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Methods

Gene prediction and verification in a compact genome with numerous small introns

¹ Laboratory for Computational Genomics and Department of Computer Science, Washington University, St. Louis, Missouri 63130, USA , ² Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA , ³ Department of Molecular Microbiology, Washington University Medical School, St. Louis, Missouri 63110-1093, USA

The genomes of clusters of related eukaryotes are now being sequenced at an increasing rate, creating a need for accurate, low-cost annotation of exon-intron structures. In this paper, we demonstrate that reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing based on predicted gene structures satisfy this need, at least for single-celled eukaryotes. The TWINSCAN gene prediction algorithm was adapted for the fungal pathogen Cryptococcus neoformans by using a precise model of intron lengths in combination with ungapped alignments between the genome sequences of the two closely related Cryptococcus varieties. This approach resulted in 60% of known genes being predicted exactly right at every coding base and splice site. When previously unannotated TWINSCAN predictions were tested by RT-PCR and direct sequencing, 75% of targets spanning two predicted introns were amplified and produced high-quality sequence. When targets spanning the complete predicted open reading frame were tested, 72% of them amplified and produced high-quality sequence. We conclude that sequencing a small number of expressed sequence tags (ESTs) to provide training data, running TWINSCAN on an entire genome, and then performing RT-PCR and direct sequencing on all of its predictions would be a cost-effective method for obtaining an experimentally verified genome annotation.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.2816704. Article published online before print in October 2004.

⁴ Present address: Dept. of Biomolecular Engineering, Univ. of California-Santa Cruz, Santa Cruz, California 95064, USA.

⁵ Corresponding author.
E-mail brent{at}cse.wustl.edu; fax (314) 935-7302.

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