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

Pius M. Brzoska, Clark Brown, Michael Cassel, Toni Ceccardi, Valentina Di Francisco, Alex Dubman, Jason Evans, Rixun Fang, Michael Harris, Jeffrey Hoover, Fangqi Hu, Charles Larry, Peter Li, Michael Malicdem, Sergei Maltchenko, Mark Shannon, Sarah Perkins, Karen Poulter, Marion Webster-Laig, Chunlin XiaoSonny Young, Gene Spier, Karl Guegler, Dennis Gilbert, Raymond R. Samaha

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)437-445
Number of pages9
JournalGenomics
Volume87
Issue number4
DOIs
StatePublished - Apr 2006

Keywords

  • Novel human genes
  • RT-PCR

ASJC Scopus subject areas

  • Genetics

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