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

Aaron E. Tenney, Randall H. Brown, Charles Vaske, Jennifer K. Lodge, Tamara L. Doering, Michael R. Brent

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

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 CESTs) 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.

Original languageEnglish
Pages (from-to)2330-2335
Number of pages6
JournalGenome research
Volume14
Issue number11
DOIs
StatePublished - Nov 1 2004

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