title: An efficient and high-throughput approach for experimental validation of novel human gene predictions.
abstract: 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.
authors.affiliation: Applied Biosystems, 850 Lincoln Center Drive, Foster City, CA 94404, USA.
authors.forename: Pius M
authors.initials: PM
authors.lastname: Brzoska
authors.forename: Clark
authors.initials: C
authors.lastname: Brown
authors.forename: Michael
authors.initials: M
authors.lastname: Cassel
authors.forename: Toni
authors.initials: T
authors.lastname: Ceccardi
authors.forename: Valentina
authors.initials: V
authors.lastname: Di Francisco
authors.forename: Alex
authors.initials: A
authors.lastname: Dubman
authors.forename: Jason
authors.initials: J
authors.lastname: Evans
authors.forename: Rixun
authors.initials: R
authors.lastname: Fang
authors.forename: Michael
authors.initials: M
authors.lastname: Harris
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Hoover
authors.forename: Fangqi
authors.initials: F
authors.lastname: Hu
authors.forename: Charles
authors.initials: C
authors.lastname: Larry
authors.forename: Peter
authors.initials: P
authors.lastname: Li
authors.forename: Michael
authors.initials: M
authors.lastname: Malicdem
authors.forename: Sergei
authors.initials: S
authors.lastname: Maltchenko
authors.forename: Mark
authors.initials: M
authors.lastname: Shannon
authors.forename: Sarah
authors.initials: S
authors.lastname: Perkins
authors.forename: Karen
authors.initials: K
authors.lastname: Poulter
authors.forename: Marion
authors.initials: M
authors.lastname: Webster-Laig
authors.forename: Chunlin
authors.initials: C
authors.lastname: Xiao
authors.forename: Sonny
authors.initials: S
authors.lastname: Young
authors.forename: Gene
authors.initials: G
authors.lastname: Spier
authors.forename: Karl
authors.initials: K
authors.lastname: Guegler
authors.forename: Dennis
authors.initials: D
authors.lastname: Gilbert
authors.forename: Raymond R
authors.initials: RR
authors.lastname: Samaha
mesh_terms: 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
chemical_list: D011506:Proteins