Gene prediction and verification in a compact genome with numerous small introns.
Journal Article (Journal Article)
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 approximately 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.
- Tenney, AE; Brown, RH; Vaske, C; Lodge, JK; Doering, TL; Brent, MR
- November 2004
Volume / Issue
- 14 / 11
Start / End Page
- 2330 - 2335
Pubmed Central ID
International Standard Serial Number (ISSN)
Digital Object Identifier (DOI)
- United States