Gene prediction and verification in a compact genome with numerous small introns.
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.
Laboratory for Computational Genomics and Department of Computer Science, Washington University, St. Louis, Missouri 63130, USA.
Aaron E
AE
Tenney
Randall H
RH
Brown
Charles
C
Vaske
Jennifer K
JK
Lodge
Tamara L
TL
Doering
Michael R
MR
Brent
D000465:Algorithms; D019295:Computational Biology; D003455:Cryptococcus neoformans / Q000235:genetics*; D016681:Genome, Fungal; D007438:Introns / Q000235:genetics*; D011237:Predictive Value of Tests; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software