An efficient and high-throughput approach for experimental validation of novel human gene predictions

• Published in: Genomics (San Diego, Calif.) Calif.), 2006-04, Vol.87 (4), p.437-445
• Main Authors: Brzoska, Pius M., Brown, Clark, Cassel, Michael, Ceccardi, Toni, Di Francisco, Valentina, Dubman, Alex, Evans, Jason, Fang, Rixun, Harris, Michael, Hoover, Jeffrey, Hu, Fangqi, Larry, Charles, Li, Peter, Malicdem, Michael, Maltchenko, Sergei, Shannon, Mark, Perkins, Sarah, Poulter, Karen, Webster-Laig, Marion, Xiao, Chunlin, Young, Sonny, Spier, Gene, Guegler, Karl, Gilbert, Dennis, Samaha, Raymond R.
• Format: Article
• Language: English
• Subjects: Algorithms; Alternative Splicing; Biological and medical sciences; Computational Biology; Fundamental and applied biological sciences. Psychology; Gene Expression Profiling; Genes. Genome; Genetics of eukaryotes. Biological and molecular evolution; Genome; Genome, Human; Humans; Molecular and cellular biology; Molecular genetics; Novel human genes; Oligonucleotide Array Sequence Analysis; Predictive Value of Tests; Proteins - classification; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RT-PCR; Sequence Analysis, DNA; Software
• ISSN: 0888-7543; 1089-8646
• DOI: 10.1016/j.ygeno.2005.11.016

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.