Jason
J
Fang
Rixun
R
Harris
Michael
M
Hoover
Jeffrey
J
Hu
Fangqi
F
Larry
Charles
C
Li
Peter
P
Malicdem
Michael
M
Maltchenko
Sergei
S
Shannon
Mark
M
Perkins
Sarah
S
Poulter
Karen
K
Webster-Laig
Marion
M
Xiao
Chunlin
C
Young
Sonny
S
Spier
Gene
G
Guegler
Karl
K
Gilbert
Dennis
D
Samaha
Raymond R
RR
D000465:Algorithms; D017398:Alternative Splicing; D019295:Computational Biology; D020869:Gene Expression Profiling; D016678:Genome; D015894:Genome, Human; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D011237:Predictive Value of Tests; D011506:Proteins / Q000145:classification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012984:Software
D011506:Proteins